An Improved Colorimetric Transaminase Assay.

W/E RECENTLY reported a new colorimetric assay for glutamic-oxalacetic transaminase which involves coupling the oxalacetate formed in the enzymatic reaction with a diazonium salt (1, 2). Because of the greater specificity of the diazonium salt for the product of the reaction and the minimum number of manipulations involved, this procedure is simpler and more accurate than previously reported procedures (3-6) using dinitrophenylhydrazine as the color reagent. The method involves incubating 0.2 ml. serum with 1 ml. of substrate for 20 mm. at 37#{176}, adding 1 ml. of diazonium salt solution, incubating an additional 10 mm., diluting with 10 ml. of water, and reading the absorbance. The color after dilution continues to increase slowly, owing to slight residual transamiriase activity and instability of the diazonium salt at the pH of the reaction. This was not felt to be a serious disadvantage and it was recommended that samples be run at 1-mm, intervals arid read immediately after dilution. However, several investigators have since indicated to us that in their hands this technic is cumbersome when many samples must be assayed. The color can he completely stabilized by adding dilute hydrochloric acid rather than water after the second incubation. Concentrations between 0.01 and 0.05N are satisfactory; we routinely use 0.02N. This lowers the pH to about 2, but decreases the total color by about 10%. This decrease in color can be prevented by incorporating a nomonie detergent in the dilute HC1 at a concentration of 0.5 to 1% (v/v). The recommended diluent is prepared as follows. To a 1-L., graduated cylinder half filled with distilled water add 1.7 ml. of concentrated hydro-