Evaluation of nitric oxide synthase activity and inhibition kinetics by chemiluminescence

The binding affinity (K-1) and inactivation rate (k(inact)) parameters of n itric oxide synthase (NOS) inhibitors are typically estimated by kinetic ac tivity studies. Methods currently used in the estimation of these parameter s frequently employ radiolabeled materials and require intensive sample pre paration. We have devised a simple, reproducible, and sensitive method for the kinetic analysis of NOS activity and inhibition kinetics using chemilum inescence. We have used this method to characterize enzyme activity for pur ified murine macrophage nitric oxide synthase (NOS II). Using this method, we have also estimated the inhibitory parameters for a series of competitiv e antagonists and mechanism-based inactivators of NOS II. The estimated par ameters are in agreement with those reported using other methods. We conclu de that the chemiluminescence method can be used for kinetic studies of NOS activity and inhibition. This method represents a more efficient means for conducting kinetic studies of NOS inhibition, (C) 2000 Academic Press.