Assay for the determination of the activity of the enzyme glutamine-alanine transaminase
Assay for the determination of the activity of the enzyme glutamine-alanine transaminase
The basic principle of the determination of glutamine-alpha transaminase activity; master the method of the determination of glutamine-alpha transaminase activity. (Source: Biochemistry and Molecular Biology Laboratory Guide, Shao Xueling et al., Wuhan University Press, 2003)
Operation method
color rendering
Principle
Serum glutamic transaminase (SGPT) catalyzes the formation of glutamic acid and pyruvic acid from alanine and ketoglutaric acid. pyruvic acid is condensed with 2,4-dinitrophenylhydrazine to form dinitrophenylhydrazone pyruvate, which is orange-red under alkaline conditions, with a coloration consistent with Beer's law and with a maximal absorption at 520 nm.The highest levels of SGPT are found in the liver, and the enzyme escapes from the bloodstream as a result of hepatocellular damage, which can significantly increase SGPT levels. Due to liver cell damage, the enzyme escapes from the blood, which can increase the SGPT level significantly. Therefore, the measurement of serum SGPT activity can be used as an important indicator of hepatotoxicity and liver disease.
Materials and Instruments
Pyruvic acid Move Solution Preparation: Caveat 1. In the color rendering reaction, 2,4-dinitrophenylhydrazine can act with compounds with ketone group to form phenylhydrazone, and α-ketoglutaric acid in the substrate can react with it to form α-ketoglutaric acid phenylhydrazone. Therefore, when making the standard curve, it is necessary to add a certain amount of substrate to offset the effect of α-ketoglutaric acid. 2. In the determination of SGPT activity, the substrate and serum should be kept at a constant temperature in a 37 ℃ water bath beforehand, and then the substrate should be added to the serum tube and timed punctually. 3. The value on the standard curve is accurate and reliable from 20 to 100 U. If it exceeds 200 U, the sample should be diluted. 4. DL-type should be used for alanine, not D-type. If L-type is used, the dosage should be reduced by half. 5. Hemolysed specimens should not be used because of the high activity of transaminases in blood cells, which will affect the results. For more product details, please visit Aladdin Scientific website.
Acetone α-ketoglutarate dl-alanine 2 4-dinitrophenylhydrazine Sodium hydroxide
Constant temperature water bath 722S spectrophotometer Pipette Test tubes Test tube racks
1. Pyruvic acid standard solution: accurately weigh 22 mg of pure sodium pyruvate, with pH 7.4 phosphate buffer solution to 100 ml.
2. SGPT substrate solution: accurately weigh 87.6 mg of α-ketoglutaric acid, dl-alanine 5.34 g, first dissolved in 90 ml 0.1 mol/L pH 7.4 phosphate buffer, and then adjusted to pH 7.4 with 20% NaOH solution, and then diluted to 300 ml with the above phosphate buffer solution, refrigerator storage can be used for one week (plus chloroform preservation).
3. 0.1 mol/L pH7.4 phosphate buffer.
4. 2,4-Dinitrophenylhydrazine solution: weigh 19.8 mg of 2,4-Dinitrophenylhydrazine in a 100 ml volumetric flask, first dissolve with 8 ml of concentrated hydrochloric acid, then dilute with water to the scale.
5. 0.4 mol/L sodium hydroxide solution.
Operating Procedure:
1. Drawing of standard curve: take 6 dry and clean test tubes, number them and add reagents as shown in the table below.
Add 0.5 ml of 2,4-dinitrophenylhydrazine to each tube, and then keep warm for 20 min, add 5 ml of 0.4 mol/L sodium hydroxide solution to each tube, and let it stand at room temperature for 10 min, adjust the zero point with distilled water, and measure the optical density at 520 nm. The optical density was measured at 520 nm. The optical density of each tube was subtracted from the optical density of the blank tube as the vertical coordinate, and the number of micrograms of pyruvic acid as the horizontal coordinate for the standard curve.
2. Determination of SGPT activity: Take 4 clean and dry test tubes, i.e. 2 test tubes and 2 control tubes, add reagents according to the following table.
After the reaction of each tube is completed, mix well, and leave it at room temperature for 10 min, then adjust the zero point with distilled water to determine the optical density. The number of micrograms of pyruvic acid was found from the standard curve, and the activity of SGPT was calculated according to the following formula: SGPT activity (unit) = (micrograms of the assay tube - micrograms of the control tube)/2.5×0.1.