[关键词]
[摘要]
α-葡萄糖苷酶抑制剂能抑制碳水化合物水解,是高血糖人群降低餐后血糖的常用物质。本文基于α-葡萄糖苷酶-PNPG体外反应体系,建立了微量、快速的α-葡萄糖苷酶抑制剂筛选模型,该模型的主要参数如下:酶浓度为0.05 U/mL;底物浓度范围为0.05~1 mM;反应温度为37 ℃;反应时间为6 min。以该模型检测了阿卡波糖对α-葡萄糖苷酶的抑制作用,并采用Lineweaver-Burk Plots、Eadie-Hofstee Plots、Hanes-Wolff Plots、Eisenthal-Cornish-Bowden Direct Plots、Non-linear Regression Analysis五种方法对该酶促反应的动力学数据进行了详细的分析。通过对数据处理的过程和结果的比较发现,该五种方法各有特点,各法所获得的Vmax、Km和Ki存在一定的差异,Non-linear-Regression Analysis法更加简便、合理及可靠,是酶动力学数据处理的首选方法。采用Non-linear-Regression Analysis法计算,该模型中酶促反应的Vmax为3.91×10-6 mmol/min,Km为0.12 mM,阿卡波糖的Ki为90 μM。
[Key word]
[Abstract]
Alpha-glucosidase inhibitors can inhibit carbohydrate hydrolysis and are commonly used to decrease postprandial blood glucose in individuals with hyperglycemia. A model for the rapid screening of trace amounts of alpha-glucosidase inhibitors was established based on the alpha-glucosidase-pNPG in vitro reaction system. The optimized parameters of this model were as follows: the enzyme concentration, substrate concentration, reaction temperature, and reaction time were 0.05 U/mL, 0.05~1 mM, 37 ℃, and 6 min, respectively. The inhibitory effect of acarbose was determined using this model, and the enzymatic reaction kinetics data were analyzed using five methods (Lineweaver-Burk plots, Eadie-Hofstee plots, Hanes-Wolff plots, Eisenthal-Cornish-Bowden direct plots, and Non-linear regression analysis). Data processing and comparison of results showed that these five methods had distinct characteristics as the Ki, Km, and Vmax calculated by these five methods differed slightly. Non-linear Regression Analysis was more convenient, reasonable, and reliable, and therefore was the first choice for processing the data of enzyme kinetics. By this model, the Vmax (?10-6 mmol/min) and Km (mM) of enzymatic reaction were calculated to be 3.91 and 0.12, respectively and the Ki (μM) of acarbose was 90.
[中图分类号]
[基金项目]
国家自然科学基金资助项目(31460436,31260368);江西省自然科学基金项目(20132BAB204004)