Titration of low K(d) binding sites: binding of arginine analogs to nitric oxide synthases

Spectrophotometrically monitored ligand titration is an important method for the determination of equilibrium dissociation constants (K(d)) from nitric oxide synthases (NOS). Low K(d) sites such as the tetrahydrobiopterin and arginine binding sites present difficulties in that experiments often require enzyme concentrations of the same magnitude as the K(d). An analytical method based on computer simulation is described that allows the estimation of K(d) values without an independent means of monitoring free ligand or without an accurate prior determination of the number of binding sites. The K(d) for arginine is approximately 0.5 microM for the tetrahydrobiopterin replete neuronal and inducible isoforms (nNOS and iNOS), while the endothelial isoform has a slightly higher K(d) (1.5 microM). N-OH-arginine (an intermediate) binds to nNOS with a K(d) of around 0.2 microM, while the inhibitors N-methyl-arginine and N-nitro-arginine bind more tightly; our best K(d) estimates are 100 nM or lower.