title: Investigation on binding of nitric oxide to horseradish peroxidase by absorption spectrometry.
abstract: Binding of nitric oxide to horseradish peroxidase (HRP) has been investigated by absorption spectrometry in 0.2M anaerobic phosphate buffer solution (pH 7.4). Based on this binding equilibrium, a model equation for evaluating the binding constant of nitric oxide to HRP is developed and the binding constant is calculated to be (1.55+/-0.06)x10(4)M(-1), indicating that HRP can form a stable complex with nitric oxide. The type of inhibition by nitric oxide is validated on the basis of studying initial reaction rates of HRP-catalyzed oxidation of guaiacol in the presence of hydrogen peroxide and nitric oxide. The inhibition mechanism is found to follow an apparent non-competitive inhibition by Lineweaver-Burk method. Based on this kinetic mechanism, the binding constant is also calculated to be (5.22+/-0.06)x10(4)M(-1). The values of the binding constant determined by the two methods are almost identical. The non-competitive inhibition model is also applicable to studying the effect of nitric oxide on other metalloenzymes, which catalyze the two-substrate reaction with the "ping-pong" mechanism.
authors: College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong, PR China.
authors: Li
authors: L
authors: Qiang
authors: Shuhua
authors: S
authors: Zhu
authors: Hongmei
authors: H
authors: Ma
authors: Jie
authors: J
authors: Zhou
mesh_terms: D000042:Absorption; D006139:Guaiacol / Q000737:chemistry / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D016877:Oxidants / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
chemical_list: D016877:Oxidants; D009569:Nitric Oxide; D006139:Guaiacol; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase