Affinity purification and sequence determination of equine relaxin.
Affinity purification and sequence determination of equine relaxin.
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DOI：
10.1210/endo-129-1-375
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发表时间：
1991-07
期刊：
影响因子：
4.8
通讯作者：
D. Stewart;B. Nevins;É. Hadas;R. Vandlen
中科院分区：
文献类型：
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作者：
D. Stewart;B. Nevins;É. Hadas;R. Vandlen
松弛素是一种通常与妊娠相关的多肽激素，已从许多物种中纯化出来，并且越来越多的物种已确定其序列。马松弛素先前已通过丙酮提取、凝胶过滤和离子交换色谱法纯化。为了开发一种更快速、更有效的松弛素纯化方法，探索了亲和层析与HPLC联用的方法。单克隆抗体提出了对高度纯化的马松弛素，大量的抗体获得腹水生产和附着到固相支持。将足月马胎盘的提取物通过亲和柱并洗涤，通过改变pH洗脱松弛素。使用C18（ODS）反相柱和25 - 30%乙腈的线性梯度通过HPLC分离马松弛素的异构体。获得了马松弛素的四种主要和几种次要异构体。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），同种型具有相似的分子量，由亚基组成（还原条件下的SDS-PAGE），并具有相似的电荷（非变性PAGE）。在小鼠耻骨间韧带生物测定中，5种亚型的生物活性检测结果为阳性。采用Edman降解法测定了马松弛素的全序列，并通过快原子轰击质谱法进行了验证。马松弛素的异构体被发现是由于B链的C-末端的异质性。马松弛素似乎是最小的松弛素测序。A链由20个残基组成，B链有28个残基，总摩尔质量为5253。马松弛素与猪松弛素具有最大的序列同源性（67%同一性）。
Relaxin, a polypeptide hormone normally associated with pregnancy, has been purified from many species, and the sequence determined for a growing number. Equine relaxin has been previously purified by acetone extraction, gel filtration, and ion exchange chromatographies. In an attempt to develop a more rapid and efficient method for relaxin purification, the use of affinity chromatography coupled with HPLC was explored. Monoclonal antibodies were raised against highly purified equine relaxin; large quantities of antibody were obtained by ascites production and attached to a solid phase support. An extract of term equine placentas was passed through the affinity column and washed, and relaxin was eluted by a change in pH. The isoforms of equine relaxin were separated by HPLC using a C18 (ODS) reverse phase column and a linear gradient of 25-30% acetonitrile. Four major and several minor isoforms of equine relaxin were obtained. The isoforms share similar mol wt by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), are composed of subunits (SDS-PAGE under reducing conditions), and have similar charges (native PAGE). Five isoforms tested positive for biological activity in the mouse interpubic ligament bioassay. Equine relaxin was sequenced by Edman degradation, and the sequence was confirmed by fast atom bombardment mass spectrometry. The isoforms of equine relaxin were found to be due to heterogeneity of the C-terminus of the B-chain. Equine relaxin appears to be the smallest relaxin sequenced. The A-chain consists of 20 residues, and the B-chain has 28 residues, with a total mol wt of 5253. Equine relaxin shares the greatest sequence homology with porcine relaxin (67% identity).