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 μM for the tetrahydrobiopterin replete neuronal and inducible isoforms (nNOS and iNOS), while the endothelial isoform has a slightly higher K_d (1.5 μM). N-OH-arginine (an intermediate) binds to nNOS with a K_d of around 0.2 μM, while the inhibitors N-methyl-arginine and N-nitro-arginine bind more tightly; our best K_d estimates are 100 nM or lower.