尿液中

张晓洁**　孟泽

摘要　为了制备淀粉酶测定的参考品，从正常人尿液中提取了α-淀粉酶(Amy)并对其特性进行了研究。该提制品淀粉酶的比活性达84.3 kU/g蛋白，几乎不含其它杂酶(测不到ALT、AST、LD、CK、GGT、ChE、ALP活性浓度)。SDS-聚丙烯酰胺凝胶电泳显示一条Amy区带。其催化特性和人血清Amy非常相似，用亚乙基封闭的G7-pNP(EPS)作底物，偶联多功能α-葡萄糖苷酶测定其米氏常数为0.189 mmol/L。利用琼脂糖凝胶电泳，碘-淀粉显色，该提取物至少被分为S2、P2两条活性区带。本研究为今后进一步研制淀粉酶测定参考品建立了基础。
关键词　α-淀粉酶；参考材料；尿

Study on purification and properties of α-amylase from human urine

ZHANG Xiaojie,MENG Zei
(Department of Medical Laboratory Science,PLA,Nanjing General Hospital of Nanjing Command,Nanjing　210002)

Abstract　For the purpose of preparing the reference material of α-Amylase,the purification and properties of Amy in human urine were studied.The purified Amy has a specific activity of 84.3 kU/g of protein.It was almost free of contaminating enzymes.After polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate,only one band of Amy could be seen.The final purified Amy was found to be very similar to the human serum Amy in catalytic properties.The apparent Michaelis constant of the purified Amy was 0.189mmol/L with ethylidence-blocked G7-pNP(EPS)as substrate and with multifunctional α-glucosidase as auxiliary enzyme.Agarose gel electrophoresis was performed and isoenzymes were developed with iodine-starch method.At least two distinct enzymatic bands,S2 and P2 were found.This research is intended to be a fundament to prepare a reference material for the measurement of α-amylase.
Key words　α-Amylase；reference material；urine

　　淀粉酶(Amy，EC3.2.1.1)属水解酶类，分为α、β两类，人体中淀粉酶属α-淀粉酶，主要由胰腺和唾液腺分泌。Amy活性的准确测定依赖于测定方法的标准化。Amy的测定方法不下200种，德国Boehringer Mannheim(BM)公司推出了新底物4，6-亚乙基-4-硝基酚-α-庚糖偶联多功能α-葡萄糖苷酶的测定方法。IFCC根据这一新体系，提出了Amy测定的参考方法。没有参考品或标准品，仍不能确定测定结果是否正确。欧共体标准局(BCR)的CRM476是从人胰腺中提制的Amy参考品，其动力学特性与人血清基本一致［1］。我国目前尚无酶参考品。由于胰腺取材困难，并且牵涉道义和法律问题，故我们采用正常人尿液提取Amy。与从胰腺中提取淀粉酶相比，不仅取材方便，而且尿液和血清中Amy的成分基本一致，为今后制备酶的参考品建立了基础。

1　材料和方法
1.1

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