Titration of Low Kd Binding Sites: Binding of Arginine Analogs to Nitric Oxide Synthases

Abstract

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