Analyses of intramolecular disulfide bonds in proteins by polyacrylamide gel electrophoresis following two-step alkylation.
Analyses of intramolecular disulfide bonds in proteins by polyacrylamide gel electrophoresis following two-step alkylation.
复制标题
两步烷基化后通过聚丙烯酰胺凝胶电泳分析蛋白质中的分子内二硫键。
DOI：
10.1016/0003-2697(88)90028-0
复制
发表时间：
1988
影响因子：
2.9
通讯作者：
E. Doi
中科院分区：
文献类型：
--
作者：
M. Hirose;N. Takahashi;H. Oe;E. Doi
建立了一种利用聚丙烯酰胺凝胶电泳分析蛋白质分子内二硫键的方法。第一步，用碘乙酸或碘乙酰胺将具有不同数目的断裂二硫键的蛋白质烷基化。剩余的二硫键被过量的二硫苏糖醇还原，新产生的游离巯基被尚未使用的试剂（碘乙酰胺、碘乙酸或乙烯基吡啶）烷基化作为第二步。这种处理使得溶菌酶（Mr，14，000; 4个二硫化物），伴清蛋白的N-末端半分子（Mr，36，000; 6个二硫化物），伴清蛋白的C-末端半分子（Mr，40，000; 9个二硫化物）和全伴清蛋白（Mr，78，000; 15个二硫键）通过酸-尿素聚丙烯酰胺凝胶电泳分离成不同的条带，这取决于裂解的二硫键的数量。该方法使我们能够确定天然蛋白质中二硫键的总数，并评估部分还原蛋白质中二硫键的裂解水平。结合放射自显影技术的两步烷基化法特别适用于分析复杂生物系统中合成的蛋白质中的二硫键。
A method that makes use of polyacrylamide gel electrophoresis was developed for the analysis of intramolecular disulfide bonds in proteins. Proteins with different numbers of cleaved disulfide bonds are alkylated with iodoacetic acid or iodoacetamide as the first step. The disulfide bonds remaining were reduced by excess dithiothreitol, and the newly generated free sulfhydryl groups were alkylated with the reagent not yet used (iodoacetamide, iodoacetic acid, or vinylpyridine) as the second step. This treatment made it possible for lysozyme (Mr, 14,000; 4 disulfides), the N-terminal half-molecule of conalbumin (Mr, 36,000; 6 disulfides), the C-terminal half-molecule of conalbumin (Mr, 40,000; 9 disulfides), and whole conalbumin (Mr, 78,000; 15 disulfides) to be separated by acid-urea polyacrylamide gel electrophoresis into distinct bands depending on the number of disulfide bonds cleaved. The method allowed us to determine the total number of disulfide bonds in native proteins and to assess the cleaved levels of disulfide bonds in partially reduced proteins. Two-step alkylation used in combination with radioautography was especially useful for the analysis of disulfide bonds in proteins synthesized in complex biological systems.