a case study catalase activity quizlet

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7 Lab 6. Enzymes – Catalase Function

Lab 6:  enzymes – catalase function.

• Practice and apply hypothesis testing.
• Practice experimental design.
• Gain a better understanding of enzymes and some conditions that affect enzyme activity and the rate of an enzyme-catalyzed reaction.
• Understand these terms: enzyme, enzyme activity, active site, substrate, enzyme-substrate complex, product, denature, competitive inhibition, and noncompetitive inhibition.

INTRODUCTION

Enzymes are proteins that increase the rate of chemical reactions.  This process is called catalysis .  All cells use enzymes for metabolism , the sum of all chemical and physical reactions that a cell uses to break down nutrients to produce energy and to build up important molecules needed for cell function.  Enzymes increase the rate of a reaction by lowering activation energy but are not themselves consumed in the reaction.  Thus, one enzyme can catalyze repeated rounds of the same reaction.

As with all proteins, an enzyme’s function depends completely on its shape.   Enzymes have a very complex three-dimensional structure consisting of one or more polypeptide chains folded to form an active site – a specific area into which the substrate (material to be acted on by the enzyme) will fit. The shape of the enzyme determines which reaction it will catalyze, because only one substrate will fit into its active site.

Changes in temperature, pH, and/or the addition of certain ions or molecules may affect the structure of an enzyme’s active site and thus the ability of the enzyme to catalyze the reaction (the enzyme’s “enzyme activity”).  Changing an enzyme’s shape so that it is no longer biologically active is called denaturation .  Enzyme inhibitors can occupy the active site (competitive inhibitors) or change the shape of the active site by binding elsewhere (noncompetitive inhibitors). Hence, these factors also affect the rate of the reaction in which the enzyme participates.  The rate of an enzymatic reaction can also be affected by the relative concentrations of enzyme and substrate.

Salt concentration: If the salt concentration is very low or zero, the charged amino acid side-chains of the enzyme will stick together. The enzyme will denature and form an inactive precipitate. If, on the other hand, the salt concentration is very high, normal interaction of charged groups will be blocked, new interactions occur, and again the enzyme will precipitate. An intermediate salt concentration such as that of blood (0.9%) or cytoplasm is optimum for most enzymes.

pH: pH is a logarithmic scale that measures the acidity or H+ concentration in a solution. As the pH is lowered, an enzyme will tend to gain H+ ions, and eventually enough side chains will be affected so that the enzyme’s shape is disrupted. Likewise, as the pH is raised, the enzyme will lose H+ ions and eventually lose its active shape. Many enzymes have an optimum in the neutral pH range and are denatured at either extremely high or low pH. Some enzymes, such as those which act in the human stomach where the pH is very low, will have an appropriately low pH optimum. A buffer is a compound that will gain or lose H+ ions so that the pH changes very little.

Temperature: All chemical reactions speed up as the temperature is raised. As the temperature increases, more of the reacting molecules have enough kinetic energy to undergo the reaction. Since enzymes are catalysts for chemical reactions, enzyme reactions also tend to go faster with increasing temperature. However, if the temperature of an enzyme-catalyzed reaction is raised still further, an optimum is reached: above this point the kinetic energy of the enzyme and water molecules is so great that the structure of the enzyme molecules starts to be disrupted. The positive effect of speeding up the reaction is now more than offset by the negative effect of denaturing more and more enzyme molecules. Many proteins are denatured by temperatures around 40-50C, but some are still active at 70-80C, and a few even withstand being boiled.

In this exercise you will study the enzyme catalase, which accelerates the breakdown of hydrogen peroxide (a common end product of oxidative metabolism) into water and oxygen, according to the summary reaction:

          2H 2 O 2 + Catalase —-> 2H 2 O + O 2 + Catalase

Catalase is found in animal and plant tissues, and is especially abundant in plant storage organs such as potato tubers, corms, and in the fleshy parts of fruits. You will isolate catalase from potato tubers and measure its rate of activity under different conditions.

The fact that one of the products of the above reaction (oxygen) forms a gas, it is convenient to observe the conversion from reactants to products. A small piece of filter paper can be soaked in a solution of catalase and immersed in a solution of hydrogen peroxide. It will immediately sink to the bottom of the vessel. As the catalase converts the hydrogen peroxide to water and oxygen, some of the oxygen gas accumulates on the disk, changing buoyancy of the disk and floating to the surface. The elapsed time from immersion to floatation is thus proportional to the rate of the reaction (the time required to produce sufficient product (O 2 ) to float the disk). This allows us to explore the effect of a number of variables on the rate of a reaction.

Part A : Extraction of Catalase

• Peel a fresh potato tuber and cut the tissue into small cubes. Weigh out 50 g of tissue.
• Place the tissue, 50 ml of cold distilled water, and a small amount of crushed ice in a pre-chilled blender.
• Homogenize for 30 seconds at high speed. From this point on, the enzyme preparation must be carried out in an ice bath.
• Filter the potato extract, then pour the filtrate into a 100-ml graduated cylinder. Add cold distilled water to bring up the final volume to 100 ml. Mix well. This extract will be arbitrarily labeled 100 units of enzyme per ml (100 units/ml). (NOTE – this will be enough catalase for all lab groups)

Work in groups of 4.

• Tape or permanent marker for labeling
• Five 50 ml beakers or large test tubes.
• Stock 3% H 2 O 2
• Catalase solution
• No2 Whatman filter paper
• Hole punchers
• Beaker with water
• Water baths at the following temperatures (4, 25, 37, 60 o C). These will be beakers on hot plates with temperature monitored by a thermometer.

PART B: Effect of Substrate Concentration

Before beginning, make a hypothesis and prediction about the scientific questions we are asking.

Q1. How does substrate concentration affect the speed at which catalase converts H 2 O 2 into water and oxygen?

Ha (Alternative hypothesis); H0 (Null hypothesis)

Write your scientific prediction (remember to use if—then—).

What are independent and dependent variables in this experiment?

• Place 20 ml stock 3% H 2 O 2 in one of the beakers or test tubes and label. Make sure your label identifies your group and concentration of H 2 O 2 .
• Dilute stock 3% H 2 O 2 to make 1.5%, 0.75%, 0.38% and 0.19%. You should figure out how to do this by serial dilution of your 3% H 2 O 2 stock. All beakers or test tubes should contain 10 ml except for the last beaker containing 0.19% H 2 O 2 .
• Make 25 small filter paper disks with a hole punch.
• With a forceps, grab a disk and immerse it in catalase solution for 3 seconds. BE PRECISE WITH THE TIME IT IS IMMERSED IN THE ENZYME. Immediately drop it into one of the solutions of H 2 O 2 and start your timer. If you look closely, you will see bubbles of O 2 forming on the disk. Stop your timer the moment the paper disk rises to the surface. Remove the paper disk and repeat the process four more times.
• Repeat step 4 for each of the concentration of H 2 O 2 .
• Record your data in a table.

DATA ANALYSIS

Graph your results by hand in your lab notebook. Plot the independent variables on X axis, dependent variable on Y axis. Use the averages of your replicates for the graph.

PART C: The effect of temperature on enzyme function and the reaction

Before beginning, make some hypothesis and predictions about the scientific questions we are asking.

Q1. How does temperature affect the speed at which catalase converts hydrogen peroxide into water and oxygen?

Ha (Alternative hypothesis)

H0 (Null hypothesis)

Write your scientific prediction here (remember to use if—then—)

• Prepare 5 test tubes with 10 ml each of 1.5% H 2 O 2 solution, and label them to identify your group and temperature. Keep one of your test tubes immersed in water at room temperature, the others in water baths at 4 o C (on ice), 37 o C, and 60 o C, respectively.
• Make 25 more filter paper disks.
• Measure the reaction rate as you did in part 1, by dipping each filter paper into catalase for 3 seconds, then recording the time it takes for a disk to rise to the top of each test tube after dropped to the bottom. As in part 1, repeat this procedure with 5 filter paper per test tube.

Post-lab Questions – Answer these questions in your lab notebook

• What were your conclusions regarding the effect of substrate concentration on catalase activity?
• What were your conclusions regarding the effect of temperature on catalase activity? Identify the optimum temperature on catalase activity, in other words, at what temperature was the amount of product produced by the enzyme-catalyzed reaction greatest ?
• If an enzyme were isolated from an organism, such as a clam, that lived in seawater that averages 14°C, what would you predict would be the optimal temperature for that enzyme, and why?
• Define enzyme denaturation in terms of protein structure.

LWTech General Biology (BIOL&160) Lab Protocols Copyright © by Lake Washington Institute of Technology is licensed under a Creative Commons Attribution 4.0 International License , except where otherwise noted.

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Optional Lab Activities

Lab objectives.

At the conclusion of the lab, the student should be able to:

• define the following terms: metabolism, reactant, product, substrate, enzyme, denature
• describe what the active site of an enzyme is (be sure to include information regarding the relationship of the active site to the substrate)
• describe the specific action of the enzyme catalase, include the substrate and products of the reaction
• list what organelle catalase can be found in every plant or animal cell
• list the factors that can affect the rate of a chemical reaction and enzyme activity
• explain why enzymes have an optimal pH and temperature to ensure greatest activity (greatest functioning) of the enzyme (be sure to consider how virtually all enzymes are proteins and the impact that temperature and pH may have on protein function)
• explain why the same type of chemical reaction performed at different temperatures revealed different results/enzyme activity
• explain why warm temperatures (but not boiling) typically promote enzyme activity but cold temperature typically
• decreases enzyme activity
• explain why increasing enzyme concentration promotes enzyme activity
• explain why the optimal pH of a particular enzyme promotes its activity
• if given the optimal conditions for a particular enzyme, indicate which experimental conditions using that particular enzyme would show the greatest and least enzyme activity

Introduction

Hydrogen peroxide is a toxic product of many chemical reactions that occur in living things. Although it is produced in small amounts, living things must detoxify this compound and break down hydrogen peroxide into water and oxygen, two non-harmful molecules. The organelle responsible for destroying hydrogen peroxide is the peroxisome using the enzyme catalase. Both plants and animals have peroxisomes with catalase. The catalase sample for today’s lab will be from a potato.

Enzymes speed the rate of chemical reactions. A catalyst is a chemical involved in, but not consumed in, a chemical reaction. Enzymes are proteins that catalyze biochemical reactions by lowering the activation energy necessary to break the chemical bonds in reactants and form new chemical bonds in the products. Catalysts bring reactants closer together in the appropriate orientation and weaken bonds, increasing the reaction rate. Without enzymes, chemical reactions would occur too slowly to sustain life.

The functionality of an enzyme is determined by the shape of the enzyme. The area in which bonds of the reactant(s) are broken is known as the active site. The reactants of enzyme catalyzed reactions are called substrates. The active site of an enzyme recognizes, confines, and orients the substrate in a particular direction.

Enzymes are substrate specific, meaning that they catalyze only specific reactions. For example, proteases (enzymes that break peptide bonds in proteins) will not work on starch (which is broken down by the enzyme amylase). Notice that both of these enzymes end in the suffix -ase. This suffix indicates that a molecule is an enzyme.

Environmental factors may affect the ability of enzymes to function. You will design a set of experiments to examine the effects of temperature, pH, and substrate concentration on the ability of enzymes to catalyze chemical reactions. In particular, you will be examining the effects of these environmental factors on the ability of catalase to convert H 2 O 2 into H 2 O and O 2 .

The Scientific Method

As scientists, biologists apply the scientific method. Science is not simply a list of facts, but is an approach to understanding the world around us. It is use of the scientific method that differentiates science from other fields of study that attempt to improve our understanding of the world.

The scientific method is a systematic approach to problem solving. Although some argue that there is not one single scientific method, but a variety of methods; each of these approaches, whether explicit or not, tend to incorporate a few fundamental steps: observing, questioning, hypothesizing, predicting, testing, and interpreting results of the test. Sometimes the distinction between these steps is not always clear. This is particularly the case with hypotheses and predictions. But for our purposes, we will differentiate each of these steps in our applications of the scientific method.

You are already familiar with the steps of the scientific method from previous lab experiences. You will need to use your scientific method knowledge in today’s lab in creating hypotheses for each experiment, devising a protocol to test your hypothesis, and analyzing the results. Within the experimentation process it will be important to identify the independent variable, the dependent variable, and standardized variables for each experiment.

Part 1: Observe the Effects of Catalase

• Obtain two test tubes and label one as A and one as B.
• Use your ruler to measure and mark on each test tube 1 cm from the bottom.
• Fill each of two test tubes with catalase (from the potato) to the 1 cm mark
• Add 10 drops of hydrogen peroxide to the tube marked A.
• Add 10 drops of distilled water to the tube marked B.
• Bubbling height tube A
• Bubbling height tube B
• What happened when H 2 O 2 was added to the potato in test tube A?
• What caused this to happen?
• What happened in test tube B?
• What was the purpose of the water in tube B?

Part 2: Effects of pH, Temperature, and Substrate Concentration

Observations.

From the introduction and your reading, you have some background knowledge on enzyme structure and function. You also just observed the effects of catalase on the reaction in which hydrogen peroxide breaks down into water and oxygen.

From the objectives of this lab, our questions are as follows:

• How does temperature affect the ability of enzymes to catalyze chemical reactions?
• How does pH affect the ability of enzymes to catalyze chemical reactions?
• What is the effect of substrate concentration on the rate of enzyme catalyzed reactions?

Based on the questions above, come up with some possible hypotheses. These should be general, not specific, statements that are possible answers to your questions.

• Temperature hypothesis
• pH hypothesis
• Substrate concentration hypothesis

Test Your Hypotheses

Based on your hypotheses, design a set of experiments to test your hypotheses. Use your original experiment to shape your ideas. You have the following materials available:

• Catalase (from potato)
• Hydrogen peroxide
• Distilled water
• Hot plate (for boiling water)
• Acidic pH solution
• Basic pH solution
• Thermometer
• Ruler and wax pencil

Write your procedure to test each hypothesis. You should have three procedures, one for each hypothesis. Make sure your instructor checks your procedures before you continue.

• Procedure 1: Temperature
• Procedure 2: pH
• Procedure 3: Concentration

Record your results—you may want to draw tables. Also record any observations you make. Interpret your results to draw conclusions.

• Do your results match your hypothesis for each experiment?
• Do the results reject or fail to reject your hypothesis and why?
• What might explain your results? If your results are different from your hypothesis, why might they differ? If the results matched your predictions, hypothesize some mechanisms behind what you have observed.

Communicating Your Findings

Scientists generally communicate their research findings in written reports. Save the things that you have done above. You will be use them to write a lab report a little later in the course.

Sections of a Lab Report

• Title Page:  The title describes the focus of the research. The title page should also include the student’s name, the lab instructor’s name, and the lab section.
• Introduction:  The introduction provides the reader with background information about the problem and provides the rationale for conducting the research. The introduction should incorporate and cite outside sources. You should avoid using websites and encyclopedias for this background information. The introduction should start with more broad and general statements that frame the research and become more specific, clearly stating your hypotheses near the end.
• Methods:  The methods section describes how the study was designed to test your hypotheses. This section should provide enough detail for someone to repeat your study. This section explains what you did. It should not be a bullet list of steps and materials used; nor should it read like a recipe that the reader is to follow. Typically this section is written in first person past tense in paragraph form since you conducted the experiment.
• Results:  This section provides a written description of the data in paragraph form. What was the most reaction? The least reaction? This section should also include numbered graphs or tables with descriptive titles. The objective is to present the data, not interpret the data. Do not discuss why something occurred, just state what occurred.
• Discussion:  In this section you interpret and critically evaluate your results. Generally, this section begins by reviewing your hypotheses and whether your data support your hypotheses. In describing conclusions that can be drawn from your research, it is important to include outside studies that help clarify your results. You should cite outside resources. What is most important about the research? What is the take-home message? The discussion section also includes ideas for further research and talks about potential sources of error. What could you improve if you conducted this experiment a second time?
• Biology 101 Labs. Authored by : Lynette Hauser. Provided by : Tidewater Community College. Located at : http://www.tcc.edu/ . License : CC BY: Attribution
• BIOL 160 - General Biology with Lab. Authored by : Scott Rollins. Provided by : Open Course Library. Located at : http://opencourselibrary.org/biol-160-general-biology-with-lab/ . License : CC BY: Attribution

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3.4: Catalase

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• Page ID 14681

• Molly Smith and Sara Selby
• South Georgia State College via GALILEO Open Learning Materials

Catalase is an enzyme that breaks hydrogen peroxide into water and oxygen. Hydrogen peroxide is a common byproduct of metabolic reactions occurring in an environment where water and oxygen are present, but it is toxic to cells. Therefore, most organisms that survive in the presence of oxygen contain enzymes to degrade the hydrogen peroxide.

• Start with a clean microscope slide and place a drop of hydrogen peroxide on it.
• Using an inoculating loop, remove some of your assigned organism and put the loop with the organism on it into the drop of hydrogen peroxide. Do not stir.
• Observe the drop of hydrogen peroxide.

Interpretation

Bubbles will form around the organism on the loop if catalase is produced

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Article Contents

Introduction, the ssa-cat assay, ethics statement, results and discussion, limitations, acknowledgements, author contributions, conflict of interest statement, data availability.

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An improved method for measuring catalase activity in biological samples

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Mahmoud Hussein Hadwan, Marwah Jaber Hussein, Rawa M Mohammed, Asad M Hadwan, Hawraa Saad Al-Kawaz, Saba S M Al-Obaidy, Zainab Abbas Al Talebi, An improved method for measuring catalase activity in biological samples, Biology Methods and Protocols , Volume 9, Issue 1, 2024, bpae015, https://doi.org/10.1093/biomethods/bpae015

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Catalase (CAT) is an important enzyme that protects biomolecules against oxidative damage by breaking down hydrogen peroxide (H 2 O 2 ) into water and oxygen. CAT is present in all aerobic microbes, animals, and plants. It is, however, absent from normal human urine but can be detected in pathological urine. CAT testing can thus help to detect such urine. This study presents a novel spectrophotometric method for determining CAT activity characterized by its simplicity, sensitivity, specificity, and rapidity. The method involves incubating enzyme-containing samples with a carefully chosen concentration of H 2 O 2 for a specified incubation period. Subsequently, a solution containing ferrous ammonium sulfate (FAS) and sulfosalicylic acid (SSA) is added to terminate the enzyme activity. A distinctive maroon-colored ferrisulfosalicylate complex is formed. The formation of this complex is a direct result of the reaction between FAS and any residual peroxide present. This leads to the generation of ferric ions when coordinated with SSA. The complex has a maximum absorbance of 490 nm. This advanced method eliminates the need for concentrated acids to stop CAT activity, making it safer and easier to handle. A comparative analysis against the standard ferrithiocyanate method showed a correlation coefficient of 0.99, demonstrating the new method’s comparable effectiveness and reliability. In conclusion, a simple and reliable protocol for assessing CAT activity, which utilizes a cuvette or microplate, has been demonstrated in this study. This interference-free protocol can easily be used in research and clinical analysis with considerable accuracy and precision.

Oxidative stress is the main mechanism implicated in numerous pathologies and toxicities caused by xenobiotics [ 1 ]. The balance between oxidation and reduction in cells affects the signaling cascades of hydrogen peroxide (H 2 O 2 ) [ 2 ]. Although H 2 O 2 has essential signaling functions, it can also be hazardous [ 3 ]. H 2 O 2 can be hazardous in high concentrations as it can undergo a Fenton reaction with transition metal ions, producing hydroxyl radicals (OH • ) that cause oxidative damage to cellular components such as proteins, lipids, and DNA [ 4 ]. This can lead to changes in their structure and function, harming biological systems. The accumulation of oxidative damage can lead to chronic inflammation, accelerate aging, and play a role in the development of diseases such as cardiovascular diseases, neurodegenerative disorders, and certain cancers [ 5 ].

CAT activity is the primary mechanism for detoxifying and regulating H 2 O 2 levels [ 6 ]. CAT is an oxidoreductase enzyme (EC1.11.1.6), part of the antioxidant enzyme group, and can be found in the cells of mammals, plants, and aerobic bacteria [ 6 , 7 ]. Based on their structure and function, CATs can be classified into three main types. The first and second groups comprise heme-containing enzymes called typical or true CATs and CAT-peroxidases. The third group comprises non-heme manganese CATs [ 8 ]. The structure of CATs is a tetramer, with monomers made up of over 500 amino acids. This tetramer contains four porphyrin heme groups, which resemble those of hemoglobin, cytochromes, chlorophylls, and nitrogen-fixing enzymes [ 9 ]. CAT is present ubiquitously but is generally found in peroxisomes and has higher activity in kidneys, red blood cells (RBCs), and the liver [ 10 ]. CAT is closely related to peroxidases, both structurally and functionally, and has two functions: it reacts “peroxidatically” at lower concentrations of peroxide and “catalytically” at higher concentrations of peroxide [ 11 ].

CATs are predominately found in the peroxisome as it is the center of H 2 O 2 production due to purine catabolism, oxidative stress, fatty acid β-oxidation, and photorespiration [ 12 ]. CATs are also found in other cellular parts like mitochondria, chloroplast, and cytosol [ 13 ]. CAT plays a crucial role in protecting HepG2 cells from ROS, and specific inhibitors that reduce CAT activity decrease their resistance to ROS. Glutathione level does not affect cellular resistance to ROS. HepG2 cells strongly resist ROS-induced apoptosis due to higher CAT activity than HeLa and other cell types [ 14 ].

CAT activity is regulated by various factors such as substrate concentration, pH, temperature, and post-translational modifications such as phosphorylation [ 15 ]. When the H 2 O 2 concentration is high, CAT can be phosphorylated. This phosphorylation can then lead to the inhibition of the enzyme’s activity [ 8 ]. This inhibition is because phosphorylation can alter the enzyme’s conformation, making it less effective in catalyzing H 2 O 2 decomposition [ 16 ]. However, the complete mechanism of phosphorylation inhibiting CAT activity is still not fully understood [ 8 , 16 ].

Urinary tract infections (UTIs) can potentially change the level of CAT activity in urine samples. The presence of CAT activity can be used as a diagnostic indicator for certain diseases or infections [ 17 ]. However, it is important to note that relying solely on CAT activity may not be enough to establish a definitive diagnosis, and it should be used together with other clinical parameters. Normally, CAT is not found in urine [ 18 ]. However, in bacterial infections, bacteria can release CAT enzymes, which can be detected in urine samples. In UTIs, immune cells such as neutrophils can be found in urine in response to the infection [ 18 , 19 ]. Also, various pathologies can result in the presence of other cells, such as epithelial cells, in urine. However, the specific types of cells found in urine can vary depending on the underlying condition, and additional diagnostic tests may be necessary for accurate identification [ 19 ].

H 2 O 2 is a chemical compound actively produced by specialized cells, such as neutrophils [ 20 ]. These cells generate H 2 O 2 as a defense mechanism against infections or as a byproduct of enzymatic activities, including those catalyzed by mitochondrial monoamine oxidases [ 21 ]. However, the main source of H 2 O 2 arises from the conversion of the superoxide anion through the action of mitochondrial superoxide dismutase (SOD2) or cytosolic SOD1, which play a role in detoxification [ 22 ]. Apart from its detoxifying properties, H 2 O 2 functions as a transcription-independent signaling molecule. It contributes to redox sensing and regulation and is as indispensable as Ca 2+ or ATP [ 23 ]. In multicellular organisms, H 2 O 2 regulates various transcriptional elements and thus plays a crucial role in many biological processes. CAT is another enzyme that is crucial in maintaining physiological levels of H 2 O 2, and plays a vital role in both preventing its cytotoxic effects and externalizing it as a threat signal [ 24 ].

Measuring CAT activity is essential for determining the redox state when assessing xenobiotic toxicity. There are several protocols available for assaying CAT activity. The first protocol involves monitoring the breakdown of H 2 O 2 by CAT using UV spectroscopy. This method, however, requires large volumes of samples and can only measure one sample at a time [ 25 ]. The second method is titrimetric, which is suitable for tissues with low CAT levels, but this method has limitations in terms of practicality due to the large volume of samples required [ 2 , 25 ]. The third method involves monitoring O 2 generation to assess CAT activity. This method is simple, quick, and economical but is not well-suited to kinetic studies, and only one sample can be measured at a time [ 26 ].

An alternative method for measuring CAT activity involves observing the H 2 O 2 breakdown using a suitable gel. This method requires less sample volume than the abovementioned methods, but it is worth noting that it only provides a qualitative result [ 27 ].

Chemiluminescence can be employed to assess CAT activity, utilizing H 2 O 2 -sensitive Cadmium telluride quantum dots (CdTe-QD). This method enables a rapid and sensitive determination of CAT activity but requires a luminescence reader and an intermediary step involving H 2 O 2 and CdTe-QDs, which may impose certain limitations. Moreover, including CdTe-QDs in the assay increases the overall cost [ 28 ].

A previous study introduced a simple method to measure CAT activity using Pyrogallol Red (PGR) as a sensitive probe to measure H 2 O 2 levels [ 29 ]. The method relied on the catalytic effects of molybdenum [ 7 ]. Spectrophotometric methods use various chemical compounds to generate colored complexes. Two examples of light absorption by chemical complexes are the carbonate cobaltate (III) ([Co(CO 3 ) 3 ]Co) complex, which absorbs light at 440 nm [ 29 ], and the peroxovanadate complex (NH 4 [VO(O 2 )SO 4 ), which absorbs light at 452 nm [ 30 ].

A high-performance liquid chromatography assay was developed to measure human erythrocytic CAT activity in a previous study. The assay relies on glutathione analysis and employs a highly stable o-phthalaldehyde (OPA) derivative sensitive to H 2 O 2 . This method demonstrates suitability for measuring CAT activity at low concentrations, but it can be influenced by glutathione-related enzymes [ 31 ]. Maral et al . [ 32 ] employed a different method of assessing erythrocyte CAT activity across various species by measuring light emission from luminol oxidation catalyzed by horseradish peroxidase. The authors established a reference value of 100 for normal human blood CAT activity and expressed CAT activity in other animal species as a percentage of this reference value.

Different methods have been developed to measure the CAT activity of bacteria. One simple method is to use H 2 O 2 to determine if CAT-positive bacteria are present. These bacteria convert H 2 O 2 into oxygen, which produces bubbles [ 33 ]. Alternatively, methods, such as colorimetric and spectrophotometric assays, are more quantitative [ 34 ], but these can be costly and have some drawbacks, including complex procedures and the need for specialized kits [ 33 ].

Various methods have been developed for measuring CAT activity spectrophotometrically, but microplate-based methods are limited in number. The literature describes three microplate-based methods for measuring CAT activity. The first two methods are similar in principle and details [ 2 , 34 ], as they depend on following the dissociation of H 2 O 2 at a wavelength of 240 nm. Another microplate method involves assessing unreacted H 2 O 2 in a ferrithiocyanate system, as explained by Cohen et al . [ 35 ]. The ferrithiocyanate method relies on unreacted peroxide oxidizing Fe(II) to Fe(III). Subsequently, a complex is formed with potassium thiocyanate, which has a peak absorbance (λmax) of 480 nm.

The current protocol describes a simple microplate assay for CAT activity based on spectrophotometric detection of unreacted H 2 O 2 . The assay incubates enzyme-containing samples with a phosphate buffer containing suitable concentrations of H 2 O 2 . After a specified incubation period, the assay introduces a mixture of sulfosalicylic acid (SSA) and ferrous ammonium sulfate (FAS) to stop the enzyme reaction. SSA binds to the ferric ions produced from the interaction of FAS and residual peroxide, creating a maroon-colored ferrisulfosalicylate complex. This complex is then measured using a spectrophotometer at 490–500 nm. The SSA-CAT assay is unaffected by different types of biomolecules and does not require strong, concentrated acids or protein precipitation to stop the enzyme reaction. It is a rapid and efficient method for measuring CAT activity.

FAS hexahydrate [(NH 4 ) 2 Fe(SO 4 ) 2 (H 2 O) 6 ; MWT: 392.14 g/mol, CAS number: 7783-85-9], SSA [C 7 H 6 O 6 S; MWT: 218.185 g/mol, CAS number: 97-05-2], hydrochloric acid (HCl, CAS number: 7647-01-0), glacial acetic acid (CH 3 COOH, CAS number: 64-19-7), H 2 O 2 (30%, CAS number: 7722-84-1), monopotassium phosphate (KH 2 PO 4 MWT: 136.09 g/mol, CAS number: 7778-77-0), sodium azide (NaN 3 , CAS number: 26628-22-8), and sodium hydroxide (NaOH, CAS number: 1310-73-2), were purchased from Thomas Baker (Chemicals) Pvt. Ltd The standard CAT was purchased from HiMedia (product code TC037; India),

UV–visible spectra were measured using a Shimadzu Spectrophotometer 1301A, which is equipped with 1 cm quartz cells. The study used a BioTek ELx800 UV-Vis reader to measure the 96-well plate accurately with Gene version 5 Software. All instruments and software were purchased from Aflo Company for Medical and Laboratory Equipment (Baghdad, Iraq).

Two solutions were prepared to create a pH 7.4, 50 mM phosphate buffer. Solution (i) is 6.81 g of KH 2 PO 4 dissolved in 1 L of distilled water (DW), and solution (ii) is 8.90 g of Na 2 HPO 4 .2H 2 O dissolved in 1 L of DW. The two solutions were mixed in a 1:1.5 ratio to create a freshly prepared phosphate buffer. To prepare a 10 mM H 2 O 2 solution, 0.34 mL of 30% (v/v) H 2 O 2 was carefully diluted with the above phosphate buffer and adjusted to a final volume of 100 mL. This solution was freshly prepared and standardized daily, employing a molar extinction coefficient of 43.6 M –1  cm –1 at 240 nm. PBS-H 2 O 2 -NaN 3 solution was prepared by dissolving 0.372 g EDTA, mM H 2 O 2 solution, and 0.6501 g of NaN 3 in 100 mL of PBS (pH 7.4, 50 mM). The final volume was adjusted to 100 mL with PBS. Standard potassium permanganate further standardized the diluted H 2 O 2 solution [ 36 ].

For the preparation of FAS (10 mM), 0.4 g of FAS was dissolved in 100 mL of 7% (v/v) glacial acetic acid solution. Similarly, to prepare SSA (10 mM), 1.09 g of SSA was dissolved in 100 mL of 7% (v/v) glacial acetic acid solution. To prepare the working solution freshly, 100 mL of FAS solution and 100 mL of SSA solution were accurately measured and thoroughly mixed together. Protein concentration was measured by Bradford Protein Colorimetric Assay Kit (Cat. No.: E-BC-K168-M).

Blood samples

Three milliliters of whole blood were collected and placed into a heparin tube to prepare erythrocyte lysates. The tube was centrifuged for 10 min at 400 ×  g to separate the plasma fractions and buffy-coat cells. The RBCs were washed thrice with 500 μL of 0.9% sodium chloride solution. After washing, 500 μL of the erythrocyte mixes were mixed with 2 mL of ice-cold double-DW. The mixture was vortexed for ten seconds and left in the dark for 15 min at 4°C. The resulting stock hemolysate was diluted further with a dilution factor 500 and resuspended in 50 mM phosphate buffer solution (PBS). Finally, the diluted hemolysate solutions were used as a source of CAT activity.

Tissue preparation

Male albino rats and mice were obtained from the Bioscience Department, University of Babylon (Iraq) animal house for the experimental investigation. The liver tissues of the animals were surgically removed before assessing CAT activity. The liver was extensively cleaned with a 0.9% (w/v) NaCl solution to ensure the elimination of blood and other contaminants. The liver was then homogenized using a glass homogenizer in cold 1.15% (w/v) KCl. The homogenate was filtered through two layers of muslin to remove cellular debris and large particles. The resulting mixture was then diluted with 50 mM PBS at a ratio of 1:500. This diluted liver homogenate was used for subsequent CAT-activity assays.

Standard methods for quantifying CAT activity

The thiocyanate method was utilized as a reference protocol [ 35 ].

The UV-kinetic method

H 2 O 2 was prepared in 50 mM phosphate buffer (pH 7.4) to create a final concentration of 5 mM. Next, 1000 μL of substrate solution was rapidly added to a cuvette with 25 μL of the sample. The cuvette was scanned in a spectrophotometer every 10 s for 5 min at 25°C using a wavelength of 240 nm. The CAT activity was calculated based on the rate of H 2 O 2 decomposition, which is proportional to the reduction of absorbance at 240 nm [ 2 , 25 ].

Cuvette spectrophotometric protocol

In a water bath, 2 mL of 10 mM peroxide and 1 mL of diluted CAT sample were incubated at 37°C for 2 min. After completing the enzymatic reaction, 100 μL aliquots were transferred to a clean test tube containing 3 mL of a working solution. The test tube was vortexed and incubated at 25°C for 5 min. Finally, the absorbance was measured at 490 nm.

In a blank test tube, DW was used instead of CAT enzyme and H 2 O 2 . In a standard test tube, DW was used instead of CAT enzyme. In a control test tube, DW was used instead of a H 2 O 2 solution.

Microplate protocol

A 96-well plate was prepared with 100 µL of 5 mM peroxide mixed with 20 µL of CAT sample. The plate was incubated at 37°C for 5 min. Following this, 130 µL of working reagent was added to each well and mixed. The plate was then incubated for a further 5 min at 25°C. Finally, the absorbance was measured at 490 nm.

In a blank well, DW was used instead of CAT enzyme and H 2 O 2 . In a standard well, DW was used instead of CAT enzyme. In a control well, DW was used instead of H 2 O 2 solution.

We used the microplate protocol for all practical experiments in this study. However, the microplate and cuvette protocols yielded identical results when practically compared.

Calculation

S°: absorbance of the standard tube.

S: absorbance of the test tube.

M: absorbance of control test (correction factor).

Vt: total volume (ml) of test tube.

Vs: volume of sample (ml).

During the procedure, it is necessary to eliminate interferences that arise from the presence of sugars, amino acids, proteins, and vitamins in the sample. We apply a correction factor known as the Control test to do this.

In this method, the absorbance observed in the test tube is attributed to two categories of substances: unreacted H 2 O 2 and interferences present in the sample. However, in the control test tube, the absorbance solely arises from the interfering compounds found in the sample. By subtracting the absorbance of the control tube from that of the test tube, we can eliminate the influence of oxidizing compounds in the sample. Consequently, the remaining absorbance exclusively corresponds to unreacted H 2 O 2 .

The interfering activity and matrix effect

The term “matrix effect” refers to the impact of other sample components on an analytical assay besides the analyte being tested [ 37 ]. For instance, in the CAT assay, the presence of glutathione peroxidase (GPx) in biological samples can potentially interfere with the results. However, a corrected CAT activity can be measured to mitigate such interference. Eliminating any matrix effect interference on CAT activity is a relatively simple process. An interfering activity test tube was incorporated into the assay design to counteract any interference caused by the GPx enzyme present in the sample being used.

The test tube’s CAT activity is the sum of the H 2 O 2 -dissociation activity of CAT activity and GPx activity. However, the interfering activity only reflects the H 2 O 2 -dissociation activity of the GPx enzyme. To ensure the accuracy of the present method, GPx activity was eliminated. This was done by subtracting the interfering H 2 O 2 -dissociation activity from the total H 2 O 2 -dissociation activity. This subtraction guarantees that the remaining H 2 O 2 -dissociation activity solely represents the precise CAT activity. Therefore, the measurement obtained is free from interfering factors.

The homogenous solution of liver tissues of the male albino rats was applied to assess precise CAT activity. The final CAT activity was adjusted to 500 katal/L using the carbonato-cobaltate complex method [ 29 ].

Interfering H 2 O 2 -dissociation activity

An above 96-well plate was applied to assess interfering activity using PBS-H 2 O 2 -NaN 3 solution instead of the original PBS-H 2 O 2 solution. Sodium azide (NaN 3 ) is added to inhibit the CAT enzyme and prevent its interaction with GPx activity. DW was used in a blank well instead of CAT enzyme and H 2 O 2 . In a standard well, DW was used instead of CAT enzyme. In a control well, DW was used instead of H 2 O 2 solution.

Precise CAT activity calculation

Precise CAT activity: the H 2 O 2 -dissociation activity of CAT activity.

Total CAT activity: the H 2 O 2 -dissociation activity of CAT activity and GPx activity.

Interfering CAT activity: the H 2 O 2 -dissociation activity of GPx activity.

Signal stability

A standard CAT solution (0.5 U/mL) was utilized to evaluate the stability of the maroon-colored chelate complex. The working solution was prepared with equal volumes of FAS and SSA. Absorbance measurements were taken at 490 nm at specific intervals, including 15, 30, 45, and 60 min, 5 h, one day, three days, and one week. This systematic method enabled us to monitor the long-term stability and persistence of the maroon-colored chelate complex.

Linearity and sensitivity

This study evaluated the linearity and sensitivity using various CAT concentrations ranging from 0 to 8.0 U/mL. To prepare the standard CAT solution, 20 mg of standard powder (HiMedia, product code TC037; India) was dissolved in 100 mL phosphate buffer (50 mM, pH 7.0). The final CAT activity was adjusted to 8 U/mL using the carbonato-cobaltate complex method [ 29 ]. To assess the linearity of the method, it was compared to unreacted H 2 O 2 using the ferrithiocyanate method [ 35 ], and the absorbance at 240 nm [ 34 ] was monitored using the UV-kinetic method. This comparison was conducted using a web-based program that estimates bias and compares analytical methods [ 38 ]. Limits of quantitation (LOQ) and detection (LOD) were estimated to determine the sensitivity of the SSA-CAT assay. These parameters are important to assess the lower limits of reliable quantification and detection of CAT activity within the assay [ 39 ].

Selectivity, reproducibility, and accuracy

The robustness of the present CATCAT method was evaluated by conducting experiments with several types of interfering biomolecules. These biomolecules were dissolved in a phosphate buffer and divided into four flasks. The first flask contained only the buffer, while the second contained ribose, sucrose, glucose, and xylose; the third flask contained histidine, leucine, valine, and methionine; and the fourth flask contained bovine serum albumin and casein. The test-method accuracy in the presence of these biomolecules was determined by obtaining assay recovery values for each mixture. The results, summarized in Table 1 , demonstrated that the CAT assay accurately measured CAT activity even in the presence of tested biomolecules. The table provides information on the correlation between the biomolecules and the observed percentage errors. The experiments used a standardized CAT activity level and involved enzymatic reactions with the biomolecule solutions.

The correlation between measured the CAT activity and the incubation.

Mean of triplicate measurement.

Biological samples from male albino rats and mice were used to assess the method’s reproducibility. The rats’ livers were surgically removed, washed, and homogenized in a cold KCl solution. The resulting liver homogenate was filtered and diluted with PBS. This diluted sample served as a source of CAT activity. The intra- and inter-day reproducibility experiments measured the variability in CAT activity within a single day and across multiple days, respectively. The results were presented in terms of the relative standard deviation (RSD).

The ferrisulfosalicylate and ferrithiocyanate methods were compared using Bland–Altman analysis [ 40 ] and Passing–Bablok regression [ 41 ]. GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA) was utilized for statistical analysis.

Ethics Committee (University of Babylon/College of Science/Iraq), Ref. no.: 2148A Date: 3/9/2023.

The Institutional Research Ethics Committee approved this research, and each participant completed an informed consent form. Ethics Committee (University of Babylon/College of Science), Reference number of approval: 2157A; Date: 23/12/2023.

Statistical analysis

Data analysis was performed using GraphPad Prism version 8 statistical software (GraphPad Software, San Diego, CA, USA). The findings were reported as mean values accompanied by standard deviations. Student’s t-tests and Pearson correlations were employed to compare the studied parameters. A significance level of P  < .05 was considered statistically significant.

Adding a reagent comprising FAS and SSA effectively terminates the CAT enzymatic reaction, as depicted in Scheme 1 . After the CAT has consumed a significant portion of the H 2 O 2 , any residual H 2 O 2 reacts with ferrous ions (Fe 2+ ), leading to oxidation to ferric ions (Fe 3+ ). Subsequently, salicylic acid chelates with the ferric ions, forming a complex known as ferrisulfosalicylate. This complex exhibits a distinctive maroon color, with its absorption reaching a maximum at 490 nm [ 42 ].

Figure 1 shows a single peak at 490–500 nm, which reflects the absorbance of ferrisulfosalicylate as a function of the residual peroxide concentration from the CAT enzyme reactions. This result confirms the correlation between the absorbance values and the residual peroxide levels and supports the assay’s ability to quantify CAT activity.

The absorbance of the ferrisulfosalicylate complex shows an inverse relationship with the activity of the CAT enzyme. The figure shows the absorption spectra of the ferrisulfosalicylate complex. The concentrations of H 2 O 2 were (a) 10 mM H 2 O 2 (0.54 katal unit), (b) 8 mM H 2 O 2 (1.48 katal unit), (c) 6 mM H 2 O 2 (2.68 katal unit), (d) 4 mM H 2 O 2 (3.7 katal unit), (e) 2 mM H 2 O 2 (5.01 katal unit), (f) and 1 mM H 2 O 2 (5.84 katal unit).

The effectiveness of the working solution in inhibiting the CAT enzymatic reaction was evaluated before starting the practical experiments. Three test tubes containing a standard CAT enzymatic activity of 8 U. mL −1 were treated with the FAS, SSA, and a combination of both (FAS/SSA) solutions. CAT activity was monitored at 240 nm. The CAT enzymatic reaction was initiated by adding freshly prepared CAT solution (8 U.mL −1 ). The results demonstrated that the FAS solution immediately stopped the CAT enzyme reaction upon addition. In comparison, the SSA solution reduced the CAT enzymatic reaction by approximately 22%. The working solution (FAS/SSA) completely inhibited the CAT reaction and exhibited similar results to the FAS solution. Figure 2 clarifies the detailed results.

Decomposition of H 2 O 2 over time using the CAT enzyme. The decrease in H 2 O 2 concentration was measured by monitoring the absorbance at a wavelength of 240 nm. Three test tubes were used, each containing 3 mL of 5 mM H 2 O 2 . The reaction was initiated by adding 50 µL of 8 katal units of CAT enzyme, and the absorbance was monitored at 240 nm for 7 min. The reaction was stopped by adding 1 mL of either SSA solution (A), FAS solution (B), or working solution (C).

This study determined the optimal incubation time for CAT through a specific experiment. The optimal incubation time ranged from 2 to 6 min, and Table 1 documents the results. These results were found to be consistent with previous studies. Li and Schellhorn [ 34 ] monitored CAT enzyme activity using 240 nm absorbance and concluded that precise results could be obtained after 1 min of incubation. Similarly, Goth [ 43 ] measured CAT activity after 1 min of incubation.

This research assessed precise CAT activity in liver tissue homogenates. The results presented in Table 2 show precise CAT activity, total H 2 O 2 -dissociation activity, and interfering H 2 O 2 -dissociation activity.

The precise CAT activity, total H 2 O 2 -dissociation activity, and interfering H 2 O 2 -dissociation activity were measured using the ferrisulfosalicylate method.

Each value was expressed as the mean of five replicates.

In animals, CAT is found in peroxisomes, while GPx is found in mitochondria and cytosol. These two enzymes play complementary roles in decomposing endogenous H 2 O 2 [ 44 ]. However, although GPx contributes to the degradation of H 2 O 2 , its interference is excluded in this study. The results presented in Table 1 indicate that the GPx enzyme interferes with the current protocol by approximately 3%. The lack of noticeable interference of GPx with CAT assessment is attributed to the outstanding catalytic efficiency of the CAT compared to the GPx enzyme.

CAT is an enzyme that has the highest turnover numbers compared to all other enzymes [ 45 ]. According to the Braunschweig Enzyme Database (BRENDA), CAT can convert over 2.8 million H 2 O 2 molecules to water and oxygen per second using only one molecule [ 46 ]. Another study even suggests that the turnover numbers for CAT can be as high as 40 million [ 47 ]. Conversely, the BRENDA reports that the turnover numbers for GPx can range from 4.7 to 727.8 molecules per second. In brief, while GPx and CAT are essential for H 2 O 2 dissociation, CAT stands out with its extraordinary turnover number, making it a main enzymatic powerhouse that dissociates H 2 O 2 .

The findings of this study align with the previous research conducted by Mueller et al . [ 48 ], which provides comprehensive insights into the decomposition of H 2 O 2 in human erythrocytes, focusing on the roles of CAT and GPx in this process. Mueller et al . [ 48 ] reveal that the degradation of H 2 O 2 by CAT exhibits a linear dependence on the concentration of H 2 O 2 . This implies that as the concentration of H 2 O 2 increases, the activity of CAT in breaking down H 2 O 2 also increases proportionally. This linear relationship suggests that the activity of CAT is directly influenced by the concentration of H 2 O 2 , and higher concentrations of H 2 O 2 result in an increased rate of CAT-mediated degradation. The study establishes CAT as the main enzyme responsible for removing H 2 O 2 in human erythrocytes, particularly at H 2 O 2 concentrations above 10 −6  mol/L. In contrast, GPx becomes saturated at concentrations of H 2 O 2 greater than 10 −6  mol/L. This means that at higher concentrations of H 2 O 2 , the activity of GPx in breaking down H 2 O 2 reaches a maximum and does not further increase with additional increments in H 2 O 2 concentration. At a concentration of 10 −6  mol/L, CAT exhibits a degradation rate for H 2 O 2 that is approximately 12.5 times faster than GPx. However, when the concentration of H 2 O 2 is increased to 10 −4  mol/L, the rate significantly escalates to become 100 times faster than GPx. Consequently, CAT contributes almost exclusively to the overall turnover of H 2 O 2 at concentrations exceeding 10 −6  mol/L.

This study observed that the colored chelate maroon complex is highly stable at room temperature. Our measurements showed that the ferrisulfosalicylate complex’s absorbance at 490 nm remained stable for over a week at 25°C. The initial absorbance was 1.6, whereas the absorbance decreased to 1.593 after a week. The data are not shown here.

Sensitivity and linearity

Figure 3 shows a strong positive correlation (0.999) between the ferrithiocyanate and ferrisulfosalicylate methods. The line equation is y = 0.99x − 0.01, where y represents CAT activity measured by the ferrisulfosalicylate method and x represents CAT activity measured by the ferrithiocyanate method. In contrast, Fig. 4 compares the ferrisulfosalicylate method with the UV-kinetic method. The plot reveals a strong positive correlation (0.998) between the UV-kinetic and ferrisulfosalicylate methods. The line equation is y = 1.0072x − 0.0264, where y represents CAT activity measured by the ferrisulfosalicylate method and x represents CAT activity measured by the UV-kinetic method.

The linearity of the CAT activity method was determined by plotting a straight line between the ferrithiocyanate and ferrisulfosalicylate methods for a series of dilutions.

The linearity of the CAT activity method was determined by plotting a straight line between the UV-kinetic method and ferrisulfosalicylate methods for a series of dilutions.

The ferrisulfosalicylate method exhibits linearity for CAT enzyme activity values ranging from 0.1 to 8.0 U/mL. The LOQ and LOD values are 0.09 U/mL and 0.022 U/mL, respectively. These low LOQ and LOD values indicate high sensitivity of the SSA-CAT assay, enabling the detection of low levels of CAT enzyme activity. The linearity of the ferrisulfosalicylate method is comparable to that of the ferrithiocyanate and UV-kinetic methods, indicating its reliability in measuring CAT enzyme activity.

Reproducibility, selectivity, and accuracy of the SSA-CAT assay

Table 3 shows the results of an experiment examining the possibility of different biomolecules interfering with the ferrisulfosalicylate method. The lack of apparent interference indicates that the presence of these biomolecules did not significantly influence or distort CAT activity assessment when using our method, which increases the usefulness of the method.

Correlation between relative percentage errors and biological interference during CAT activity assessment utilizing the ferrisulfosalicylate method.

Flasks 1–4 are described in detail in the materials and methods above.

This research examined CAT activity in liver tissue homogenates. The results presented in Table 4 indicate that the CAT activity assessed using the ferrisulfosalicylate method corresponded to the levels obtained with the thiocyanate method. Furthermore, the assay’s intra-day precision was satisfactory, with RSD% values ranging from 3.49% to 3.86% ( Table 3 ). Similarly, the ferrisulfosalicylate assay’s inter-day precision assessment, which examines reproducibility across samples on various days, was considered satisfactory, with RSD% values ranging from 3.8% to 4.4% ( Table 3 ). These data validate the assay’s accuracy and precision under different experimental conditions. The low RSD% results for intra-day and inter-day precision indicate that the ferrisulfosalicylate method is accurate and precise for assessing hepatic CAT activity in liver tissue homogenates.

Comparison of CAT activities in diluted liver tissue homogenates (at a ratio of 1:500) using the ferrisulfosalicylate and thiocyanate methods.

Liver tissue homogenates of male albino rats (A) and mice (B).

The CAT activity can be used to assess the liver’s ability to reduce oxidative stress. Furthermore, the oxidant/antioxidant balance has been determined by several systematic investigations that have measured CAT activity in the livers of laboratory animals [ 49 , 50 ].

Validation and method comparison

The effectiveness of this assay for measuring CAT activities was verified by conducting Bland–Altman plot analyses using GraphPad Software in San Diego, CA, USA. To compare CAT activities, this study used ferrisulfosalicylate and ferrithiocyanate assays with paired enzymatic samples. The Bland–Altman plot in Fig. 5 shows the differences and the mean relative bias between the two methods. The correlation coefficient of 0.9968 between the ferrisulfosalicylate and the ferrithiocyanate methods confirms that this assay is as accurate as the reference method, as shown in Fig. 6 . The Passing–Bablok correlation analysis demonstrates a good agreement between the two methods, as shown in Fig. 5 . Pearson correlation also proved the correlation, with a Pearson r of more than 0.99 between the ferrisulfosalicylate method and the ferrithiocyanate method’s results for different samples.

Bland–Altman plot demonstrates the differences between ferrisulfosalicylate and ferrithiocyanate methods, including their mean relative bias. %Difference = ([The ferrithiocyanate method (katal unit)−The ferrisulfosalicylate method (katal unit)]/average) × 100; average =  [(The ferrithiocyanate method (katal unit) + The ferrisulfosalicylate method (katal unit)]/2.

CAT activity results were determined utilizing the ferrisulfosalicylate and ferrithiocyanate methods at various enzyme dilutions.

The reaction between ferric ion and SSA to form ferrisulfosalicylate complex.

Application I

This study also conducted experiments to assess CAT activity in lysates derived from five distinct bacterial laboratory strains. The primary aim was to explore further potential applications of the SSA-CAT method. The results revealed that the ferrisulfosalicylate method yielded CAT enzyme activities comparable to those obtained through the thiocyanate method across the bacterial strains. Our findings indicated that Staphylococcus aureus exhibited a noticeably higher CAT enzyme activity than other bacterial strains. For in-depth information and specific data, please refer to Table 5 .

Comparison of the SSA-CAT and thiocyanate methods for bacterial CAT activities (katal unit).

Application II

Analysis was performed on 100 urine samples obtained from patients who visited Prof. Dr Abdul Razzaq Alsalman’s private medical, Infertility, and Urology Clinic in Babylon Governorate, Hilla City, Iraq, between December 2023 and January 2024. The participants underwent a physical examination and provided a complete medical history. The Institutional Research Ethics Committee approved the study, and all participants signed an informed consent form.

Midstream urine samples were collected in sterilized, airtight plastic containers labeled with participant-specific codes. The samples were stored in a cold box during transportation to the laboratory. In the laboratory, the samples were subjected to urine analysis and urine culture using the method described by Berger et al .[ 51 ]. CAT activity was measured using the SSA-CAT assay.

Out of the 100 samples, 38 were positive for colony count and CAT determination (group I), indicating the presence of bacteria. Additionally, 57 samples that were negative for colony count also tested negative for CAT determination (group II). It was observed that the presence of RBCs in 5 urine samples led to a false positive result in CAT activity (group III) [ 46 ]. In conclusion, the SSA-CAT method proved to be a successful screening test for significant bacteriuria in CAT determinations. Table 6 shows detailed information and the CAT activity.

The urine CAT activities (katal unit) were obtained using the SSA-CAT method.

The samples were diluted ten times with PBS (pH 7.4, 50 mM).

Application III

The study comprised 100 male students from the College of Science at the University of Babylon, Iraq. The participants had an average age of 22.0 ± 2 years and a body mass index of 22.86 ± 1.2 kg/m 2 . Informed written consent was obtained from all volunteers after providing them with a clear explanation of the study’s purpose. The participants were then categorized into two groups: smokers and controls. The control group consisted of individuals with no smoking history, while the smokers had been smoking an average of 20 ± 5 cigarettes per day for over 2 years. All participants were non-alcoholics and were not afflicted with any chronic diseases. The institutional ethics committee approved the study before its initiation.

After an overnight fast, 5 mL of venous blood containing heparin was drawn. The blood was centrifuged at 3000 rpm for 10 min to separate the plasma from the erythrocytes. To obtain packed erythrocytes, the erythrocytes were washed multiple times with a 0.9% NaCl solution until a colorless supernatant was observed. To obtain erythrocyte hemolysate, 500 µL of packed erythrocytes were lysed by adding four volumes of cold redistilled water. The resulting mixture was centrifuged twice to remove all cell membranes: first, it was centrifuged for 10 min in a tube centrifuge at 3500 rpm at 4°C and then in an Eppendorf centrifuge at 7800 rpm for 5 min at 4°C [ 52 ]. The resulting clear supernatant was obtained as hemolysate for determining CAT activity.

According to Table 7 , the CAT activity in erythrocytes is significantly lower in smokers than in nonsmokers ( P  < .05). This finding suggests that tobacco smoking is associated with a reduction in CAT activity within erythrocytes. Smoking introduces harmful substances into the body, such as reactive oxygen species (ROS) and free radicals. These substances can induce oxidative stress, overpowering the body’s antioxidant defense systems, including CAT. Continuous exposure to tobacco smoke can disrupt the balance between ROS production and CAT’s ability to neutralize them, resulting in a decrease in CAT activity. These results align with previous studies [ 53 , 54 ] that have reported similar findings.

Comparison of erythrocyte CAT activity of tobacco smokers and non-smokers.

This study has limitations, such as the lack of kinetic information about the studied enzyme, including its kinetic parameters, enzyme-substrate binding affinity, and turnover numbers. These parameters help us understand the enzyme’s behavior and predict the effects of experimental conditions or modulators on the reaction. However, previous studies have provided all the necessary kinetic information about the CAT enzyme. The proposed method can be used to gain more insights into enzyme behavior and predict the effects of experimental conditions on the CAT enzymatic reaction.

The above protocol details a method for fast and precise measurement of CAT activity. This technique is not limited to microorganisms but can also be potentially used to estimate CAT activity in animal tissues, animal fluids, and plant tissues. Therefore, it is a versatile tool. The protocol involves the use of a microplate reader. The chemicals used in this method are more environmentally friendly than those used in the past, particularly the ferrithiocyanate method, which has high toxicity and environmental risks. By replacing thiocyanate with SSA, this method aligns with the principles of green chemistry.

The authors would like to express their gratitude to the University of Babylon (Iraq) for their financial and moral support, which played a significant role in completing the protocol. Additionally, the authors would like to thank the Dean of the College of Science at University College (Iraq) for their support and encouragement throughout the completion of the protocol.

Mahmoud Hussein Hadwan (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Funding acquisition [equal], Investigation [equal], Methodology [equal], Project administration [equal], Resources [equal], Software [equal], Supervision [equal], Validation [equal], Visualization [equal], Writing—original draft [equal], and Writing—review & editing [equal]), Marwah Jaber Hussein (Data curation [equal], Investigation [equal], Methodology [equal], Software [equal], and Writing—review & editing [equal]), Rawa M Mohammed (Data curation [equal], Investigation [equal], Methodology [equal], and Writing—review & editing [equal]), Asad Hadwan (Data curation [equal], Formal analysis [equal], Investigation [equal], Methodology [equal], Validation [equal], and Writing—review & editing [equal]), Saba S. M. Al-Obaidy (Data curation [equal], Investigation [equal], Methodology [equal], Validation [equal], and Writing—review & editing [equal]), Zainab Abbas Al Talebi (Formal analysis [equal], Investigation [equal], Methodology [equal], Validation [equal], and Writing—review & editing [equal]), and Hawraa Saad Al-Kawaz (Investigation [equal], Methodology [equal], Validation [equal], and Writing—review & editing [equal]).

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

The authors declare that all data supporting the findings of this study can be found within the article. Additional data supporting the findings of this study are available from the corresponding author upon request.

El-Demerdash FM , Tousson EM , Kurzepa J et al.  Xenobiotics, oxidative stress, and antioxidants . Oxid Med Cell Longev 2018 ; 2018 : 9758951 .

Google Scholar

Grilo LF , Martins JD , Cavallaro CH et al.  Development of a 96-well based assay for kinetic determination of catalase enzymatic-activity in biological samples . Toxicol in Vitro 2020 ; 69 : 104996 .

Di Marzo N , Chisci E , Giovannoni R. The role of hydrogen peroxide in redox-dependent signaling: homeostatic and pathological responses in mammalian cells . Cells 2018 ; 7 : 156 .

Yang H , Tai F , Wang T et al.  Hydrogen peroxide and iron ions can modulate lipid peroxidation, apoptosis, and the cell cycle, but do not have a significant effect on DNA double-strand break . Biochem Biophys Res Commun 2023 ; 651 : 121 – 6 .

Singh A , Kukreti R , Saso L et al.  Oxidative stress: a key modulator in neurodegenerative diseases . Molecules 2019 ; 24 : 1583 .

Bukowska B , Chajdys A , Duda W et al.  Catalase activity in human erythrocytes: effect of phenoxyherbicides and their metabolites . Cell Biol Int 2000 ; 24 : 705 – 11 .

Farman AA , Hadwan MH. Simple kinetic method for assessing catalase activity in biological samples . MethodsX 2021 ; 8 : 101434 .

Glorieux C , Calderon PB. Catalase, a remarkable enzyme: targeting the oldest antioxidant enzyme to find a new cancer treatment approach . Biol Chem 2017 ; 398 : 1095 – 108 .

Nagaraja P , Chamaraja N , Krishna H. Development and kinetic validation of bio-catalatic pathway for the quantification of catalase activity using 3-methyl-2 benzothiazolinehydrazone hydrochloride and pyrocatechol as a chromogenic co-substrates . Int J Bio-Technol Res 2014 ; 3 : 9 – 22 .

Kadhum MA , Hadwan MH. A precise and simple method for measuring catalase activity in biological samples . Chem Pap 2021 ; 75 : 1669 – 78 .

Wang G , Fung D. Significance of Bacterial Catalase In Food Microbiology: a Review 1 . J Food Saf 1986 ; 8 : 47 – 67 .

Suman S , Bagal D , Jain D et al.  Biotic stresses on plants: reactive oxygen species generation and antioxidant mechanism. Frontiers in Plant-Soil Interaction . Cambridge, MA : Academic Press , 2021 , 381 – 411 .

Google Preview

Baker A , Lin CC , Lett C et al.  Catalase: a critical node in the regulation of cell fate . Free Radic Biol Med 2023 ; 199 : 56 – 66 .

Zhao MX , Wen JL , Wang L et al.  Intracellular catalase activity instead of glutathione level dominates the resistance of cells to reactive oxygen species . Cell Stress Chaperones 2019 ; 24 : 609 – 19 .

Tessier SN , Breedon SA , Storey KB. Modulating Nrf2 transcription factor activity: revealing the regulatory mechanisms of antioxidant defenses during hibernation in 13‐lined ground squirrels . Cell Biochem Funct 2021 ; 39 : 623 – 35 .

Yano S , Yano N. Regulation of catalase enzyme activity by cell signaling molecules . Mol Cell Biochem 2002 ; 240 : 119 – 30 .

Reiner K. Catalase test protocol . Am Soc Microbiol 2010 ; 11 : 1 – 6 .

Hasandka A , Singh AR , Prabhu A et al.  Paper and thread as media for the frugal detection of urinary tract infections (UTIs) . Anal Bioanal Chem 2022 ; 414 : 847 – 65 .

Bilsen MP , Jongeneel RM , Schneeberger C et al.  Definitions of urinary tract infection in current research: a systematic review . Open Forum Infect. Dis. 2023 ; 10 : ofad332 . US: Oxford University Press.

Martemucci G , Costagliola C , Mariano M et al.  Free radical properties, source and targets, antioxidant consumption and health . Oxygen 2022 ; 2 : 48 – 78 .

Wang Y , Branicky R , Noë A , Hekimi S. Superoxide dismutases: dual roles in controlling ROS damage and regulating ROS signaling . J Cell Biol 2018 ; 217 : 1915 – 28 .

Van der Vliet A , Janssen‐Heininger YM. Hydrogen peroxide as a damage signal in tissue injury and inflammation: murderer, mediator, or messenger? J Cell Biochem 2014 ; 115 : 427 – 35 .

Marinho HS , Real C , Cyrne L et al.  Hydrogen peroxide sensing, signaling and regulation of transcription factors . Redox Biol 2014 ; 2 : 535 – 62 .

Nandi A , Yan LJ , Jana CK et al.  Role of catalase in oxidative stress-and age-associated degenerative diseases . Oxid Med Cell Longev 2019 ; 2019 : 9613090 .

Aebi H. [13] Catalase in vitro. Methods in Enzymology , Vol. 105 . Cambridge, MA : Academic Press , 1984 , 121 – 6 .

Teixeira JA , Mota M. Determination of catalase activity and its inhibition by a simple manometric method . Biochemical Education 1992 ; 20 : 174 – 5 .

Weydert CJ , Cullen JJ. Measurement of superoxide dismutase, catalase and glutathione peroxidase in cultured cells and tissue . Nat Protoc 2010 ; 5 : 51 – 66 .

Ghavamipour F , Sajedi RH , Khajeh K. A chemiluminescence-based catalase assay using H 2 O 2 -sensitive CdTe quantum dots . Microchim Acta 2018 ; 185 : 1 – 8 .

Hadwan MH. Simple spectrophotometric assay for measuring catalase activity in biological tissues . BMC Biochem 2018 ; 19 : 7 – 8 .

Hadwan MH , Abed HN. Data supporting the spectrophotometric method for the estimation of catalase activity . Data Brief 2016 ; 6 : 194 – 9 .

Böhmer A , Jordan J , Tsikas D. High-performance liquid chromatography ultraviolet assay for human erythrocytic catalase activity by measuring glutathione as o-phthalaldehyde derivative . Anal Biochem 2011 ; 410 : 296 – 303 .

Maral J , Puget K , Michelson AM. Comparative study of superoxide dismutase, catalase and glutathione peroxidase levels in erythrocytes of different animals . Biochem Biophys Res Commun 1977 ; 77 : 1525 – 35 .

Iwase T , Tajima A , Sugimoto S et al.  A simple assay for measuring catalase activity: a visual approach . Sci Rep 2013 ; 3 : 3081 – 4 .

Li Y , Schellhorn HE. Rapid kinetic microassay for catalase activity . J Biomol Tech 2007 ; 18 : 185 – 7 .

Cohen G , Kim M , Ogwu V. A modified catalase assay suitable for a plate reader and for the analysis of brain cell cultures . J Neurosci Methods 1996 ; 67 : 53 – 6 .

Carneiro TS , Favoreto MW , Bernardi LG et al.  Application tip and concentration of a self-mixing bleach: hydrogen peroxide inside the pulp chamber, color change, and amount of bleaching gel used . Oper Dent 2023 ; 48 : 146 – 54 .

Azeez AM , Hadwan MH. Simple assay for quantifying xanthine oxidase activity . Anal Biochem 2023 ; 673 : 115192 .

Bahar B , Tuncel AF , Holmes EW et al.  An interactive website for analytical method comparison and bias estimation . Clin Biochem 2017 ; 50 : 1025 – 9 .

Armbruster DA , Pry T. Limit of blank, limit of detection and limit of quantitation. The clinical biochemist reviews . Clin Biochem Rev 2008 ; 29 : S49 – 52 .

Doğan NÖ. Bland-Altman analysis: a paradigm to understand correlation and agreement . Turk J Emerg Med 2018 ; 18 : 139 – 41 .

Mayer B , Gaus W , Braisch U. The fallacy of the Passing-Bablok-regression . Jokull 2016 ; 66 : 95 – 106 .

Khalifa HH , Hadwan MH. Simple method for the assessment of peroxiredoxin activity in biological samples . Chemical Data Collections 2020 ; 27 : 100376 .

Goth L. A simple method for determination of serum catalase activity and revision of reference range . Clinica Chimica Acta 1991 ; 196 : 143 – 51 .

Katiyar SK , Afaq F , Mukhtar H. Effects of solar radiation on detoxification mechanisms in the skin. Comprehensive Series in Photosciences , Vol. 3 . Amsterdam, Netherlands : Elsevier , 2001 , 419 – 36 .

Takio N , Yadav M , Yadav HS. Catalase-mediated remediation of environmental pollutants and potential application–a review . Biocatalysis and Biotransformation 2021 ; 39 : 389 – 407 .

Smejkal GB , Kakumanu S. Enzymes and their turnover numbers . Expert Rev Proteomics 2019 ; 16 : 543 – 4 .

Tehrani HS , Moosavi-Movahedi AA. Catalase and its mysteries . Prog Biophys Mol Biol 2018 ; 140 : 5 – 12 .

Mueller S , Riedel HD , Stremmel W. Direct evidence for catalase as the predominant H2O2-removing enzyme in human erythrocytes . Blood 1997 ; 90 : 4973 – 8 .

Sriset Y , Chatuphonprasert W , Jarukamjorn K. Bergenin attenuates sodium selenite-induced hepatotoxicity via improvement of hepatic oxidant-antioxidant balance in HepG2 cells and ICR mice . J Biol Act Prod Nat 2021 ; 11 : 97 – 115 .

Abdelazeim SA , Shehata NI , Aly HF et al.  Amelioration of oxidative stress-mediated apoptosis in copper oxide nanoparticles-induced liver injury in rats by potent antioxidants . Sci Rep 2020 ; 10 : 10812 .

Berger SA , Bogokowsky B , Block C. Rapid screening of urine for bacteria and cells by using a catalase reagent . J Clin Microbiol Journal of clinical microbiology 1990 May; 28 : 1066 – 7 .

Martín Mateo MC , Martín B , Beneit MS et al.  Catalase activity in erythrocytes from colon and gastric cancer patients. Influence of nickel, lead, mercury, and cadmium . Biol Trace Elem Res 1997 ; 57 : 79 – 90 .

Fernández-Torres J , Aztatzi-Aguilar OG , Zamudio-Cuevas Y et al.  Effect of smoking on the redox status of knee osteoarthritis: A preliminary study . Exp Biol Med (Maywood) 2023 ; 248 : 1754 – 67 .

Shafagoj Y , Al-Qudah T , Al-Essa MK et al.  Levels of selective oxidative stress markers and antioxidant enzymes in blood of Hubble-Bubble smokers . Jordan Med J 2023 ; 57 : 1 – 11 .

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• Published: 30 October 2013

A Simple Assay for Measuring Catalase Activity: A Visual Approach

• Tadayuki Iwase 1 ,
• Akiko Tajima 1 ,
• Shinya Sugimoto 1 ,
• Ken-ichi Okuda 1 ,
• Ippei Hironaka 2 ,
• Yuko Kamata 3 ,
• Koji Takada 4 &
• Yoshimitsu Mizunoe 1  

Scientific Reports volume  3 , Article number:  3081 ( 2013 ) Cite this article

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• Bacterial techniques and applications
• Biochemical assays

In this study, an assay that combines the ease and simplicity of the qualitative approach for measuring catalase activity was developed. The assay reagents comprised only hydrogen peroxide and Triton X-100. The enzyme-generated oxygen bubbles trapped by Triton X-100 were visualized as foam, whose height was estimated. A calibration plot using the defined unit of catalase activity yielded the best linear fit over a range of 20–300 units (U) (y = 0.3794x − 2.0909, r 2 = 0.993). The assay precision and reproducibility at 100 U were 4.6% and 4.8%, respectively. The applicability of the assay for measuring the catalase activity of various samples was assessed using laboratory strains of Escherichia coli , catalase-deficient isogenic mutants, clinically isolated Shiga toxin-producing E. coli , and human cells. The assay generated reproducible results. In conclusion, this new assay can be used to measure the catalase activity of bacterial isolates and human cells.

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Introduction.

Catalase is a ubiquitous antioxidant enzyme that degrades hydrogen peroxide into water and oxygen 1 . Several pathogens produce catalase in order to defend themselves against attacks by hydrogen peroxide, a weapon commonly used by the host's immune system, in addition to oxidative stress. A previous report has in fact demonstrated that a catalase-deficient mutant pathogen was more susceptible than its wild-type strain to the oxidative stress induced by hydrogen peroxide and immune cell attacks (which involve hydrogen peroxide) 2 . It is thus useful to measure the catalase activity of pathogens in order to gain a better understanding of the underlying mechanisms of their pathogenicity, including their resistance towards oxidative stress.

Escherichia coli has 2 catalase enzymes, hydroperoxidase I (HPI) and HPII, which catalyze the dismutation of hydrogen peroxide to water and oxygen 1 . The katG gene product, HPI, is transcriptionally induced during logarithmic growth in response to low concentrations of hydrogen peroxide 3 . However, HPII, encoded by katE and positively regulated by rpoS , is not peroxide inducible and is a key player in survival during the stationary phase and other stresses 4 , 5 . Several studies have focused on these 2 catalases, as they play pivotal roles in protecting cells against the effects of oxidative stress 6 . Because HPII activity varies among clinical isolates, it is necessary to distinguish between the activities of HPI and HPII in order to investigate the activity of the RpoS-encoded stress response gene rpoS .

To date, several methods have been described for measuring catalase activity in bacteria. One of the simplest qualitative procedures involves determination of the enzyme's presence in the test bacterial isolate by using hydrogen peroxide, which is broken down to bubble-producing O 2 by catalase-positive bacteria 7 , 8 . On the other hand, quantitative approaches focusing on careful measurements include colorimetric and spectrophotometric assays 9 , 10 , 11 , 12 , 13 , 14 . There are several limitations inherent in these methods, including cumbersome procedures and high cost, despite the availability of convenient kits. Furthermore, during quantitative analyses, although the reaction occurs spontaneously, the enzyme increases the reaction rate considerably. Therefore, while processing a large number of samples, the initial and final reaction rates differ among different samples, thus producing ambiguous results. This necessitates the development of a simple and cost-effective method for measuring catalase activity.

In order to address these challenges, in this study, we developed an assay that combines the ease and simplicity of the qualitative approach for measuring catalase activity. The assay uses simple and readily available reagents, namely hydrogen peroxide, Triton X-100, and catalase. We applied this assay to clinical isolates and laboratory strains of E. coli and its derivatives carrying mutations in the catalase genes or in their regulatory factors, and human cells.

The underlying principle of this approach is that the oxygen bubbles generated from the decomposition of hydrogen peroxide by catalase are trapped by the surfactant Triton X-100. The trapped oxygen bubbles are then visualized as foam, the test-tube height of which is measured to quantify the catalase activity.

In the present study, by employing our novel approach, we measured the catalase activity by quantifying the trapped oxygen gas, which is visualized as foam. The oxygen gas generated by the catalase–hydrogen peroxide reaction in test tubes is shown in Fig. 1A , where the height of foam developed in the tubes was measured. The calibration curve plotted using the defined unit of catalase activity is shown in Fig. 1B . A linear regression on the pooled data yielded the best linear fit over a range of 20–300 units (U) of catalase activity (y = 0.3794x − 2.0909, r 2 = 0.993); hence, all the measurements were carried out in that range. The assay precision (intraassay coefficient of variation) and reproducibility (interassay coefficient of variation) at 100 U were determined to be 4.6% and 4.8%, respectively. The assay generated reproducible results ( Fig. 1B and 1C ).

(A) Image of test tubes showing foam developed as a result of catalase activity. Each solution of concentration of catalase (100-μL) was added in a Pyrex tube (13 mm diameter × 100 mm height, borosilicate glass; Corning, USA). Subsequently, 100 μL of 1% Triton X-100 and 100 μL of undiluted hydrogen peroxide (30%) were added to the solutions and mixed thoroughly and were then incubated at room temperature. Following completion of the reaction, the height of O 2 -forming foam in the test tube was measured using a ruler. (B) The best linear fit between the foam heights and catalase activity was observed over a range of 20–200 U (y = 0.3794x − 2.0909, r 2 = 0.993). Mean values are shown (n = 3). Error bars represent the standard deviation. (C) The height of foam generated after mixing catalase, Triton X-100, and H 2 O 2 .

No reagents were used to stop the reaction, as the generation of oxygen stops naturally within 5 min. After the reaction, the generation of bubbles ceased completely, despite an additional 100 μL of hydrogen peroxide being added to all the test tubes. To verify the specificity of the assay for measuring catalase activity, the catalase inhibitor azide was used and, as expected, foam formation was inhibited.

By applying the developed method to E. coli strains carrying mutations in the catalase genes or in their regulatory factors, we were able to determine the catalase activity in the katE , katG , and rpoS deletion mutants, as well as in the wild type ( Fig. 2A ), and to demonstrate the ability of the assay to accurately discriminate between the activities of HPI and HPII. Only HPI or HPII activity was observed in the katE and katG deletion mutants, respectively, and the rpoS deletion mutant showed only HPI activity. In contrast, both HPI and HPII activities were noted in the wild-type strain.

Catalase activities of (A) laboratory strains of E. coli and (B) clinical isolates. Mean values are shown (n = 3). Error bars represent the standard deviation.

Furthermore, different clinical isolates were analysed for their catalase activity ( Fig. 2B ). HPI activity was detected in all the isolates tested in the study. However, the activity of HPII varied among the isolates.

In addition, we applied the developed method to assay the catalase activity of LNCaP and PC-3 cells, which are human prostate cancer cell lines commonly used in the field of oncology. A linear regression on the pooled data yielded the best linear fit over a range of 2.5 × 10 6 to 1 × 10 7 LNCaP and PC-3 cells ( Fig. 3A and B ). The catalase activity of LNCaP cells was higher than that of PC-3 cells ( Fig. 3A and B ).

In this experiment, 10 5 –10 7 cells were used. (A) Image of test tubes showing foam developed as a result of the catalase activity of LNCaP and PC-3 cells. (B) A plot of the data (n = 3).

Using the method, the limit of detection for the assay was higher than that of other previous methods 9 , 10 , 11 , 12 , 13 , 14 , but the linearity, intraassay precision, and interassay precision were similar. In terms of handling, the method was simpler than other quantitative methods.

In this study, the catalase activity of various samples was measured by using the new method. When the assay was applied to E. coli strains carrying mutations in the catalase genes or in their regulatory factors, the expected results were obtained. When the assay was also applied to various clinical isolates of Shiga toxin-producing E. coli , the activity of HPII (which is encoded by katE and is positively regulated by rpoS ) was found to vary among the isolates. These findings are consistent with previous reports 7 , 15 , 16 , 17 , 18 . In addition, the developed method could measure the catalase activity in human cells, where the activity in LNCaP was higher than that in PC-3. This result is also in accord with a past report 19 . Our method will be useful for easy measurement of catalase activity in various samples.

With regard to the technical aspects, the choice of surfactant was a key point for measuring the catalase activity in this assay. We tested 3 common surfactants, namely Triton X-100, Tween-20, and sodium dodecyl sulphate. Three surfactants produced similar results. Triton X-100 formed fine foam, however, other surfactants, especially SDS, formed rough foam. Therefore, we chose Triton X-100 in the assay. Next, we confirmed that the use of clean (unstained) new test tubes ensured reproducible results. Finally, this assay comprises simple procedures, in that it involves only the mixing of the bacterial suspension, Triton X-100, and hydrogen peroxide solution, without any other procedures such as the preparation of bacterial extract. In terms of costs, the cost per test of reagents used in this method is close to being gratis, and no machinery or equipment is required for the assay.

In conclusion, we have developed a simple and cost-effective assay and demonstrated its feasibility in determining the catalase status of bacterial and human cells.

Bacterial strains and culture conditions

Various pathogenic Escherichia coli strains 20 were employed: 96-7 (STEC), 98E2 (STEC), 98E5 (STEC), CL-49 (STEC), 467 (STEC), 468 (STEC), E333 (STEC), 98E6 (STEC), E32511 (STEC), E07781 (VT2 + ), E07794 (VT2 + ), and E07868 (VT1 + and VT2 + ). In addition, deletion mutants of katE (which encodes heat-stable HPII), katG (which encodes heat-labile HPI), and rpoS (which positively regulates the expression of katE catalase), and the wild-type (BW25113) strain were also studied. All strains were obtained from the National Institute of Genetics (NIG, Mishima, Japan). The bacteria were propagated in Luria-Bertani (LB; BD Biosciences, USA) medium at 37°C, with shaking under aerobic conditions. The bacterial culture was inoculated into 10 mL of LB medium and cultured for 16 h at 37°C with shaking.

The LNCaP and PC-3 cell lines used in this study were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 containing 10% foetal bovine serum and Antibio-Antimyco (Life Technologies, Carlsbad, CA, USA). The cells were collected using trypsin, washed with PBS, and then used for the assay.

Reagents and sample preparations

The reagents used were hydrogen peroxide solution (30% (w/w) in H 2 O; Sigma-Aldrich, USA), 1% Triton X-100 (Sigma-Aldrich), catalase from bovine liver (2950 units/mg solid; 65% protein; 4540 units/mg protein, the product number C1345, Sigma-Aldrich), and 10 μM azide (an inhibitor of catalase activity 21 ; Sigma-Aldrich).

Catalase powder, which was purchased from Sigma-Aldrich, was solved in 100-μL distilled pure water for preparing each concentration of catalase standards. Bacteria samples were prepared using overnight-cultured bacteria. Bacterial cells (10 mg) were suspended in 100-μL of physiological saline (diluted, if necessary). Human cells (10 5 –10 7 cells) were suspended in 100-μL of phosphate buffered saline.

Quantification of catalase activities

In the present method, to quantify the catalase activity, a calibration curve was plotted with the defined unit of catalase activity. Each catalase solution (100-μL) or bacterial suspension (100-μL) was added in a Pyrex tube (13 mm diameter × 100 mm height, borosilicate glass; Corning, USA). Subsequently, 100 μL of 1% Triton X-100 and 100 μL of undiluted hydrogen peroxide (30%) were added to the solutions and mixed thoroughly and were then incubated at room temperature. Following completion of the reaction, the height of O 2 -forming foam that remained constant for 15 min in the test tube was finally measured using a ruler.

Discrimination of HPI and HPII activities

In order to accurately distinguish between the activities of the heat-labile catalase HPI and heat-stable catalase HPII in E. coli , the bacterial suspension was divided into 2 identical aliquots. One aliquot of the bacterial suspension was subjected to heat treatment (55°C, 15 min). Finally, the residual catalase activity following heat treatment was subtracted from the total catalase activity to determine the activity of the heat-labile catalase.

Inhibition of catalase activity

To test the specificity of the assay for catalase, the 100 μL of physiological saline solution in the enzyme assay was replaced with 100 μL of 10 μM azide to inhibit catalase activity 21 .

Loewen, P. C., Switala, J. & Triggs-Raine, B. L. Catalases HPI and HPII in Escherichia coli are induced independently. Arch. Biochem. Biophys. 243 , 144–149 (1985).

Article   CAS   Google Scholar  

Day, W. A., Jr, Sajecki, J. L., Pitts, T. M. & Joens, L. A. Role of catalase in Campylobacter jejuni intracellular survival. Infect. Immun. 68 , 6337–6345 (2000).

Triggs-Raine, B. L. & Loewen, P. C. Physical characterization of katG , encoding catalase HPI of Escherichia coli . Gene 52 , 121–128 (1987).

Loewen, P. C. & Switala, J. Purification and characterization of catalase HPII from Escherichia coli K12. Biochem. Cell Biol. 64 , 638–646 (1986).

Schellhorn, H. E. Regulation of hydroperoxidase (catalase) expression in Escherichia coli . FEMS Microbiol. Lett. 131 , 113–119 (1995).

Farr, S. B. & Kogoma, T. Oxidative stress responses in Escherichia coli and Salmonella typhimurium . Microbiol. Rev. 55 , 561–585 (1991).

CAS   PubMed   PubMed Central   Google Scholar  

Subbarayan, P. R. & Sarkar, M. A comparative study of variation in codon 33 of the rpoS gene in Escherichia coli K12 stocks: implications for the synthesis of sigma(s). Mol. Genet. Genomics 270 , 533–538 (2004).

Schellhorn, H. E. & Stones, V. L. Regulation of katF and katE in Escherichia coli K-12 by weak acids. J. Bacteriol. 174 , 4769–4776 (1992).

Goldblith, S. A. & Proctor, B. E. Photometric determination of catalase activity. J. Biol. Chem. 187 , 705–709 (1950).

CAS   PubMed   Google Scholar  

Lemberg, R. & Foulkes, E. C. Reaction between catalase and hydrogen peroxide. Nature 161 , 131 (1948).

Article   ADS   CAS   Google Scholar  

Chance, B. The reaction of catalase and cyanide. J. Biol. Chem. 179 , 1299–1309 (1949).

Chance, B. The reactions of catalase in the presence of the notatin system. Biochem. J. 46 , 387–402 (1950).

Beers, R. F., Jr & Sizer, I. W. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195 , 133–140 (1952).

Li, Y. & Schellhorn, H. E. Rapid kinetic microassay for catalase activity. J. Biomol. Tech. 18 , 185–187 (2007).

PubMed   PubMed Central   Google Scholar  

Jishage, M. & Ishihama, A. Variation in RNA polymerase sigma subunit composition within different stocks of Escherichia coli W3110. J. Bacteriol. 179 , 959–963 (1997).

Visick, J. E. & Clarke, S. RpoS- and OxyR-independent induction of HPI catalase at stationary phase in Escherichia coli and identification of rpoS mutations in common laboratory strains. J. Bacteriol. 179 , 4158–4163 (1997).

Ferenci, T. The spread of a beneficial mutation in experimental bacterial populations: the influence of the environment and genotype on the fixation of rpoS mutations. Heredity 100 , 446–452 (2007).

Article   Google Scholar  

Chiang, S. M., Dong, T., Edge, T. A. & Schellhorn, H. E. Phenotypic diversity caused by differential RpoS activity among environmental Escherichia coli isolates. Appl. Environ. Microbiol. 77 , 7915–7923 (2011).

Jung, K. et al. Antioxidant enzymes in malignant prostate cell lines and in primary cultured prostatic cells. Free Radical Biol. Med. 23 , 127–133 (1997).

Iida, K., Mizunoe, Y., Wai, S. N. & Yoshida, S. Type 1 fimbriation and its phase switching in diarrheagenic Escherichia coli strains. Clin. Diagn. Lab. Immunol. 8 , 489–495 (2001).

Klots, M. G., Kim, Y. C., Katsuwon, J. & Anderson, A. J. Cloning, characterization and phenotypic expression in Escherichia coli of catF , which encodes the catalytic subunit of catalase isozyme CatF of Pseudomonas syringae . Appl. Microbiol. Biotechnol. 43 , 656–666 (1995).

Google Scholar  

Download references

Acknowledgements

We thank our laboratory colleagues for their comments and help regarding our study. A part of the study was supported by The Jikei University Research Fund, The Jikei University Graduate Research Fund, Grant-in-Aid for Young Scientists (A), The Uehara Memorial Foundation, The MEXT-supported Program for the Strategic Research Foundation at Private Universities, Science Research Promotion Fund of The Promotion and Mutual Aid Corporation for Private Schools of Japan (non-profit organization).

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T.I. designed the study. T.I., Y.K., A.T., S.S., K.O., I.H., K.T., Y.M. carried out experiments and discussed in detail about the obtained results. T.I. wrote the manuscript.

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Iwase, T., Tajima, A., Sugimoto, S. et al. A Simple Assay for Measuring Catalase Activity: A Visual Approach. Sci Rep 3 , 3081 (2013). https://doi.org/10.1038/srep03081

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5.1: Catalase and Peroxidase

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Description of the Enzymes

Catalase and peroxidase are heme enzymes that catalyze reactions of hydrogen peroxide. 94,95 In catalase, the enzymatic reaction is the disproportionation of hydrogen peroxide (Reaction 5.82) and the function of the enzyme appears to be prevention of any buildup of that potentially dangerous oxidant (see the discussion of dioxygen toxicity in Section III).

\[2H_{2}O_{2} \xrightarrow{catalase} 2H_{2}O + O_{2} \tag{5.82}\]

Peroxidase reacts by mechanisms similar to catalase, but the reaction catalyzed is the oxidation of a wide variety of organic and inorganic substrates by hydrogen peroxide (Reaction 5.83).

\[H_{2}O_{2} + AH_{2} \xrightarrow{peroxidase} 2H_{2}O + A \tag{5.83}\]

(The catalase reaction can be seen to be a special case of Reaction 5.83 in which the substrate, AH 2 , is hydrogen peroxide.) Some examples of peroxidases that have been characterized are horseradish peroxidase, cytochrome c peroxidase, glutathione peroxidase, and myeloperoxidase. 94,95

X-ray crystal structures have been determined for beef-liver catalase 80 and for horseradish peroxidase 96 in the resting, high-spin ferric state. In both, there is a single heme b group at the active site. In catalase, the axial ligands are a phenolate from a tyrosyl residue, bound to the heme on the side away from the active-site cavity, and water, bound to heme within the cavity and presumably replaced by hydrogen peroxide in the catalytic reaction. In horseradish peroxidase, the axial ligand is an imidazole from a histidyl residue. Also within the active-site cavity are histidine and aspartate or asparagine side chains that appear to be ideally situated to interact with hydrogen peroxide when it is bound to the iron. These residues are believed to play an important part in the mechanism by facilitating O—O bond cleavage (see Section VI.B below).

Three other forms of catalase and peroxidase can be generated, which are referred to as compounds I, II, and III. Compound I is generated by reaction of the ferric state of the enzymes with hydrogen peroxide. Compound I is green and has spectral characteristics very similar to the Fe IV (P •- )(O) + complex prepared at low temperatures by reaction of ferric porphyrins with single-oxygenatom donors (see Section V.C.1.a). Titrations with reducing agents indicate that it is oxidized by two equivalents above the ferric form. It has been proposed (see 5.84) that the anionic nature of the tyrosinate axial ligand in catalase may serve to stabilize the highly oxidized iron center in compound I of that enzyme, 80 and furthermore that the histidyl imidazole ligand in peroxidase may deprotonate, forming imidazolate, 52,97 or may be strongly hydrogen bonded, 98 thus serving a similar stabilizing function for compound I in that enzyme.

Reduction of compound I by one electron produces compound II, which has the characteristics of a normal ferryl-porphyrin complex, analogous to 2 , i.e., (L)Fe IV (P)(O). Reaction of compound II with hydrogen peroxide produces compound III, which can also be prepared by reaction of the ferrous enzyme with dioxygen. It is an oxy form, analogous to oxymyoglobin, and does not appear to have a physiological function. The reactions producing these three forms and their proposed formulations are summarized in Reactions (5.85) to (5.88).

\[Fe^{III}(P)^{+} + H_{2}O_{2} \rightarrow Fe^{IV}(P^{-})(O)^{+} + H_{2}O \tag{5.85}\]

\[ferric\; form \quad \qquad \qquad Compound\; I \qquad \qquad \]

\[Fe^{IV}(P^{\cdotp -})(O)^{+} + e^{-} \rightarrow Fe^{IV}(P)(O) \tag{5.86}\]

\[Compound\; I \qquad \qquad Compound\; II \quad \]

\[Fe^{IV}(P)(O) + H_{2}O_{2} \rightarrow Fe(P)O_{2} + H_{2}O \tag{5.87}\]

\[Compound\; II \qquad \qquad Compound\; III \qquad \qquad \]

\[Fe{II}(P) + O_{2} \rightarrow Fe(P)O_{2} \tag{5.88}\]

\[ferrous\; form \qquad Compound\; III\]

The accepted mechanisms for catalase and peroxidase are described in Reactions (5.89) to (5.94).

\[Fe^{III}(P)^{+} + H_{2}O_{2} \rightarrow Fe^{III}(P)(H_{2}O_{2})^{+} \rightarrow Fe^{IV}(P^{\cdotp -})(O)^{+} + H_{2}O \tag{5.89}\]

\[\qquad \qquad \qquad \qquad \qquad \qquad \qquad \qquad \qquad \qquad \qquad \qquad \qquad Compound\; I\]

catalase: \[ Fe^{IV}(P^{\cdotp -})(O)^{+} + H_{2}O_{2} \rightarrow Fe^{III}(P)^{+} + H_{2}O + O_{2} \tag{5.90}\]

\[Compound\; I \qquad \qquad \qquad \qquad \qquad \qquad \qquad \qquad \]

peroxidase: \[ Fe^{IV}(P^{\cdotp -})(O)^{+} + AH_{2} \rightarrow Fe^{IV}(P)(O) +HA^{\cdotp} + H^{+} \tag{5.91}\]

\[Compound\; I \qquad \qquad \qquad \qquad Compound\; II\]

\[Fe^{IV}(P)(O) + AH_{2} \rightarrow Fe^{III}(P)^{+} +HA^{\cdotp} + OH^{-} \tag{5.92}\]

\[Compound\; II \qquad \qquad \qquad \qquad \qquad \qquad \qquad \qquad \]

\[2HA^{\cdotp} \rightarrow A + AH_{2} \tag{5.93}\]

\[2HA^{\cdotp} \rightarrow HA - AH \tag{5.94}\]

In the catalase reaction, it has been established by use of H 2 18 O 2 that the dioxygen formed is derived from hydrogen peroxide, i.e., that O—O bond cleavage does not occur in Reaction (5.90), which is therefore a two-electron reduction of compound I by hydrogen peroxide, with the oxo ligand of the former being released as water. For the peroxidase reaction under physiological conditions, it is believed that the oxidation proceeds in one-electron steps (Reactions 5.91 and 5.92), with the final formation of product occurring by disproportionation (Reaction 5.93) or coupling (Reaction 5.94) of the one-electron oxidized intermediate. 94,95

Comparisons of Catalase, Peroxidase, and Cytochrome P-450

The proposal that these three enzymes all go through a similar high-valent oxo intermediate, i.e., 3 or compound I, raises two interesting questions. The first of these is why the same high-valent metal-oxo intermediate gives two very different types of reactions, i.e., oxygen-atom transfer with cytochrome P-450 and electron transfer with catalase and peroxidase. The answer is that, although the high-valent metal-oxo heme cores of these intermediates are in fact very similar, the substrate-binding cavities seem to differ substantially in how much access the substrate has to the iron center. With cytochrome P-450, the substrate is jammed right up against the location where the oxo ligand must reside in the high-valent oxo intermediate. But the same location in the peroxidase enzymes is blocked by the protein structure so that substrates can interact only with the heme edge. Thus oxidation of the substrate by electron transfer is possible for catalase and peroxidase, but the substrate is too far away from the oxo ligand for oxygen-atom transfer. 99,124

The second question is about how the the high-valent oxo intermediate forms in both enzymes. For catalase and peroxidase, the evidence indicates that hydrogen peroxide binds to the ferric center and then undergoes heterolysis at the O—O bond. Heterolytic cleavage requires a significant separation of positive and negative charge in the transition state. In catalase and peroxidase, analysis of the crystal structure indicates strongly that amino-acid side chains are situated to aid in the cleavage by stabilizing a charge-separated transition state (Figure 5.14).

In cytochrome P-450, as mentioned in Section V.C.1, no such groups are found in the hydrophobic substrate-binding cavity. It is possible that the cysteinyl axial ligand in cytochrome P-450 plays an important role in O—O bond cleavage, and that the interactions found in catalase and peroxidase that appear to facilitate such cleavage are therefore not necessary.

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• v.2019; 2019

Role of Catalase in Oxidative Stress- and Age-Associated Degenerative Diseases

Ankita nandi.

1 Department of Biotechnology, Visva-Bharati University, Santiniketan, West Bengal 731235, India

Liang-Jun Yan

2 Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, TX 76107, USA

Chandan Kumar Jana

3 Department of Chemistry, Purash-Kanpur Haridas Nandi Mahavidyalaya, P.O. Kanpur, Howrah, West Bengal 711410, India

Nilanjana Das

Associated data.

Reactive species produced in the cell during normal cellular metabolism can chemically react with cellular biomolecules such as nucleic acids, proteins, and lipids, thereby causing their oxidative modifications leading to alterations in their compositions and potential damage to their cellular activities. Fortunately, cells have evolved several antioxidant defense mechanisms (as metabolites, vitamins, and enzymes) to neutralize or mitigate the harmful effect of reactive species and/or their byproducts. Any perturbation in the balance in the level of antioxidants and the reactive species results in a physiological condition called “oxidative stress.” A catalase is one of the crucial antioxidant enzymes that mitigates oxidative stress to a considerable extent by destroying cellular hydrogen peroxide to produce water and oxygen. Deficiency or malfunction of catalase is postulated to be related to the pathogenesis of many age-associated degenerative diseases like diabetes mellitus, hypertension, anemia, vitiligo, Alzheimer's disease, Parkinson's disease, bipolar disorder, cancer, and schizophrenia. Therefore, efforts are being undertaken in many laboratories to explore its use as a potential drug for the treatment of such diseases. This paper describes the direct and indirect involvement of deficiency and/or modification of catalase in the pathogenesis of some important diseases such as diabetes mellitus, Alzheimer's disease, Parkinson's disease, vitiligo, and acatalasemia. Details on the efforts exploring the potential treatment of these diseases using a catalase as a protein therapeutic agent have also been described.

1. Introduction

Reactive species (RS) are highly active moieties, some of which are direct oxidants, and some have oxygen or oxygen-like electronegative elements produced within the cell during cellular metabolism or under pathological conditions. Some of the reactive species are free radicals such as the hydroxyl radical and the superoxide radical, and some are nonradicals such as hydrogen peroxide. Free radicals are any independent species which consist of one or more unpaired electrons in their atomic or molecular orbital. They are generally unstable, short lived, but usually chemically reactive. They can react with any molecule either by oxidizing it or by causing any other kind of chemical modification. Free radicals can potentially oxidize all cellular biomolecules including nucleic acids, proteins, and lipids. For example, peroxidation of omega-6 polyunsaturated fatty acid (such as arachidonic acid and linoleic acid) leads to the production of 4-hydroxynonenal (HNE), which is one of the main reactive aldehydes produced by oxidative stress [ 1 ]. There are many reactive species and free radicals [ 2 ] which are listed in Table 1 .

Examples of the various free radicals and other oxidants in the cell [ 2 ].

These free radicals are formed in the cell during normal cellular metabolism as mitochondrial electron transport chain, β -oxidation of fatty acids, and cytochrome P450-mediated reactions and by the respiratory burst during immune defense. For example, autooxidation of some biologically important substances such as FADH 2 and tetrahydropteridines can yield O 2 · – in the presence of oxygen [ 3 ]. The imbalance between production and quenching of these reactive substances through antioxidant mechanisms causes oxidative stress. The loss of functionality and adaptability of important biomolecules due to oxidative stress are two interdependent biological processes, which are among the important factors that mediate aging. The free radical hypothesis, also known as oxidative stress hypothesis, is one of the strongly supported theories which can define the causes behind the aging process.

Oxidative stress has been implicated in many metabolic and neurologic degenerative disorders. Degenerative diseases, where the function and structure of a tissue or organs deteriorate over time such as in Alzheimer's disease, Parkinson's disease, diabetes, cataracts, cancer, and cardiovascular disease, have been attributed to oxidative stress conditions and the process of natural aging. Thus, oxidative stress, aging, and degenerative diseases are interconnected.

The body has a defense mechanism against oxidative stress in which both enzymatic and nonenzymatic molecules are the two prime components. This antioxidant defense system consists of some enzymes, some proteins, and a few low molecular weight molecules. The antioxidant enzymes can catalytically remove the reactive species. For example, superoxide dismutase dismutates superoxide into hydrogen peroxide which is in turn degraded by catalase or by glutathione peroxidase. The relationship between the different antioxidant enzymes is depicted graphically in Figure 1 . Transferrin, metallothionein, and caeruloplasmin are some of the proteins which can reduce the availability of prooxidants such as transition metal ions like iron ions and copper ions which can produce a hydroxyl radical from hydrogen peroxide by the Fenton reaction. The low molecular weight antioxidants include ascorbic acid, α -tocopherol, glutathione, and uric acid, which neutralize the RS by scavenging the whole molecule or its byproducts, by reducing it by or participating in any form of chemical reaction leading to complete or partial destruction of it or its byproducts. The interaction of catalase with other antioxidants and proteins can be predicted by the STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis [ 4 , 5 ]. STRING is a biological database used to study protein-protein interaction. The STRING network analysis of catalase's interaction with other proteins has been categorized into two distinct modules ( Figure 2 ). Module 1 contains four proteins which are basically involved in the pathways of peroxisomes including CAT and three proteins of module 2 such as SOD1 (superoxide dismutase 1), SOD2 (superoxide dismutase 2), and PRDX5 (peroxiredoxin 5) [ 6 – 8 ] (Supplementary ). In module 1, ACOX1 (peroxisomal acyl coenzyme A oxidase), HSD17B4 (peroxisomal multifunctional enzyme), and HAO1 (hydroxyacid oxidase 1) are involved in the fatty acid oxidation pathway in the peroxisome while the protein DAO (D amino acid oxidase) is involved in the amino acid metabolism pathway in the peroxisome [ 6 – 8 ] (Supplementary ). All the components of module 1 are involved in different metabolic pathways. The proteins in module 2 are mainly involved in responses against oxidative stress. All the proteins have antioxidant activity except AKT1 (RAC-alpha serine-threonine protein kinase). AKT1 is a serine-threonine protein kinase which is involved in cell survival, metabolism, growth, and angiogenesis. All the proteins of both modules 1 and 2 including CAT have catalytic activity and are located in the lumen of intracellular organelles. SOD2 and AKT1 of module 2 including CAT were involved in the longevity regulating pathway and FOXO signaling pathway in mammals [ 6 – 8 ] (Supplementary Figures and ). But in multiple other species, SOD1 and SOD3 (superoxide dismutase 3) were also involved along with SOD2, AKT1, and CAT [ 6 – 8 ] (Supplementary ). Among the reactive species, hydrogen peroxide is freely diffusible and is relatively long-lived. It acts as a weak oxidizing as well as reducing agent; however, it is not very reactive, but it is the progenitor of many other reactive oxygen species (ROS). It has been demonstrated to oxidatively modify glyceraldehyde-3-phosphate dehydrogenase by oxidation of the labile essential thiol groups at the active site of this enzyme [ 2 ]. In most cellular injuries, this molecule is known to play an indirect role. One of the most important products is the formation of a more reactive free radical · OH radical in the presence of transition metal ions such as Fe 2+ by means of the Fenton reaction.

Relationship between catalase and other antioxidant enzymes.

STRING analysis of interaction of catalase with other proteins.

There are many enzymes that are able to neutralize hydrogen peroxide. These enzymes include catalase, glutathione peroxidase, and other peroxidases such as cytochrome c peroxidase and NADH peroxidase [ 2 ]. Catalase is a key enzyme which uses hydrogen peroxide, a nonradical ROS, as its substrate. This enzyme is responsible for neutralization through decomposition of hydrogen peroxide, thereby maintaining an optimum level of the molecule in the cell which is also essential for cellular signaling processes. The importance of the enzyme can be gauged from the fact of its direct and indirect involvement in many diseases and infections. In this review, an attempt has been made to correlate the role of catalase with the pathogenesis and progression of oxidative stress-related diseases. A brief account of catalase, its isoforms, structure, and reaction mechanism, and its relation with some common important disorders is described in this review article.

2. Catalase

A catalase (E.C. 1.11.1.6) is one of the most important antioxidant enzymes. It is present in almost all aerobic organisms. Catalase breaks down two hydrogen peroxide molecules into one molecule of oxygen [ 9 ] and two molecules of water in a two-step reaction [ 10 ]. The same is represented in Figure 3 as derived from Ivancich et al. [ 11 ] and Lardinois [ 12 ]. The first step of the reaction mechanism involves formation of a spectroscopically distinct intermediate compound I ( Figure 3(a) ) which is a covalent oxyferryl species (Fe IV O) having a porphyrin π -cation radical, through the reduction of one hydrogen peroxide molecule [ 11 ]. In the second step reaction ( Figure 3(b) ), compound I is reduced through redox reactions by a two-electron transfer from an electron donor (the second molecule of hydrogen peroxide) to produce the free enzyme, oxygen, and water [ 10 ].

Steps in catalase reaction: (a) first step; (b) second step.

In 1937, the protein was first crystallized from bovine liver at Sumner and Dounce's laboratory [ 13 ]. The first prokaryotic catalase was purified from an aerobic bacterium, Micrococcus lysodeikticus , in 1948 [ 14 ]. The gene coding for catalase is the CAT gene which is positioned in chromosome 11 in humans. In the following decades, several studies have been carried out on prokaryotic catalase and also on the lower eukaryotic catalase. In particular, research on catalase from Saccharomyces cerevisiae has generated data and information on the evolution of the enzyme at the molecular level. It has also been reported that catalase is an important enzyme implicated in mutagenesis and inflammation conditions as well as during the suppression of apoptosis [ 15 – 18 ] which are all known to be associated with oxidative stress conditions.

Catalase has been characterized from many eukaryotic as well as prokaryotic organisms. Table 2 summarizes some basic physiochemical information available in the literature to date on catalase from different organisms. Based on the differences in their sequence and structure, there are three different types of catalase. The monofunctional heme-containing enzyme is the most widespread one. It is present in all aerobic organisms. The bifunctional catalase-peroxidase belongs to the second class, which is relatively less abundant in nature. This enzyme also contains a heme group. It is closely related to the plant peroxidases with structural and sequence similarities. The third class belongs to the Mn-containing catalase group which lacks the heme group.

Physicochemical characteristics of catalase from various sources.

Humans possess a typical monofunctional heme-containing catalase having a prosthetic group of ferric protoporphyrin IX which reacts with hydrogen peroxide. Located in the peroxisomes, the enzyme has a molecular mass of approximately 220-240 kDa [ 19 ]. It is a tetrameric protein with each subunit divided into four domains, the N-terminal threading arm, C-terminal helices, wrapping loop, and β barrel [ 20 ] ( Figure 4 ). Each subunit has a hydrophobic core comprising eight stranded β barrels surrounded by α -helices. These β barrels are antiparallel with each other. The heme distal side of the subunit is made up of the first four β strands ( β 1- β 4) of the β barrel domain and the remaining four strands ( β 5- β 8) play a part in the NADPH binding pocket. The N-terminal threading arm of a subunit (residues 5-70) intricately connects two subunits by hooking through a long wrapping loop (residues 380-438). Finally, a helical domain at one face of the β barrel is composed of four C-terminal helices. Tetramerization forces the N-terminal threading arms from the arm-exchanged dimer to cover the heme active site for the other pair of dimers and suggests that catalase fits the more general pattern of domain swapping with the arm-exchange being a later, tetramer-dependent elaboration. Throughout the protein, water fills in packing defects between the four domains of the subunit and between subunits within the tetramer. Only the hydrophobic β barrel and the immediate vicinity of the active site are substantially devoid of these structural water molecules. From XRD studies, the root mean square deviation (r.m.s.d.) coordinate differences between the four subunits were found to be 0.156 Å for the backbone atoms, 0.400 Å for the side chains, and 0.125 Å for the heme groups [ 21 ]. The 3D structure of the enzyme at 1.5 Å was elucidated in 2001 [ 22 ]. The crystal structures of human catalase show that the active site iron is pentacoordinated. The negatively charged heme carboxylate radical forms salt bridges to three arginine residues (Arg72, Arg117, and Arg365) which likely aid in heme burial and help increase the redox potential of the compound I porphyrin radical and are conserved in bacterial, fungal, plant, and animal catalase. Besides the heme group, the active conformation of the enzyme consists of one tightly bound NADPH molecule in each subunit. There are various reports on the role of this NADPH molecule. It has been demonstrated to obstruct the formation of Fe (IV)oxo-ligated porphyrin, an inactive form of catalase—by hydrogen peroxide, and to also slowly induce the removal of inactive catalase [ 19 , 23 ].

Crystal structure of human erythrocytic catalase [ 20 ] PDB ID: 1F4J.

3. Catalase-Related Diseases

Catalase deficiency or malfunctioning is associated with many diseases such as diabetes mellitus, vitiligo, cardiovascular diseases, Wilson disease, hypertension, anemia, some dermatological disorders, Alzheimer's disease, bipolar disorder, and schizophrenia [ 24 – 26 ] ( Figure 5 ). It has been reported that an anomaly of catalase activity is inherited in acatalasemia which is a rare genetic disorder (also known as Takahara disease) [ 27 ]. It is an autosomal recessive trait and characterized by a reduced level of catalase. Catalase has a prime role in regulating the cellular level of hydrogen peroxide [ 28 , 29 ], and its hydrogen peroxide catabolism protects the cells from oxidative assault, for example, by securing the pancreatic β cells from hydrogen peroxide injury [ 30 , 31 ]. Low catalase activities have been reported in schizophrenic patients such as also in patients with atherosclerosis [ 32 ].

List of some diseases linked to catalase deficiency.

Genetic variations in the catalase gene and in its promoter region also play a role in the pathogenesis of various diseases which is depicted in Figure 6 . Several studies have investigated CAT polymorphism and its involvement in the development of various diseases as well as its role as regulator in the CAT gene expression. Single nucleotide polymorphisms of the CAT gene in the promoter region possibly affect the transcription frequencies resulting in low CAT expression [ 33 , 34 ]. The most common polymorphisms that influence the transcription of the CAT gene and also affect catalase activity are -262C/T and -844G/A or -844C/T in the promoter region [ 35 ]. There are many other polymorphisms involved in the development of numerous diseases which varies amongst populations. CAT -262C/T polymorphism is related to type 1 diabetes and breast cancer [ 36 – 38 ]. Two single nucleotide polymorphisms of CAT gene, viz., 1167T/C and -262C/T, have been reported to have a strong association with type 1 diabetes mellitus [ 36 ]. The functional consequence of this 1167T/C polymorphic positioned in exon 9 is not known. But in case of -262C/T, the variation shows significant functional significance. It influences the AP-2 and Sp-1 (nuclear transcriptional factors) binding and also effects the expression as well as the level of catalase in the red blood cells [ 36 ]. In Swedish populations, the concentration of erythrocytic catalase in individuals carrying the TT genotype was high compared to those of the CC genotype [ 39 ]. In Russian populations—on the other hand—the individuals carrying the CC genotype have a higher risk of developing type 1 diabetes than those carrying the TT genotype [ 36 ]. The blood catalase level was found to be low in CC individuals which results in oxidative stress conditions, thereby promoting type 1 diabetes [ 36 ]. Another single nucleotide polymorphism of the CAT gene 111C/T in exon 9 was examined among different forms of diabetes and showed a very poor association [ 40 ]. The CAT -262C/T polymorphism has an association with breast cancer. The CC genotype showed higher catalase activity in red blood cell as compared to TT and TC genotypes with a correlated reduced risk of breast cancer by 17% [ 38 ]. However, it must be noted here that this population study was performed with a much lesser number of individuals. Studies have shown that the level of -262C/T polymorphism effects not only the transcriptional activity but also the level of catalase in red blood cells [ 37 ]. Another common CAT polymorphism is -844C/T or -844G/A which might result in a lower catalase level by influencing the transcription frequency. CAT -844C/T polymorphism has a strong association with hypertension among the Chinese population [ 41 ]. Hypertension is a multifactorial complex lifestyle disease. Among Japanese populations, this -844C/T polymorphism has been reported to show a strong association with hypertension [ 42 ]. But the functional relationship is not very clear. The CAT -844G/A, -89A/T, and -20T/C polymorphisms have been shown to be associated with malnutrition [ 43 ]. This polymorphism might affect the transcription rates thereby lowering the catalase level. The -89A/T polymorphism has also been reported to exhibit an association with vitiligo and osteonecrosis [ 44 , 45 ]. The variant with CAT -89A/T has been reported to be associated with a significantly reduced level of catalase with a correlation with developing vitiligo in the Chinese population [ 44 ]. The CAT 389C/T genotype has no reported association with vitiligo in the Chinese population, but a connection has been established in North America and the United Kingdom [ 44 , 46 , 47 ]. The relation of these genotypes with vitiligo pathogenesis is discussed in a later section. The CAT -89A/T, -20T/C, +3033C/T, +14539A/T, +22348C/T, and +24413T/C polymorphisms might be involved in osteonecrosis amongst Korean populations [ 45 ]. Data from all the studies show different polymorphisms of the CAT gene among different populations in various regions of the world. Further population-based research across the world is required to gain a clear idea about the association of the CAT gene in different diseases. In the future, this might unlock new therapeutic approaches by regulating the CAT gene.

Association of catalase polymorphism with risk of some widespread diseases.

3.1. Diabetes Mellitus

Diabetes mellitus is a common disease nowadays. They are caused by a bundle of metabolic disorders, distinguished by high levels of glucose in the blood due to improper secretion of insulin or its activity or both. It can lead to other secondary afflictions such as nerve damage, blindness, heart disease, stroke, and kidney disease. There has been a significant rise in the diabetes-affected population in recent years. It is estimated that, worldwide, the number of diabetes-affected adults will increase more than twofold from the 135 million affected in 1995 to approximately 300 million by 2025 [ 48 ] and 629 million by 2045 [ 49 ], and the majority of increment will be from developing countries such as India [ 48 ].

The 2018 data from the diabetes country profile from the World Health Organization (WHO) [ 50 ] is depicted in Figure 7 which shows the prevalence of diabetes amongst both genders in different countries classified according to their economic status by a United Nation's report. The disease seems more prevalent in the developed nations, and the percentage of the affected population seems to show a more or less uniform level in all these countries. A lot of discrepancies in the level are observed amongst the developing nations with the highest percentage of the population being affected in Egypt. Less prevalence is observed amongst the least developing nations indicating that lifestyle and diet play a major role in development of the disease as observed amongst the developed nations. Gender also seems to play a role with more prevalence of the disease among the females than the males in the developing nations indicating that societal norms may also play a role. In contrast, males seem more susceptible in developed nations, which indicates a possible genetic and lifestyle role in development of the disease.

Prevalence of diabetes amongst males and females in some countries in 2018 (data source: World Health Organization-Diabetes Country Profile 2018).

There are two general forms of diabetes mellitus, type 1 and type 2. Type 1 diabetes mellitus is a juvenile form and insulin-dependent diabetes which accounts for approximately 10% of all cases, but it may also develop in adults [ 51 ]. In this case, pancreatic β cells are destroyed by autoantibodies rendering the cells incapable of producing insulin. This autoimmune disease has a correlation between immunologic and genetic factors. There are three major types of autoantibodies found in type 1 diabetes such as GDP65, IA2, and insulin autoantibodies, but antibodies against insulin can be identified mostly in young patients and may be lacking in adults [ 52 , 53 ]. These antibodies bind mainly to the conformational epitopes on the B chain of insulin. The genetic feature shows a relationship between type 1 diabetes and some alleles of the HLA complex. There is a strong connection between the progression of type 1 diabetes and the presence of HLA class II alleles.

Type 2 diabetes mellitus is the most common form of the disease, accounting for approximately 90% of all diabetes cases. It occurs primarily due to low production of insulin and secondarily also due to insulin resistance by the body's cells. The β cells of islets of Langerhans become damaged which make them unable to produce insulin. Oxidative stress has been demonstrated to be an important factor responsible for the advancement of type 2 diabetes. It has been demonstrated that hydrogen peroxide acts as an oxidant and damages the β cell interrupting the signaling pathway of insulin production [ 30 , 54 , 55 ]. According to a study from Prof. Kassab's laboratory, a four-fold increase in the concentration of hydrogen peroxide was observed in type 2 diabetes mellitus patients than in the healthy controls [ 56 ]. This observation was corroborated with observations of low catalase activity in the β cells in hyperglycemic mice models [ 57 ].

Another form of diabetes known as pancreatogenic diabetes has been classified as type 3c diabetes mellitus (T3cDM). T3cDM is the result of pancreatitis (both acute and chronic), cystic fibrosis in the tissue of pancreas, inflammation, and damage of pancreatic tissue [ 58 , 59 ]. The damage of exocrine pancreatic peptide (PP) and pancreatic enzymes occurs at the early phase of pancreatic diabetes. The reduction of the glycogen level due to damage of α cells occurs at a late phase of pancreatic diabetes. The resultant elevated level of glucagon can lead to hyperglycemia in diabetes mellitus [ 60 ]. There are many aspects associated with the pathophysiology of pancreatic diabetes. Immunopathogenesis is one of the important aspects which contribute to the development of pancreatic diabetes. Different proinflammatory cytokines like tumor necrosis factor α , interferon γ , and interleukin 1 β are involved in the pathogenesis of pancreatic diabetes [ 60 ]. Higher concentration of cytokines leads to the dysfunction of the β cells at an early stage of chronic pancreatitis [ 61 ]. At higher concentration, interleukin 1 β induces the apoptosis of β cells by the NF κ B pathway [ 62 ]. Higher concentration of interferon γ diminishes the translocation of pancreatic and duodenal home box 1 (PDX1), a transcription factor. PDX1 is important for the development of pancreatic cells through maturation of β cells and also via duodenal differentiation [ 63 ]. Reduction of survivability and differentiation of β cells occur in patients with chronic pancreatitis due to loss of PDX1. Hydrogen peroxide plays a central role in this pathway as a signaling molecule [ 64 ]. At lower concentration, hydrogen peroxide plays as a signaling molecule while it becomes toxic at higher concentration [ 65 ] and catalase plays an important role in maintaining homeostasis of the cells by degrading hydrogen peroxide. The activity of catalase in the serum was observed to be high in acute pancreatitis [ 66 ] and persists at its elevated level for as long as 10 to 14 days [ 66 ]. Therefore, the high catalase activity may contribute to the pathogenesis of T3cDM in an indirect way by maintaining the hydrogen peroxide concentration which would induce the synthesis of proinflammatory cytokines resulting in pancreatic diabetes.

Gestational diabetes mellitus (GDM) is another common form of diabetes among pregnant women. The pathogenesis of GDM is very similar to type 2 diabetes mellitus. There are several factors including ethnicity, maternal age, hypertension, obesity, and polycystic ovary syndrome (PCOS) which are associated with the possibility of developing GDM [ 67 , 68 ]. Pregnant women with GDM have higher risk of developing type 2 diabetes mellitus after pregnancy [ 68 ]. The offspring of gestational diabetic mothers are prone to development of different diseases like hypertension, different metabolic syndrome, and chronic kidney disease [ 69 , 70 ]. These birth defects might be due to higher concentration of reactive oxygen species and lowering of the antioxidant defense which in turn make the cell more susceptible to oxidative insults [ 70 , 71 ]. GDM usually develops in the second and third trimesters of the pregnancy period. Reports on the link of catalase with GDM are very conflicting. It has been reported that oxidative stress is high in the second and third trimesters of pregnancy and the catalase activity was also low during this period [ 72 , 73 ]. The blood catalase activity has been reported to be low in pregnant women with GDM compared to nonpregnant and pregnant nondiabetic healthy control women [ 72 ]. However, the blood catalase activity was observed to increase in the third trimester than in the second trimester in pregnant individuals with GDM [ 72 ]. In another study, low blood catalase activity has been observed in pregnant women with GDM [ 40 ]. As already mentioned, there is poor association between 111C/T polymorphism and different forms of diabetes mellitus which include GDM [ 40 ]. The mRNA expression of the CAT gene in the placenta of gestational diabetic pregnant women was found to be higher in comparison to that in normal pregnant women [ 74 ]. So it may be concluded from the above that catalase might have a relation with GDM pathophysiology during pregnancy, but further research to establish the facts is needed.

Hydrogen peroxide has been implicated to act as a cellular messenger in the signaling pathway for insulin secretion by inactivating tyrosine phosphatase [ 65 , 75 – 78 ]. It has been postulated that catalase in the liver may confer cellular protection by degrading the hydrogen peroxide to water and oxygen [ 28 – 31 ]. Lack of catalase can contribute to the development of diabetes mellitus [ 76 , 79 ] with a positive correlation being observed between diabetes mellitus in acatalasemic patients. It is estimated that approximately 12.7% of acatalasemic/hypocatalesemic patients are also affected by diabetes mellitus [ 79 ]. It was proposed that catalase deficiency may be responsible for the development of diabetes mellitus in an indirect way [ 24 ]. The β cells are known to be oxidant sensitive. These cells are not only deprived of catalase but also have a higher concentration of mitochondria [ 80 ] which is one of the major sources of superoxide and hydrogen peroxide in the cell through the electron transport pathway. Therefore, in acatalasemic/hypocatalesemic patients, a low amount of oxidative stress over a long period of time may result in the accumulation of oxidative damage in the β cells that results in the onset of diabetes [ 76 , 79 ].

There are many vascular complications in diabetes mellitus including microvascular complications (diabetic retinopathy, nephropathy, neuropathy, etc.) and cardiovascular complications [ 81 ]. Oxidation plays an important role in different complications which occur in both type 1 and type II diabetes. Due to the low expression levels or activity of catalase, the concentration of hydrogen peroxide may increase in the cells creating oxidative stress conditions causing the progression of different types of complications. In the case of diabetes retinopathy, the retina is damaged by retina neovascularization where new vessel origination from existing veins extends to the retinal inner cells [ 82 ] leading to blindness [ 83 ]. Vascular endothelial growth factor (VEGF) is a prime inducer of angiogenesis, a procedure of new vessel development. Nox4, a major isoform of NADPH oxidase, is predominant in the endothelial cells of the retina. It causes the generation of hydrogen peroxide instead of other reactive species [ 84 ]. Hydrogen peroxide may have a role as a signaling molecule in the VEGF signaling molecule. An upregulation of the Nox4 expression with downregulation of the catalase expression and/or activity in diabetes increases the hydrogen peroxide concentration which promotes retinal neovascularization through the VEGF signaling pathway [ 82 ]. In a study on a diabetic rat model, high concentration of hydrogen peroxide was observed in the retinal cells, creating oxidative stress conditions within the cell [ 85 ]. Since retinal cells have high content of polyunsaturated fatty acid content [ 86 ], they can be oxidized by the hydroxyl radicals generated from hydrogen peroxide by the Fenton reaction. High levels of lipid peroxides and oxidative DNA damage have been observed in diabetic retinopathy [ 87 – 90 ].

In a recent study, researchers have been able to distinguish five distinct clusters of diabetes by combining parameters such as insulin resistance, insulin secretion, and blood sugar level measurements with age of onset of illness [ 91 ]. Group 1 essentially corresponds to type 1 diabetes while type 2 diabetes is further subdivided into four subgroups labelled as group 2 to group 5. Individuals with impaired insulin secretion and moderate insulin resistance are labelled under group 2 (the severe insulin-deficient diabetes group) while in group 3, the severe insulin-resistant diabetes patients with obesity and severe insulin resistance are included. Group 4 is composed of the mild obesity-related diabetes patients who are obese and fall ill at a relatively young age while the largest group of patients is in group 5 with mild age-related diabetes in mostly elderly patients. A relationship between this new classification of diabetes with catalase expression levels or its activity has still not been probed for a link, if any, and needs further research.

3.2. Neurological Disorders

3.2.1. alzheimer's disease.

Alzheimer's disease is one of the onset of dementia diseases in adults [ 92 ]. According to the report of the Alzheimer's Association, approximately 5.5 million people in the United States of America were suffering from Alzheimer's disease in 2017. It is estimated that by 2050, the prevalence of Alzheimer's diseases will increase immensely from 4.7 million in 2010 to an estimated 13.8 million in 2050 [ 93 ].

Many factors including smoking and diabetes are associated with a higher risk of dementia. Alzheimer's disease is characterized by deposition of senile plaques of amyloid β peptides in the brain [ 94 , 95 ]. There are several studies which demonstrate that amyloid β peptides are toxic to neurons in culture [ 96 – 109 ]. Amyloid β , an amyloid precursor protein processing (APP) product, is a soluble component of the plasma and cerebrospinal fluid (CSF). In all cases of Alzheimer's disease, it has been observed that the soluble amyloid β is converted to insoluble fibrils in senile plaques through formation of protein-protein adducts [ 96 – 99 , 101 ].

It has been observed using in vitro cell culture studies that the nascent amyloid β is nontoxic but aged amyloid β becomes toxic to neurons [ 110 ]. It has been observed that amyloid β peptide is responsible for hydrogen peroxide accumulation within the cultures of neuroblastoma and hippocampal neurons [ 111 , 112 ] probably by the direct binding of amyloid β to catalase leading to decreased enzyme activities [ 26 ]. These findings led to development of the hypothesis that the catalase-amyloid β interaction may play a significant role in the increment of hydrogen peroxide in the cells linking the accretion of amyloid β and development of oxidative stress conditions in Alzheimer's disease [ 26 ]. So the current hypothesis regarding the mechanism of amyloid β -stimulated oxidative damage in cells is that amyloid β directly interacts with catalase by binding with the protein and deactivating its catalytic activity thereby creating oxidative stress conditions. In addition, full-length amyloid β peptides bind to Cu 2+ at their N-terminal section of the peptide and reduce it to the Cu + form [ 113 ]. It has been reported that amyloid β -Cu + complex can lead to hydrogen peroxide production [ 114 , 115 ]. Therefore, catalase has both a direct and an indirect relationship with the pathogenesis of Alzheimer's disease.

3.2.2. Parkinson's Disease

Parkinson's disease is an age-associated neurological disorder with the initial symptom as a simple tremor of the hand which gradually affects the whole body movement diminishing the quality of life severely with the advancement of the disease. Its clinical manifestations include bradykinesia, rigidity, resting tremor, and postural instability. It starts with rhythmic tremor of limbs especially during periods of rest or sleep. At the developing stage of the disease, patients face difficulties in controlling movement and muscle rigidity. Due to this muscular rigidity, slowness of movement and slowness of initiation of movement occur.

The disease is characterized by the exhaustion of dopamine due to damage of dopamine-producing neurons in the substantia nigra pars compacta (SNpc) [ 116 – 118 ]. It has been demonstrated that Parkinson's disease-affected patients suffer from 100-200 SNpc neuronal damages per day [ 119 ]. As various factors such as genetic inheritance, environmental toxins, oxidative stress, and mitochondrial dysfunction are probably involved in the pathogenesis of the disease, it is very challenging to understand the pathogenesis of Parkinson's disease.

It has been demonstrated that a protein, alpha ( α ) synuclein, is closely related to the cytopathology and histopathology of Parkinson's disease [ 120 ]. It has been observed that mutation in a gene responsible for the production of α -synuclein results in the production of a mutant protein that can promote the deposition of dopamine in the cytoplasm of neurons [ 121 ]. The small neurotransmitter molecules like dopamine are synthesized in the cytoplasm and are transferred to small vesicles as it becomes oxidized at the physiological pH. Mutant α -synuclein permeabilizes these vesicles causing leakage of the dopamine into the cytoplasm where it autooxidizes producing hydrogen peroxide, superoxide molecules, and toxic dopamine-quinone species creating oxidative stress conditions [ 122 ]. Mutant α -synuclein protein is also known to inhibit the expression and activity of catalase [ 123 ]. Arrest of catalase activity by α -synuclein is probably by hindering the peroxisome proliferator-activated receptor γ (PPAR γ ) transcription activity, which regulates the CAT gene expression [ 123 ]. Based on such experiments, it may be concluded that the low catalase activity and high hydrogen peroxide production in Parkinson's disease might be due to (the indirect) inhibition of catalase expression by the α -synuclein molecule.

3.3. Vitiligo

Vitiligo is one of the chronic pigmentary disorders where skin melanocyte cells—the pigment responsible for the color of the skin—are damaged or are unable to produce melanin. Various studies have shown that the catalase levels in the epidermis of vitiligo patients are lower as compared to those of the healthy control subjects [ 124 , 125 ] with a resultant increase in the concentration of hydrogen peroxide. In the cell, hydroxyl radicals can be produced spontaneously from hydrogen peroxide through photochemical reduction, i.e., the Haber-Weiss reaction [ 15 ]. These hydroxyl radicals are able to oxidize lipids in the cell membrane. This may be the cause behind damage of keratinocytes and melanocytes in the epidermal layer of the skin in such patients [ 126 – 130 ]. Moreover, the inhibitory effect of hydrogen peroxide or allelic modification of the CAT gene results in low catalase activity. However, it has been observed that there is an erratic relationship between catalase polymorphism and vitiligo. The 389C/T polymorphisms of exon 9, codon 389, and -89A/T of the promoter region were studied in vitiligo patients [ 34 , 44 , 46 , 47 , 131 , 132 ]. But the results were not observed to be consistent. Amongst the Chinese population, an association was observed in AT and TT genotypes with the increased risk of vitiligo whereas no association was observed between vitiligo and the -89A/T CAT polymorphism in the Korean population [ 34 , 44 ]. In the case of 389C/T polymorphism, several studies showed no difference between the controls and vitiligo patients [ 34 , 44 , 46 , 131 ] although contrary results have also been obtained in a few studies [ 47 , 131 ]. It has been reported that a mutation in the CAT gene might change the gene expression and/or cause structural changes in the keratinocytes and/or melanocytes [ 46 ]. Though the results are inconsistent from population studies, an interconnection between the pathogenesis and catalase may still be possible as scattered demonstrations are reported in the literature. Therefore, further studies to understand the link is necessary.

3.4. Acatalasemia

Acatalasemia (AC) is a hereditary disorder which is linked with the anomaly of catalase enzyme affecting its activity. In 1948, Takahara, a Japanese otolaryngologist, first reported this disorder [ 133 , 134 ]. He found that four out of seven races in Japan had the same genetic flaw [ 135 ]. His ex vivo experiments consisted of filling the mouth ulcer of a diseased patient with hydrogen peroxide. Since no bubble formation was observed, he concluded that a catalase or its enzymatic activity is absent in the saliva of the patients. In honor of his primary findings, this disease was christened as the Takahara disease. Acatalasemia and hypocatalasemia signify homozygotes and heterozygotes, respectively. The heterozygote of acatalasemia shows half of the catalase activity than normal and this phenotype is known as hypocatalasemia [ 136 ]. Depending on the geographical location from where it has been first studied, there are different types of acatalasemia described as Japanese, Swiss, Hungarian, German, and Peruvian types. Approximately 113 acatalasemic patients have been reported to date from all over the world.

Two kinds of mutations in the catalase gene have been reported to be involved in the Japanese acatalasemia. A splicing mutation has been held responsible for Japanese acatalasemia I where a substitution of a guanine residue with adenine residue at position 5 of intron 4 disturbed the splicing pattern of the RNA product producing a defective protein [ 137 ]. In Japanese acatalasemia II, a frame shift mutation occurs due to the deletion of thymine in position 358 of exon 4 which modifies the amino acid sequence and produces a new TGA (stop) codon at the 3′ terminal. Translation of this mutated strand produces a polypeptide of 133 amino acid residues. This is a truncated protein that is unstable and nonfunctional [ 138 ].

Aebi et al. first described Swiss acatalasemia [ 139 – 141 ]. The study on the fibroblast from Swiss acatalasemia patients suggests that structural mutations in the CAT gene are responsible for inactivation of catalase [ 142 ]. Goth, a Hungarian biochemist, first described Hungarian acatalasemia in 1992 after studying the disease in two Hungarian sisters. He found that the catalase activities in the blood of these two acatalasemic sisters were 4.4% and 6.7% of the reference catalase activity in the healthy population whereas the level of activity in hypocatalasemic patients was 38.9% [ 24 ]. Studies at his laboratory led Goth to suggest that mutations of the CAT gene and resultant structural changes in the catalase protein are responsible for Hungarian acatalasemia. This laboratory also reported that there was a risk of diabetes mellitus amongst the Hungarian acatalasemic family members though further biochemical and genetic analysis needs to be performed to validate the hypothesis that acatalasemic patients have more chance of developing diabetes mellitus [ 79 ]. There are generally four types of Hungarian acatalasemia which varies according to the (different) site of gene mutation in the DNA. The same is represented in Table 3 .

Four different types of Hungarian acatalasemia.

4. Therapeutic Role of Catalase

Catalase is one of the most important antioxidant enzymes. As it decomposes hydrogen peroxide to innocuous products such as water and oxygen, catalase is used against numerous oxidative stress-related diseases as a therapeutic agent. The difficulty in application remains in delivering the catalase enzyme to the appropriate site in adequate amounts. Poly(lactic co-glycolic acid) nanoparticles have been used for delivering catalase to human neuronal cells, and the protection by these catalase-loaded nanoparticles against oxidative stress was evaluated [ 143 ]. It was observed that the efficiency of the encapsulation of catalase was very high with approximately 99% enzymatic activity of encapsulated catalase along with significantly sustained activity over a month. The nanoparticle-loaded catalase showed significant positive effect on neuronal cells preexposed to hydrogen peroxide reducing the hydrogen peroxide-mediated protein oxidation, DNA damage, mitochondrial membrane transition pore opening, and loss of membrane integrity. Thus, the study suggests that nanoparticle-loaded catalase may be used as a therapeutic agent in oxidative stress-related neurological diseases [ 143 ]. Similar research has been conducted using EUK 134 which is a class of synthetic superoxide dismutase/catalase mimetic as an effective therapeutic agent in stroke [ 144 ]. EUK 134 is a salen-manganese complex which has both high catalase and superoxide dismutase activity. It was concluded from these studies on the rat stroke model that EUK 134 may play a protective role in management of this disease.

Studies using Tat-CAT and 9Arg-CAT fusion proteins as therapeutic agents have also been carried out with encouraging results [ 145 ]. To study the effect of these fusion proteins under oxidative stress conditions, mammalian cell lines (HeLa, PC12) were transduced with purified fusion Tat-CAT and 9Arg-CAT protein and these cells were exposed to hydrogen peroxide. It was found that the viability of the transduced cells increased significantly. It was also observed that when the Tat-CAT and 9Arg-CAT fusion proteins were sprayed over animal skin, it could penetrate the epidermis and dermis layers of the skin. The fusion proteins transduced in mammalian cells were active enzymatically for over 60 h after which they became unstable. This study suggests that these fusion proteins can be potentially used as protein therapeutic agents in catalase-related disorders [ 145 ].

Amyotrophic lateral sclerosis (ALS) is one of the most common types of progressive and fatal neurological disorders which results in loss of motor neurons mostly in the spinal cord and also to some extent in the motor cortex and brain stem. Amongst the two distinct types of ALS, the familial form (FALS) accounts for 10% of all ALS cases and 15 to 20% of FALS cases are related to the SOD1 gene mutation, an antioxidant enzyme which scavenges the superoxide radical. In some of the FALS cases, it has been found that the mutation in the SOD1 gene is not linked to a lowered activity of SOD1. Rather, the mutated SOD1 has toxic properties with no lowering of the enzymatic activity. This mutated SOD1 protein reacts with some anomalous substrates such as hydrogen peroxide using it as a substrate and produces the most reactive hydroxyl radical which can severely damage important biomolecules [ 146 ]. Mutated SOD1 also has the potential to use peroxynitrite as an atypical substrate leading to the formation of 3-nitrotyrosine which results in the conversion of a functional protein into a nonfunctional one [ 147 ]. Catalase can reduce the hydrogen peroxide concentration by detoxifying it. Therapeutic approaches using putrescine-modified catalase in the treatment of FALS have also been attempted [ 148 ]. It was found that putrescine-catalase—a polyamine-modified catalase—delayed the progression of weakness in the FALS transgenic mouse model [ 148 ]. Thus, the delay in development of clinical weakness in FALS transgenic mice makes the putrescine-modified catalase a good candidate as a therapeutic agent in diseases linked with catalase anomaly. In this connection, it must be mentioned that the putrescine-modified catalase has been reported to exhibit an augmented blood-brain barrier permeability property while maintaining its activity comparable to that of native catalase with intact delivery to the central nervous system after parenteral administration [ 148 ]. Therefore, further studies with this molecule seem to be warranted.

Investigations using synthetic SOD-catalase mimetic, increase in the lifespan of SOD2 nullizygous mice along with recovery from spongiform encephalopathy, and alleviation of mitochondrial defects were observed [ 149 ]. These findings lead the authors to hypothesize that the SOD-catalase mimetic could be used as a potential therapy for different neurological diseases related to oxidative stress such as Alzheimer's disease and Parkinson's disease [ 149 ].

Studies using type 1 and type 2 diabetic mice models with 60-fold upregulated catalase expression showed amelioration in the functioning of the cardiomyocytes [ 150 ]. Cardiomyopathy is related to improper functioning of heart muscles where the muscles become enlarged, thick, or stiff. It can lead to irregular heartbeats or heart failure. Many diabetic patients suffer from cardiomyopathy with structural and functional anomalies of the myocardium without exhibiting concomitant coronary artery disease or hypertension [ 151 ].

As already discussed, catalase is interconnected to diabetes mellitus pathogenesis. It has been observed that a 60-fold increase of catalase activity could drastically reduce the usual features of diabetic cardiomyopathy in the mouse model [ 150 ]. Due to catalase overexpression, the morphological impairment of mitochondria and the myofibrils of heart tissue were prevented. The impaired cardiac contractility was also inhibited with decrease in the production of reactive oxygen species mediated by high glucose concentrations [ 150 ]. So this approach could be an effective therapeutic approach for the treatment of diabetic cardiomyopathy.

5. Future Perspective

This review summarizes a relation between catalase and the pathogenesis of some critical diseases such as diabetes, Parkinson's disease, acatalasemia, vitiligo, and Alzheimer's disease. An increase in focus on the role of catalase in the pathogenesis of oxidative stress-related diseases and its therapeutic approach is needed.

Catalase plays a significant role in hydrogen peroxide metabolism as a key regulator [ 28 , 29 , 152 – 154 ]. Some studies have also shown the involvement of catalase in controlling the concentration of hydrogen peroxide which is also involved in the signaling process [ 155 – 158 ]. Acatalasemia is a rare genetic disorder which is not as destructive as other diseases discussed here, but it could be a mediator in the development of other chronic diseases due to prolonged oxidative stress on the tissues.

We have also discussed the risk of type 2 diabetes mellitus among acatalasemic patients. But more research on the biochemical, molecular, and clinical aspects of the disease is necessary. There are many more questions about acatalasemia and its relation to other diseases which need to be answered. Therefore, further studies are needed to focus on catalase gene mutations and its relationship to acatalasemia and other diseases with decreased catalase activity so that the link can be understood more completely.

The therapeutic approaches using catalase needs more experimental validation so that clinical trials can be initiated. Use of catalase as a medicine or therapy may be a new and broad field of study. Any novel finding about therapeutic uses of catalase will have a huge contribution in medical science. Positive findings can direct towards its possible use for treatment of different oxidative stress-related diseases.

6. Conclusion

Catalase is one of the crucial antioxidant enzymes which plays an important role by breaking down hydrogen peroxide and maintaining the cellular redox homeostasis. Diabetes, Alzheimer's disease, Parkinson's disease, etc. are currently becoming common diseases. While there are many factors involved in the pathogenesis of these diseases, several studies from different laboratories have demonstrated that catalase has a relationship with the pathogenesis of these diseases. Research in this area is being carried out by many scientists at different laboratories exploring different aspects of these diseases, but with an ever-increasing aging population, much remains to be achieved. On the other hand, the potential of catalase as a therapeutic drug in the treatment of several oxidative stress-related diseases is not adequate and is still being explored. Additional research is needed to confirm if catalase may be used as a drug in the treatment of various age-related disorders.

Acknowledgments

The authors thank the Council of Scientific and Industrial Research (CSIR), India (Project no. 38(1343)/12/EMR-II) for EMR grants. AN is grateful to CSIR for the project fellowship and the University Grants Commission (UGC), India, for the non-NET fellowship.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Supplementary Materials

(Supplementary Figure 1). In module 1, ACOX1 (peroxisomal acyl coenzyme A oxidase), HSD17B4 (peroxisomal multifunctional enzyme), and HAO1 (hydroxyacid oxidase 1) are involved in the fatty acid oxidation pathway in the peroxisome while the protein DAO (D amino acid oxidase) is involved in the amino acid metabolism pathway in the peroxisome [ 4 – 6 ] (Supplementary Figure 1). All the components of module 1 are involved in different metabolic pathways. The proteins in module 2 are mainly involved in responses against oxidative stress. All the proteins have antioxidant activity except AKT1 (RAC-alpha serine-threonine protein kinase). AKT1 is a serine-threonine protein kinase which is involved in cell survival, metabolism, growth, and angiogenesis. All the proteins of both modules 1 and 2 including CAT have catalytic activity and are located in the lumen of intracellular organelles. SOD2 and AKT1 of module 2 including CAT were involved in the longevity regulating pathway and FOXO signaling pathway in mammals [ 4 – 6 ] (Supplementary Figures 2 and 3). But in multiple other species, SOD1 and SOD3 (superoxide dismutase 3) were also involved along with SOD2, AKT1, and CAT [ 4 – 6 ] (Supplementary Figure 4). Among the reactive species, hydrogen peroxide is freely diffusible and is relatively long-lived. It acts as a weak oxidizing as well as reducing agent; however, it is not very reactive, but it is the progenitor of many other reactive oxygen species (ROS). It has been demonstrated to oxidatively modify glyceraldehyde-3-phosphate dehydrogenase by oxidation of the labile essential thiol groups at the active site of this enzyme [ 2 ]. In most cellular injuries, this molecule is known to play an indirect role. One of the most important products is the formation of a more reactive free radical · OH radical in the presence of transition metal ions such as Fe 2+ by means of the Fenton reaction.