A colorimetric 96-well microtiter plate assay for the determination of enzymatically formed citrulline

L-Citrulline constitutes a product of a number of enzymatic reactions. In t he past a number of colorimetric methods for the determination of L-citrull ine, upon its chemical modification with diacetyl monoxime at 95 degreesC, have been reported. However, all these methods are time- and material-consu ming. In this work, using the same chemical reaction, a new method for the use in 96-well polystyrene microtiter plates was developed. The method is f ast and requires substantially less material as the enzymatic reaction is p erformed in a volume of 60 mul. The applicability of this enzymatic assay w as established using L-N-omega,N-omega-dimethylarginine dimethylaminohydrol ase, which generates L-citrulline from side-chain methylated derivatives of L-arginine. The detection limit for L-citrulline is about 0.2 nmol, In add ition, our studies show that most commonly used biochemical buffers and buf fer additives do not affect the assay. This method may prove useful in the studies of other L-citrulline producing enzymes including nitric oxide synt hase, (C) 2000 Academic Press.