A simple procedure for isolating microgram quantities of biologically active bikunin from human urine.
A simple procedure for isolating microgram quantities of biologically active bikunin from human urine.
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从人尿中分离微克量的生物活性比库宁的简单程序。
DOI：
10.1111/j.1464-410x.2005.05700.x
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Marengo,SusanRuth
中科院分区：
文献类型：
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作者：
Yang,Lizhu;Resnick,MartinI;Marengo,SusanRuth
目的报道一种简单、相对快速的方法，利用离子交换胰蛋白酶亲和色谱法从人尿液中分离生物活性bikunin。Bikunin是一种蛋白酶抑制剂，已被证明在各种过程中发挥作用，包括抑制草酸钙结晶、调节增殖和调节致癌作用。纯化的蛋白质的不可用性阻碍了bikunin在这些processing.MATERIALS和METHODSFEMALE人尿的扩大作用的研究透析（15 kDa阈值）和粗分馏与双饱和硫酸铵沉淀。 第一次沉淀用35%饱和硫酸铵，收获上清液，第二次沉淀用90%饱和硫酸铵，收集沉淀。然后使蛋白质混合物通过串联连接的Sepharose SP-快速流动阳离子交换柱和Sepharose Q-快速流动阴离子交换柱。最终纯化采用选择性结合bikunin的胰蛋白酶亲和柱。该程序可回收1 µg bikunin/2 mL尿液，最终产物基本上不含污染的α-胰蛋白酶抑制剂间重链或bikunin-重链缀合物。 通过双向聚丙烯酰胺凝胶电泳结合银染色或蛋白质印迹法确认产品纯度。所有分离物均含有17 kDa最低限度糖基化/硫酸化形式的bikunin和28 kDa形式的bikunin。 一些制剂还含有33-48 kDa形式的bikunin。 通过质谱和Western blot分析，证实这三种蛋白的核心蛋白均为bikunin。收获的bikunin保留其胰蛋白酶抑制活性（l-苯甲酰精氨酸-对硝基苯胺测定）。含有33-45 kDa形式的制剂比不含此条带的制剂具有2 - 3倍的胰蛋白酶抑制活性。 本品由生物活性bikunin的三种异构体组成，不含污染重链或bikunin-重链结合物。纯化的bikunin的现成可用性应有利于未来的研究bikunin的尿石症，增殖和致癌作用的新兴作用。
OBJECTIVETo report a simple, relatively rapid protocol to isolate biologically active bikunin from human urine using ion‐exchange‐trypsin affinity chromatography. Bikunin is a protease inhibitor which has been shown to play a role in various processes, including inhibition of calcium oxalate crystallization, the regulation of proliferation and modulation of carcinogenesis. The unavailability of the purified protein has hampered studies on bikunin's expanding role in these processes.MATERIALS AND METHODSFemale human urine was dialysed (15 kDa threshold) and crudely fractionated with a double‐saturated ammonium sulphate precipitation. The first precipitation was with 35% saturated ammonium sulphate, and the supernatant was harvested, and the second with 90% saturated ammonium sulphate, and the precipitate collected. The protein mixture was then passed over Sepharose SP‐fast‐flow cation exchange and Sepharose Q‐fast‐flow anion exchange columns connected in series. The final purification was with a trypsin‐affinity column which selectively bound bikunin.RESULTSThis procedure could recover 1 µg of bikunin per 2 mL of urine, and the final product was essentially free of contaminating inter‐α‐trypsin inhibitor heavy chains or bikunin‐heavy chain conjugates. Product purity was confirmed by two‐dimensional polyacrylamide gel electrophoresis combined with silver staining or Western blot. All isolations contained the 17 kDa minimally glycoslyated/sulphated form of bikunin and the 28 kDa form of bikunin. Some preparations also contained 33–48 kDa forms of bikunin. The protein cores of all three proteins were confirmed to be bikunin by mass spectrometry and Western blot. Harvested bikunin retained its trypsin inhibitory activity (l‐benzoylarginine‐p‐nitroanilide assay). Preparations containing the 33–45 kDa form had two to three times more trypsin inhibitory activity than preparations without this band.CONCLUSIONSThis novel ion exchange‐trypsin affinity chromatography protocol uses only two chromatographic steps. The product consists of three isomers of biologically active bikunin, free of contaminating heavy chains or bikunin‐heavy chain conjugates. The ready availability of purified bikunin should facilitate future studies of bikunin's emerging role in urolithiasis, proliferation and carcinogenesis.