盐酸小檗碱对人血红蛋白拟过氧化物酶活性的影响

Effect of berberine hydrochloride on peroxidase-like activity of human hemoglobin

目的明确盐酸小檗碱(berberine hydrochloride,BH)对人血红蛋白(hemoglobin,Hb)拟过氧化物酶(peroxidase,POD)活性的影响并分析其作用机制。方法采用紫外吸收光谱法测定Hb拟POD活性,确定最适pH和最适温度。采用不同浓度BH与Hb相互作用后测定POD活性,并通过Lineweaver-Burk双倒数作图判定其作用机制。采用荧光光谱法测定BH与Hb相互作用后荧光淬灭常数,判定荧光淬灭方式,计算结合位点数和结合常数。结果Hb拟POD活性发挥的最适pH为6.37,最适温度为38.4℃。BH与Hb相互作用后POD活性的米氏常数K_(m)和最大反应速度V_(max)均变小,K_(m)/V_(max)比值不变。荧光淬灭常数随温度升高而变小,即BH可静态淬灭Hb荧光。BH与Hb具有1个结合位点,在25、30、37℃下的结合常数分别为为8.63×103、8.25×10^(3)、7.39×10^(3)L/mol。结论BH可反竞争性抑制Hb拟POD活性。

Objective To determine the effect of berberine hydrochloride(BH)on peroxidase(POD)-like activity of human hemoglobin(Hb)and its mechanism.Methods The POD-like activity of Hb was determined by ultraviolet absorption spectroscopy to defined the optimum pH and optimum temperature.The POD activity was measured using different conentrations of BH mixed with Hb,and the mechanism of action was revealed by Lineweaver-Burk double reciprocal plot.Fluorescence spectroscopy was used to determine the fluorescence quenching constant after BH and Hb interaction for identifying the fluorescence quenching mode and calculating the number of binding sites and the binding constant.Results The optimum pH and optimum temperature of POD-like activity exhibited by Hb were 6.37 and 38.4℃,respectively.The Michaelis constant K_(m) and maximum reaction rate V max of POD activity decreased after the interaction of BH and Hb,and the ratio of K_(m)/V_(max) did not change.The fluorescence quenching constant decreases with increaing temperature,indicating that BH could statically quench the Hb fluorescence.BH had only one binding site with Hb,and the binding constants were 8.63×10^(3)、8.25×10^(3) and 7.39×10^(3) L/mol at 25、30 and 37℃,respectively.Conclusion BH could inhibit the POD-like activity of Hb in an anti-competitive manner.