Titration of Low Kd Binding Sites: Binding of Arginine Analogs to Nitric Oxide Synthases
作者： S.M.E. Smith , C. Sham , L. Roman , P. Martasek , J.C. Salerno
关键词: Nitric oxide 、 Binding site 、 Arginine binding 、 Biochemistry 、 Ligand (biochemistry) 、 Arginine 、 Stereochemistry 、 Dissociation constant 、 Chemistry 、 Titration 、 Tetrahydrobiopterin
摘要: Abstract Spectrophotometrically monitored ligand titration is an important method for the determination of equilibrium dissociation constants ( K d ) from nitric oxide synthases (NOS). Low sites such as tetrahydrobiopterin and arginine binding present difficulties in that experiments often require enzyme concentrations same magnitude . An analytical based on computer simulation described allows estimation values without independent means monitoring free or accurate prior number sites. The approximately 0.5 μM replete neuronal inducible isoforms (nNOS iNOS), while endothelial isoform has a slightly higher (1.5 μM). N -OH-arginine (an intermediate) binds to nNOS with around 0.2 μM, inhibitors -methyl-arginine -nitro-arginine bind more tightly; our best estimates are 100 nM lower.
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