Titration of low K-d binding sites : Binding of arginine analogs to nitricoxide synthases

Spectrophotometrically monitored ligand titration is an important method fo r the determination of equilibrium dissociation constants (K-d) from nitric oxide synthases (NOS). Low K-d sites such as the tetrahydrobiopterin and a rginine binding sites present difficulties in that experiments often requir e enzyme concentrations of the same magnitude as the K-d. An analytical met hod based on computer simulation is described that allows the estimation of K-d values without an independent means of monitoring free ligand or witho ut an accurate prior determination of the number of binding sites. The K-d for arginine is approximately 0.5 muM for the tetrahydrobiopterin replete n euronal and inducible isoforms (nNOS and MOS), while the endothelial isofor m. has a slightly higher K-d (1.5 muM). N-OH-arginine (an intermediate) bin ds to nNOS with a K-d of around 0.2 muM, while the inhibitors N-methyl-argi nine and N-nitro-arginine bind more tightly; our best Kd estimates are 100 nM or lower. (C) 2001 Academic Press.