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Assay of Serum Glutamate Oxaloacetate Transferase Protocol
To determine i.e, glutamate oxaloacetate transaminase in given serum sample.
Aminotransferases are the enzymes which catalyse the transfer of aminogroup from an amino donar compound to the carbonyl position of an amino acceptor compound. Usually, an aminoacid acts as aminogroup donar and Ketoacid acts as aminogroup acceptor. In some cases aldehydes may serve as aminogroup acceptor and amines may acts as donars. The enzymatic transfer of aminogroups paly an important role in many metholic process, where the interconversion of nitrogen containing molecules is involved. Nitrogen following its initial assimilation into glutamine and glutamate can be disturbed to many other compounds by reaction of aminotransferases. Glutamate is often the aminogroup donar in many biosynthetic transamination reactions. This reaction regenerate alpha ketogluterate for necessary ammonia assimilation through dehydrogenase and glutamine synthetase reaction.
SGOT catalyses the reversible interconversion between gltamate and aspartate and their keto analogues. The oxaloacetate is measured calorimetrically by the reaction with 2,4-dinitrophenyl hydrazine giving a brown coloured complex after the addition of 0.4 N NaOH. The coloured compoud which is estimated calorimetrically at 440nm.
1. PHOSPHATE BUFFER :(0.1 N, pH - 7.4) :
5.965 gms of disodium hydrogen phosphate and 1.09 gms of potassium dihydrogen phosphate was dissolved in distilled water and diluted to 500 ml with distilled water.
2. STANDARD PYRUVATE SOLUTION :
0.22gms of sodium pyruvate was dissolved in 100ml of phosphate buffer.
3. GOT Substrates :
0.146 gms of alpha Ketogluterate and 13.3gms of aspartate was transferred to a beaker and the pH is adjusted to 7.4 by adding 1 N NaOH. The solution is then diluted to 500ml with buffer. This solution is stable under refrigeration.
1. 0.01-0.05 ml of the standard pyruvate solution was pipetted out into 5 different testtubes.
2. The volume was made upto 0.6ml with distilled water in each testtube.
3. The 0.5ml of dinittophenyl hydrazine was added to all the tubes and let to stand for 2'.
4. Then 5ml of 0.4 N NaOH was added, mixed well and let to stand for 5'.
5. Read the intensity of colour at 440nm.
1. Pipette out 0.6ml of distilled water into a testtube.
2. 0.5ml of dinitrophenyl hydrazine was added and let to stand for 2'.
3. Then 5ml of 0.4N NaOH was added,mixed well and let to stand for 5' and then used as blank.
1. 0.5ml of substrate was taken in a testtube and test tube was placed in a constant temperature water bath at 37C for 5'.
2. Then 0.1ml of serum was added and mixed well and incubated for 60'.
3. Then 0.5ml of 2,4 dinitrophenyl hydrazine was added, mixed well and let to stand for 2'.
4. The intensity of colour was read at 440nm against blank.