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The Ultimate Antioxidation and Detoxification Machine: Catalase

Catalase is one of the most efficient enzymes known, with reaction rates approaching 200,000 catalytic events/second/subunit, which is near the diffusion-
controlled limit. Keep in mind that the rate of any enzyme-mediated reaction is determined by the frequency with which enzymes and substrates collide. It is
calculated that the enzyme catalyzes a reaction almost every time it collides with a substrate molecule. Some even claims that catalase is at the upper limit of how
efficient an enzyme can be. Two other enzymes that are nearly as efficient are fumarase (TCA cycle) and acetylcholine esterase (degradation of acetylcholine).

So what is the significance of catalase behaving like the ultimate enzyme machine? Well, take a look at some of its substrates: hydrogen peroxide, alcohol, phenols,
formic acid, and formaldehyde. Get the picture? These substrates are pretty nasty chemicals that can make your life miserable. Take hydrogen peroxide, for
instance, it is a byproduct of aerobic metabolism normally produced by the mitochondrial electron transport chain. Excess hydrogen peroxide can damage nucleic
acids, proteins, and lipids, causing cell injury or death, meaning that catalase has an important physiological role in protecting the cell. It turns out that intracellular
hydrogen peroxide is predominantly generated in an organelle called peroxisome, which is present in all eukaryotic cells. In addition, activated phagocytes are the
major contributor of extracellular hydrogen peroxide.

Several peroxisomal enzymes use molecular oxygen to remove hydrogen atoms from specific organic substances in an oxidative reaction, thus producing hydrogen
peroxide (RH2 + O2 => R + H2O2). Catalase, being an peroxisomal enzyme, then utilizes hydrogen peroxide to oxidize alcohol and other organic compounds in a
“peroxidative” reaction (H2O2 + R’H2 => 2H2O + R’). When H2O2 accumulates in the cell, the enzyme acts solely on this reactive oxygen species by breaking it
down to water and molecular oxygen (2H2O2 => 2H2O + O2). Another enzyme that also utilizes H2O2 as substrate is glutathione peroxidase, also an important anti-
oxidation defense system. Liver, kidney, and erythrocytes contain the highest amounts of catalase. Interestingly and importantly, a recent study by Schriner et al.
has shown that the life span of rodents could be extended by overexpression of catalase in mitochondria. This finding not only confirms the long-held belief that
aging is mainly caused by reactive oxygen species, it also offers a potential molecular strategy through which humans might more effectively fight against oxidative
stress.

Although catalase activity can be directly measured in the ultraviolet region, this method is not applicable to monitoring cellular catalase activity due to UV
absorption by protein and other molecules present in biological samples. We have developed a unique and simple colorimetric method to directly assay catalase
activity in crude cell extracts. Since the rate of decomposition of hydrogen peroxide to water and oxygen is proportional to catalase activity, the first step of the
assay system, which is carried out at 37°C for 30 min, is designed to consume a fixed amount of hydrogen peroxide by catalase. The second step is then designed
to assay for the amount of unconsumed hydrogen peroxide based on hydrogen peroxide-mediated oxidation of an azo chromogen probe, which is carried out at 50°
C for another 30 min. Oxidation of the chromogen probe yields a yellowish color measurable by spectroscopy or more conveniently by a microplate reader. Thus,
you’ll be able to determine in a semi-quantitative fashion in 1 hour how diligently your cells might fight against oxidative stress.
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