新色原底物的合成及α-L岩藻糖苷酶速率检测法  

Synthesis of a novel substrate and kinetic rate assay for alphaLfucosidase.

作　　者：李兴民[1,2] 司伊康[1,2] 杨树德[1,2] 

机构地区：[1]北京医院卫生部临床检验中心 [2]中国医学科学院药物研究所

出　　处：《中华医学检验杂志》1999年第3期157-160,共4页

摘　　要：目的合成αＬ岩藻糖苷酶新色原底物２氯４硝基苯αＬ岩藻糖苷，并以此底物为基础建立αＬ岩藻糖苷酶的速率检测法。方法以Ｌ岩藻糖为原料，采用ＷｅｓｔｐｈａｌＦｅｉｅｒ合成法制备底物２氯４硝基苯αＬ岩藻糖苷；研究αＬ岩藻糖苷酶作用于此底物的动力学性质，建立αＬ岩藻糖苷酶速率检测法。结果成功地制备了αＬ岩藻糖苷酶的新底物２氯４硝基苯αＬ岩藻糖苷，首次建立了αＬ岩藻糖苷酶速率检测法。血清αＬ岩藻糖苷酶作用于此底物的最适ｐＨ为４．８，米氏常数为０．２３ｍｍｏｌ／Ｌ，至少在６分钟观测时间内吸光度变化与时间成比例，相当高浓度的抗坏血酸、血红蛋白、胆红素对酶活性测定不产生干扰，与对硝基苯αＬ岩藻糖苷终点法的相关系数为ｒ＝０．９５０１（ｎ＝３２），批内精密度相对标准差≤２．６％，正常参考值为（２７．１±１２．８）Ｕ／Ｌ（ｘ±２ｓ）。结论以新底物为基础建立的αＬ岩藻糖苷酶速率检测法克服了以往检测法的缺陷，操作简便，适用于各种类型的自动生化分析仪，便于临床应用。Objective To synthesize a novel substrate of alphaLfucosidase (AFU) and developa kinetic rate assay method for alphaLfucosidase by the novel substrate. Methods The novel substrate was prepared by the method of Westphal & Feier and AFU kinetic characters on the new substrate were studied. The kinetic rate assay method was evaluated. Results The kinetic rate assay method for determining alphaLfucosidase was developed. Optimum pH was 4.8; michaelsMeten constant (Km) was 0.23 mmol/L for serum sample and 0.28 mmol/L for AFU from human placenta. The course of zero order reaction sustained at 6 minutes. The concentration of ascorbic acid up to 6 g/L had no effect on the determination of AFU activity, nor on hemoglobin up to 230 mg/L or bilirubin up to 250 mg/L. The reference interval ( 2s) for healthy subjects was 14.339.9 U/L. AFU activity was determined in a wide range (0244.5 U/L) and with high sensitivity. Conclusion The kinetic method surmounted defects of previous methods for determining AFU activity. Unlike conventional methods, this method does not require any blank determination, and it can be performed with various types of automated analyzers.

关 键 词：肝肿瘤 肝细胞肿瘤 新色原底物 岩藻糖苷酶 

分 类 号：R735.704[医药卫生—肿瘤]