Detection of dehydrogenase (xanthine oxidase, aldehyde dehydrogenase, EC 1.1.3.22) in milk (Schardinger reaction).

Work 8. Oxidoreductases

Oxidoreductases include enzymes that catalyze oxidation-reduction reactions:

Substrate + Acceptor Substrate (oxidized) + Acceptor (reduced)

Aerobic dehydrogenases or oxidases catalyze the transfer of protons (electrons) directly to oxygen, anaerobic dehydrogenase - to the intermediate substrate, but not oxygen; cytochromes catalyzes the transfer of only electrons. This class also includes hem-containing enzymes catalase and peroxidase, which catalyze reactions involving hydrogen peroxide.

A characteristic property of the oxidoreductases in cells is their ability to form chains of redox enzymes. They perform multi-stage transfer of hydrogen atoms or electrons from the substrate to the terminal acceptor, which is usually oxygen. The result is water formation. Another important property of oxidoreductases is that they accelerate chemical reactions accompanied by release of energy.

Oxidoreductases contain nicotinamide adenine dinucleotide, or NAD⁺ as an active group. Other coenzymes are nicotinamide adenine dinucleotide phosphate (NADP⁺). Their partners in oxidoreductase chain are flavoproteins - flavin mononucleotide FMN and flavin adenine dinucleotide FAD. Coenzymes of oxidoreductases are also quinones. Cytochrome system is formed by several oxidoreductases having ferroprotoporphyrins as prosthetic groups.

1. Detection of peroxidase (donor: H₂O₂ - oxidoreductase, EC 1.11.1.7.) in horseradish.

To 1 ml of an aqueous extract of horseradish add 5 drops of an alcoholic solution of benzidine and then 2 drops of 0.5% solution of hydrogen peroxide. The dark blue color is observed due to the oxidation of benzidine to p, p'-diiminodiphenilquinon:

Detection of dehydrogenase (xanthine oxidase, aldehyde dehydrogenase, EC 1.1.3.22) in milk (Schardinger reaction).

Pour 2 ml of fresh cow's milk into 3 test tubes Boil one sample for 2-3 minutes and cool. Add 0.5 ml of 0.4% aqueous solution of formaldehyde to the boiled sample and one unboiled sample, and 0.5 ml of water to in another unboiled sample. Then add 2 drops of 0.01% solution of methylene blue to all three tubes. Shake test tubes well and add 4 drops of mineral oil to each. Put all samples in a water bath heated to 40°C. After time, the liquid in the boiled sample containing substrate, discolored due to the formation of the reduced form of methylene blue:

If a colorless solution of methylene blue is shaked whith the air, it becomes blue:

MB×H₂ + O₂ ® MB + H₂O₂

This reaction can be used to distinguish between raw milk from boiled milk.

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