Human plasma prekallikrein: a rapid high-yield method for purification.
Human plasma prekallikrein: a rapid high-yield method for purification.
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人血浆前激肽释放酶：一种快速高产的纯化方法。
DOI：
10.1111/j.1432-1033.1979.tb02035.x
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发表时间：
1979
期刊：
影响因子：
--
通讯作者：
R. Colman
中科院分区：
文献类型：
--
作者：
C. Scott;C. Liu;R. Colman
描述了用于纯化人血浆前激肽释放酶（丝氨酸蛋白酶激肽释放酶的前体蛋白）的程序。将人血浆与等体积的在含有溴化己二甲双胍的磷酸钠（10 mM）缓冲液（pH 8.0）中平衡的prewollen季氨乙基-Sephadex混合30分钟，然后过滤。将滤液再循环两次，并将最终滤液在pH 8.0的磺丙基-Sephadex柱上层析。将浓缩的流出物在pH 5.2的磺丙基-Sephadex柱上进行色谱分离。纯化的最后一步是在偶联至人IgG的抗血清的琼脂糖上进行亲和层析以除去污染的IgG。出现与前激肽释放酶抗原一致的蛋白质的单峰，代表起始材料的39%的总产率。用纯化的Hageman因子片段活化终产物后，产生激肽释放、精氨酸酯酶和酰胺分解活性。活化的酶水解合成底物l-脯氨酰-l-苯丙氨酰-l-精氨酸-p-硝基苯胺中的酰胺键，Km为0.225 mM. This 1800倍纯化的前激肽释放酶出现在碱性圆盘/聚丙烯酰胺凝胶电泳上的主要和次要条带，并且每个组分与前激肽释放酶凝血活性相关。在十二烷基硫酸盐凝胶电泳上，观察到一条单一条带，其Mr为88000。当前激肽释放酶被Hageman因子片段激活为激肽释放酶时，这种激活的酶进行还原，两个亚基的Mr为55000和33000是明显的。
A procedure is described for the purification of human plasma prekallikrein, the precursor protein of the serine protease kallikrein. Human plasma was mixed for 30 min with an equal volume of preswollen quaternaryaminoethyl-Sephadex equilibrated in sodium phosphate (10 mM) buffer pH 8.0 containing hexadimethrine bromide, and then filtered. The filtrate was cycled two more times and the final filtrate was chromatographed on a sulphopropyl-Sephadex column at pH 8.0. The concentrated effluent was chromatographed on a sulphopropyl-Sephadex column at pH 5.2. The final step of purification was affinity chromatography on agarose coupled to antiserum to human IgG to remove contaminating IgG. A single peak of protein emerged coincident with prekallikrein antigen representing an overall yield of 39% of the starting material. After activation of the final product with purified Hageman factor fragments, kinin-releasing, arginine esterase and amidolytic activity developed. The activated enzyme hydrolyzed the amide bond in the synthetic substrate l-prolyl-l-phenylalanyl-l-arginine-p-nitroanilide with a Km of 0.225 mM. This 1800-fold purified prekallikrein appeared as a major and minor band on alkaline disc/polyacrylamide gel electrophoresis and each component was associated with prekallikrein coagulant activity. On dodecyl sulfate gel electrophoresis, a single band was observed with a Mr of 88000. When prekallikrein was activated by Hageman factor fragments to kallikrein, and this activated enzyme was subjected to reduction, two subunits with Mr of 55000 and 33000 were apparent.