Catalase Enzyme Activity...UV Assay

240nm (UV) method with sensitivity of 0.3 U catalase/mL reaction mixture. Recommended for researchers wishing to assay catalase activity in tissue homogenates, cell lysates & other biological fluids or extracts where catalase enzyme might be present. Recommended for model systems where higher sensitivity may be required.

Summary

Hydrogen peroxide (H₂O₂) is formed in cells by controlled pathways and elicits a broad spectrum of cellular response ranging from mitogenic growth stimulation to 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 mmole of hydrogen peroxide per minute at pH 7.0 and 25C, 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 concentartion 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™ Catalase activity assay method is essentially that described by Beers and Sizer, 1952 in which the decomposition of peroxide is monitored at 240 nm, with modifications to increase robustness and convenience. Our method uses a certified standard with known catalase activity units so that calibration of precise H₂O₂ concentration is not necessary in our assay. Similarly, experiments can be carried out at room temperature under conditions that are more accurate and convenient. Modifications are also made in our formulations to overcome problems associated with instability of diluted hydrogen peroxide and diluted enzyme standards at the room temperature so there is no need to keep them on ice and no time wasted to bring them to the assay temperature before each individual assay.

The assay principal is summarized in the reaction scheme below:

CAT 01 Reaction

The absorbance of hydrogen peroxide at 240 nm is measured directly to calculate the reaction rate since water and oxygen does not absorb at this wavelength. In the presence of catalase, the reaction rate is proportionally (linearly) enhanced.