嘌呤核苷磷酸化酶检测体系优化及芒果苷对其活性的影响

目的优化体外嘌呤核苷磷酸化酶(purine nucleoside phosphorylase,PNP)活性检测体系,研究芒果苷及芒果苷元对PNP活性的影响。方法采用紫外分光光度法,通过考察酶浓度、底物浓度、二硫苏糖醇(DTT)浓度对酶促反应的影响,确定检测酶活性的最适反应条件,以此为基础研究芒果苷和芒果苷元对PNP活性的影响。结果成功建立并优化了体外PNP活性检测体系,反应温度为25℃时,在磷酸钾盐缓冲液中,当PNP浓度为250 U·L-1、底物鸟苷为400μmol·L-1、DTT为0.5 mmol·L-1的反应条件下于波长258nm处动态监测15 min,芒果苷及其代谢产物苷元在浓度高达100μmol·L-1时,对PNP活性无明显抑制作用。结论以优化的PNP活性检测体系研究发现芒果苷及芒果苷元体外对PNP活性无影响。

On detection system for the activity of PNP and effect of mangiferin on PNP activtity

Aim To optimize the detection system for the activity of purine nucleoside phosphorylase(PNP),and to investigate the effects of mangiferin and its aglycone,norathyriol,on the activity of PNP in vitro.Methods The optimal conditions of assaying the activity of PNP were selected by observing the influencing factors of the kinetics of enzyme-catalyzed reaction,such as the concentration of enzyme,substrate and DTT,with the use of ultraviolet spectrophotometry in vitro.The effects of mangiferin and its aglycone on the activity of PNP were studied in the optimized detection system.Results The enzymatic reaction to assaying the activity of PNP in vitro was monitored in the potassium phosphate buffer containing 250 U·L-1 PNP,400 μmol·L-1 guanosine and 0.5 mmol·L-1 DTT at 25℃ for 15 min at 258 nm.No inhibition of mangiferin and its aglycone on the activity of PNP was observed in the condition,when the concentration of mangiferin and its aglycone were up to 100 μmol·L-1.Conclusion Mangiferin and its aglycone do not inhibit the activity of PNP in vitro by the optimized detection system.