PURIFICATION AND STRUCTURAL-ANALYSIS OF 4TH COMPONENT OF HUMAN COMPLEMENT
PURIFICATION AND STRUCTURAL-ANALYSIS OF 4TH COMPONENT OF HUMAN COMPLEMENT
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DOI：
10.1021/bi00628a039
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发表时间：
1977-01-01
期刊：
影响因子：
2.9
通讯作者：
PRAHL, J
中科院分区：
文献类型：
--
作者：
BOLOTIN, C;MORRIS, S;PRAHL, J
以5-12%聚乙二醇沉淀物为原料，用亲和吸附剂除去纤溶酶原，从新鲜血浆中纯化人补体第4组分（C4），产率为20%。在DEAE纤维素、QAE[季铵乙基]-Sephadex和DEAE-Bio-Gel A上的连续离子交换色谱法通过免疫学标准和聚丙烯酰胺凝胶电泳得到均质的C4，最后一个色谱步骤实现了天然与灭活C4的分离。用20 mM二硫苏糖醇在37 ℃还原2小时。C在0.25 M 2-氨基-2-羟甲基-1，3-丙二醇盐酸盐（pH 8.6）中，可使链间二硫键断裂。C4的3链结构得到证实，MW估计为93，000 ±。九千三百，七万五千。7500和30，000.+-. 3000确定为α，.β的和γ链，分别。研究了已知的C4失活剂对C4链的作用，证实了Cl.hivin s [C1的活化S片段]和胰蛋白酶的失活伴随着α链的断裂。链肼灭活C4产生的链大小没有检测到的变化。在1 M乙酸存在下通过凝胶过滤完成链的分离。进行天然C4和组成性链的氨基酸组成，并通过自动Edman降解建立后者的N-末端序列。
The 4th component of human complement (C4) was purified in 20% yield from fresh plasma using as starting material the 5-12% poly(ethylene glycol) precipitate which was depleted of plasminogen by an affinity adsorbent. Sequential ion-exchange chromatography on DEAE cellulose, QAE[quaternary ammonium ethyl]-Sephadex and DEAE-Bio-Gel A resulted in C4 homogeneous by immunological criteria and by polyacrylamide gel electrophoresis, the last chromatographic step achieved separation of native from inactivated C4. Reduction with 20 mM dithiothreitol for 2 h at 37.degree. C in 0.25 M 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloride, pH 8.6, effected cleavage of the interchain disulfide bonds. A 3-chain structure for C4 was confirmed, and MW estimates of 93,000 .+-. 9300, 75,000 .+-. 7500 and 30,000 .+-. 3000 determined for the .alpha.,.beta. and .gamma. chains, respectively. The effects of known inactivators of C4 upon the chains of C4 were investigated, confirming that the inactivations by Cl.hivin.s [activated S fragment of C1] and trypsin were accompanied by the fragmentation of the .alpha. chain. Inactivation of C4 by hydrazine produced no detectable change in chain size. Separation of the chains was acommplished by gel filtration in the presence of 1 M acetic acid. Amino acid compositions of native C4 and the constitutive chains were performed, and N-terminal sequences of the latter established by automated Edman degradation.