Marinkovic D V, Marinkovic J N, Tang J
Prep Biochem. 1975;5(3):257-80. doi: 10.1080/00327487508061576.
Thermal fragmentation of the beta-galactosidase was studied in different buffer solutions and at different temperatures. Fragmentation of the subunits in small size polypeptides could be observed directly. The fragmentation proceeded in buffer solution, pH 7.0, at either 75 degrees C or 100 degrees C in the presence of sodium dodecyl sulfate. The elevated temperature appeared to accelerate this process. At 100 degrees C, pH 7.2, the fragmentation proceeded in the absence of sodium dodecyl sulfate, but in the presence of 8 M urea. Molecular weights, determined by sodium dodecyl sulfate disc gel electrophoresis were from 130,000 to about 20,000. Multiple bands were observed. After dissociation was complete at 37 degrees C, the fragments were purified by ion-exchange column chromatography. Of the five fragments thus obtained, four were homogeneous by disc-gel electrophoresis. Molecular weight of the homogeneous fragments were found to be near 25,000. The fifth comprised a mixture of four fragments having molecular weights from 29,000 to 72,000. Two of the fragments were active as the alpha-donor in in vitro complementation with mutant M15, which contains a deletion in the alpha-region of the z gene.
在不同缓冲溶液和不同温度下研究了β-半乳糖苷酶的热片段化。可以直接观察到亚基在小尺寸多肽中的片段化。在十二烷基硫酸钠存在下，片段化在pH 7.0的缓冲溶液中于75℃或100℃进行。升高的温度似乎加速了这个过程。在100℃、pH 7.2时，片段化在没有十二烷基硫酸钠但有8M尿素存在的情况下进行。通过十二烷基硫酸钠圆盘凝胶电泳测定的分子量为130,000至约20,000。观察到多条带。在37℃解离完成后，通过离子交换柱色谱法纯化片段。在由此获得的五个片段中，四个通过圆盘凝胶电泳是均一的。发现均一片段的分子量接近25,000。第五个片段包含分子量为29,000至72,000的四个片段的混合物。其中两个片段在与z基因α区域缺失的突变体M15的体外互补中作为α供体具有活性。
