A method for the separation of hybrids of chromatographically identical oligomeric proteins. Use of 3,4,5,6-tetrahydrophthaloyl groups as a reversible "chromatographic handle".
A method for the separation of hybrids of chromatographically identical oligomeric proteins. Use of 3,4,5,6-tetrahydrophthaloyl groups as a reversible "chromatographic handle".
复制标题
一种分离色谱相同的寡聚蛋白杂合体的方法。
作者：
I. Gibbons;H. K. Schachman
与寡聚蛋白变体的杂交实验通常提供有关亚基结构、功能和相互作用的重要信息。然而，在一些系统中，变异体在电泳和层析上如此相似，以至于对单个杂交种进行纯化是不可行的。为此，提出了用3，4，5，6-四氢邻苯二甲酸酐作为蛋白质氨基的可逆酰化试剂制备杂化产物的方法。该技术包括在pH 8下将大约30%的氨基酰化得到净电荷显著改变的衍生物，与未修饰和修饰物种形成杂化集，通过离子交换层析分离单独的组分，最后通过在pH 6和室温下孵育约1天从所需杂化中去除四氢邻苯二甲酰基。用模型化合物和两种酶进行的实验表明，该酸酐是氨基专一性的。通过在含8M尿素的聚丙烯酰胺凝胶中250 nm处的光谱变化、游离氨基的损失和多肽链的电泳率的变化来衡量蛋白质的修饰程度。修饰的、失活的醛缩酶和天冬氨酸氨基转移酶的催化亚基的脱酰化导致了未修饰蛋白质的酶活性和电泳率的恢复。利用四氢邻苯二甲酰基作为可逆的“色谱柄”，制备和分离了天冬氨酸氨基转移酶的亚基内和亚基间杂交物。脱酰化前，含有一个酰化催化亚基和一个天然催化亚基(以及负调节亚基)的亚基间杂化没有表现出同质协同性，脱酰后，酶的特征变构性质得以恢复。同样，与变构转变相关的配体促进的构象变化在每个催化亚基中含有一个酰化链和两个天然链的亚基内杂交物的脱酰化时被重新定位。描述了可逆“色谱柄”在杂交实验中必须满足的条件，结果表明，尽管3，4，5，6-四氢邻苯二甲酸酐与蛋白质氨基反应有一定的不均一性，但它在低聚蛋白质的研究中仍有相当大的前景。
Hybridization experiments with variants of an oligomeric protein often provide important information regarding subunit structure, function, and interactions. In some systems, however, the variants are so similar electrophoretically and chromatographically that purification of individual hybrids is not feasible. Therefore a method was developed for preparing hybrids by using 3,4,5,6-tetrahydrophthalic anhydride as a reversible acylating agent for protein amino groups. The technique involved acylating about 30% of the amino groups at pH 8 to give a derivative with a markedly altered net charge, formation of the hybrid set with unmodified and modified species, separation of the individual components by ion-exchange chromatography, and finally removal of the tetrahydrophthaloyl groups from the desired hybrid by incubation for about 1 day at pH 6 and room temperature. Experiments with model compounds and two enzymes showed that the anhydride was sepcific for amino groups. The extent of modification of proteins was measured by the spectral change at 250 nm, the loss of free amino groups, and the change in electrophoretic mobility of the polypeptide chains in polyacrylamide gels containing 8 M urea. Deacylation of modified, inactive aldolase and the catalytic subunit of aspartate transcarbamylase led to the restoration of the enzyme activity and electrophoretic mobility of the unmodified proteins. Both intra- and inter-subunit hybrids of aspartate transcarbamylase were prepared and isolated by using the tetrahydrophthaloyl groups as a reversible "chromatographic handle". Prior to deacylation the inter-subunit hybrid containing one acylated and one native catalytic subunit (and negative regulatory sub-units) exhibited no homotropic cooperativity and after deacylation the characteristic allosteric properties of the enzyme were regained. Similarly the ligand-promoted conformational changes associated with the allosteric transition were resotred upon deacylation of the intra-subunit hybrid containing one acylated and two native chains in each catalytic subunit. Criteria are described which must be satisfied if a reversible "chromatographic handle" is to be effective in hybridization experiments and it is shown that, despite some heterogeneity in its reaction with protein amino groups, 3,4,5,6-tetrahydrophthalic anhydride shows considerable promise for studies of oligomeric proteins.