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 1.0mmole 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.
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:
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.
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