Investigation on binding of nitric oxide to horseradish peroxidase by absorption spectrometry.
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.
Qiang
Li
L
College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong, PR China.
Zhu
Shuhua
S
Ma
Hongmei
H
Zhou
Jie
J
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
D016877:Oxidants; D009569:Nitric Oxide; D006139:Guaiacol; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase