Journal
ANALYTICAL BIOCHEMISTRY
Volume 286, Issue 2, Pages 257-264Publisher
ACADEMIC PRESS INC
DOI: 10.1006/abio.2000.4805
Keywords
arginine deiminase; argininosuccinate synthase (ASS); citrulline (Cit); diacetyl monoxime (DAMO); dimethylargininase (DDAH); enzymatic assay; nitric oxide synthase (NOS); ornithine carbamoyl transferase (OCT)
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L-Citrulline constitutes a product of a number of enzymatic reactions. In the past a number of colorimetric methods for the determination of L-citrulline, upon its chemical modification with diacetyl monoxime at 95 degreesC, have been reported. However, all these methods are time- and material-consuming. 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 fast and requires substantially less material as the enzymatic reaction is performed in a volume of 60 mul. The applicability of this enzymatic assay was established using L-N-omega,N-omega-dimethylarginine dimethylaminohydrolase, which generates L-citrulline from side-chain methylated derivatives of L-arginine. The detection limit for L-citrulline is about 0.2 nmol, In addition, our studies show that most commonly used biochemical buffers and buffer additives do not affect the assay. This method may prove useful in the studies of other L-citrulline producing enzymes including nitric oxide synthase, (C) 2000 Academic Press.
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