Qualitative assays for aminotransfer reactions can be used to: detect the presence of aminotransferases.

Operation method

Qualitative Identification of Aminotransfer Reactions

Principle

Aminotransferase, also known as transaminase, catalyzes the interchange of the amino group of α-amino acids with the α-keto group of α-keto acids, an action known as aminotransferase. It plays a very important role in intermediate metabolism such as protein synthesis and catabolism in living organisms, and in the interconnection and interconversion of the metabolism of the three types of substances, namely sugar, fat and protein. The transamination of any amino acid is catalyzed by its specialized transaminase. Their optimal pH is close to 7.4. Among the various transaminases, glutamic acid-oxaloacetic acid transaminase (GOT) and glutamic acid-pyruvic acid transaminase (GPT) are the most active.

Materials and Instruments

Rabbit
Phosphate buffer Glutamic acid solution Pyruvic acid solution Potassium bicarbonate solution Iodine acetic acid solution Trichloroacetic acid solution Alanine solution Standard glutamic acid solution Hydrated ninhydrin ethanol solution Phenol solvent
Anatomical instruments Centrifuges Constant temperature baths Drying boxes Scales Chromatography systems

Move

The rabbit was stunned, quickly dissected, the liver was removed and sheared at low temperature. After weighing the homogenate, the supernatant was centrifuged to obtain the prepared enzyme solution. In vitro amino transfer reaction. Paper chromatography was used to check 4 spots per person (standard alanine solution, standard glutamate solution, control supernatant, sample supernatant). Spot samples were placed in a chromatography hood. With an aqueous solution saturated with phenol, after equilibration, 25 ml of saturated phenol reagent was added from the chromatographic hood to the chromatographic cylinder using a glass tube with a small funnel. Then, immediately cover the plug tightly, when the solvent front reaches about 2.5 cm from the upper end of the filter paper, remove the filter paper, dry or blow dry to remove the phenol solvent, spray 0.1% ninhydrin ethanol solution, put into the oven to develop the color of the amino acids reacted with ninhydrin and showed a purplish-red spot on the filter paper.

Caveat

1, Take fresh tissue of animals to prepare enzyme solution to prevent enzyme inactivation.

2， Add iodized acetic acid to inhibit the enzymatic action.

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