A continuous spectrophotometric method based on enzymatic cycling for determining L-glutamate.

A continuous spectrophotometric assay for determining low levels of L-glutamate is described. The assay, which involves the enzymes L-glutamate oxidase and glutamic-pyruvic transaminase, is based on the recycling of L-glutamate into alpha-ketoglutarate, with the concomitant appearance 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 substrate. In this way the interference observed in the cyclic assay using glutamic-oxalacetic transaminase, which is due to the fact that L-aspartate is also a substrate of L-glutamate oxidase, is eliminated. A kinetic study of the system is presented, with the accumulation of chromophore being seen to undergo a transient phase, which is dependent both on the cycling rate and on the auxiliary enzyme concentration. The kinetic 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. Copyright 1998 Academic Press.