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 con version 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. T he NOS-catalytic activity is found to decrease with time on stream in a flo w enzymatic reactor but the activity is recovered by proper treatment with restoring solution. For on-line detection of NO formed in-situ, using how i njection with CL detection, the response is found more sensitive if a pulse d sampling procedure is employed rather than a continuous one; the sample i n the former is small and is injected periodically between regeneration cyc les. The optimal pH, temperature and flow rate were determined. The calibra tion graph under optimal conditions is linear for arginine concentration; t he relative standard deviation is <1% and the effect of interferents presen t in biological fluids is found to be much different to those for the nativ e enzyme in solution. The immobilized NOS-reactor was long lived. (C) 2000 Elsevier Science B.V. All rights reserved.