A CONTINUOUS SPECTROPHOTOMETRIC METHOD BASED ON ENZYMATIC CYCLING FORDETERMINING L-GLUTAMATE

A continuous spectrophotometric assay for determining low levels of L- glutamate is described. The assay, which involves the enzymes L-glutam ate oxidase and glutamic-pyruvic transaminase, is based on the recycli ng of L-glutamate into alpha-ketoglutarate, with the concomitant appea rance of one molecule of hydrogen peroxide in each turn of the cycle. This is subsequently reduced by means of a peroxidase-coupled reaction , using 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) as substr ate. In this way the interference observed in the cyclic assay using g lutamic-oxalacetic transaminase, which is due to the fact that L-aspar tate is also a substrate of L-glutamate oxidase, is eliminated. A kine tic study of the system is presented, with the accumulation of chromop hore being seen to undergo a transient phase, which is dependent both on the cycling rate and on the auxiliary enzyme concentration. The kin etic parameters characterizing the system have been determined, making it possible to optimize costs with respect to the enzymes involved in the cycle, since the minimum amount needed for a given rate constant of the cycle can be calculated. (C) 1998 Academic Press.