On-line detection of nitric oxide generated by the enzymatic action of nitric oxide synthase on L-arginine using a flow injection manifold and chemiluminescence detection

A study of the catalytic activity of nitric oxide synthase (NOS) on the conversion of arginine to NO and citrulline is made. The target of this report is to establish a method for on-line monitoring the reaction process using the chemiluminescence (CL) generated from NO in the luminol-H2O2 system. The NOS-catalytic activity is found to decrease with time on stream in a flow enzymatic reactor but the activity is recovered by proper treatment with restoring solution. For on-line detection of NO formed in-situ, using how injection with CL detection, the response is found more sensitive if a pulsed sampling procedure is employed rather than a continuous one; the sample in the former is small and is injected periodically between regeneration cycles. The optimal pH, temperature and flow rate were determined. The calibration graph under optimal conditions is linear for arginine concentration; the relative standard deviation is <1% and the effect of interferents present in biological fluids is found to be much different to those for the native enzyme in solution. The immobilized NOS-reactor was long lived. (C) 2000 Elsevier Science B.V. All rights reserved.