Summary

Hydrogen peroxide (H₂O₂) is formed in cells by controlled pathways and elicits a broad spectrum of cellular response ranging from mitogenic growth stimulationto apoptosis to necrosis at different concentration levels. Locally intense amounts of H₂O₂ can also be produced by inflammatory cells to kill pathogens. H₂O₂ at high concentration is deleterious to cells and its accumulation causes oxidation of cellular targets such as proteins, lipids and DNA leading to mutagenesis and cell death. Removal of H₂O₂ from cells is therefore necessary for protection against oxidative damage.

The enzyme catalase is an endogenous antioxidant present in all aerobic cells helping to facilitate the removal of hydrogen peroxide. The enzyme consists of 4 subunits of the same size, each of which contains a heme active site to accelerate the decomposition of H₂O₂ to water and oxygen. Catalase activity varies greatly between tissues with highest activities in the liver, kidney and erythrocyte, and lowest activity present in connective tissues. In eukaryotic cells the enzyme is concentrated in sub-cellular peroxisome organelles.

The most common definition of one catalase unit is the amount of enzyme decomposing 1.0 umole of hydrogen peroxide per minute at pH 7.0 and 25°C, with initial H₂O₂ concentration of 10.3 mM. Catalase exhibits an unusual kinetic behavior in that it is not possible to saturate the enzyme with H₂O₂ substrate up to 5M catalase concentration but there is a rapid inactivation of the enzyme at substrate concentrations above 0.1 M H₂O₂. Therefore, the activity assay is typically carried out with 10-50 mM H₂O₂. Since H₂O₂ substrate must be present at substantially less than saturated concentration, the enzyme activity is dependent on precise concentration of H₂O₂.

Test Principle

The NWLSS™ NWK-CAT02 assay utilizes an assay cocktail containing H₂O₂ that is incubated with catalase samples or standards for exactly two minutes. The reaction of catalase with H₂O₂ is quenched by adding an inhibitor. H₂O₂ remaining in the reaction mixture is then allowed to react with a chromagen in the presence of Horseradish Peroxidase (HRP). The HRP catalyzed reaction results in formation of a blue colored chromophore measurable at 650 nm. Catalase concentration in samples is determined by comparing the absorbances at 650 nm to a standard curve generated using a certified catalase standard provided with the kit. The HRP-catalyzed reaction is optimized in our kit with respect to amount of H₂O₂ used in the catalase reaction in order to yield a linear response between the absorbance at 650 nm and the amount of H₂O₂ remaining. The resulting standard curve has an inversely linear correlation with the amount of catalase activity in the original sample. Using this method there is no need for precise and tedious calibration of H₂O₂ concentration. Modifications have also been made in the reagent formulations to overcome problems associated with the instability of diluted enzyme standards at the room temperature allowing experiments to be carried out at room temperature under conditions that are more convenient while still providing accurate results. The basic assay principal is summarized in the figure below: