Ishikawa K, Iwai K
J Biochem. 1975 Feb;77(2):391-8. doi: 10.1093/oxfordjournals.jbchem.a130737.
Affinity chromatography based on the reaction between SH groups in protein and +HgC6H4CO groups in the p-mercuribenzoylaminoethyl derivative of Sepharose 4B was examined with a crude preparation of calf thymus cysteine-containing histone. Adsorption of the histone onto the column by specific coupling was found to be optimal in 0.1 M citrate buffer, pH 5.5, containing 5M urea to prevent any aggregation of histones and their non-specific adsorption onto the column, and elution from the column was successfully performed by cleavage of the resulting S-Hg bond with urea-buffer solution containing 0.05 M 2-mercaptoethanol. Under these conditions both the adsorption and elution were quantitative; no adsorption was observed when either SH-blocked histone or unsubstituted Sepharose was used. The cysteine-containing histone thus recovered, after further purification by Bio-Gel P-60 chromatography to remove some cysteine-containing nonhistone proteins contaminating the starting material, showed a single band on polyacrylamide gel electrophoresis and an amino acid composition agreeing with the known sequence of this histone.
基于蛋白质中的SH基团与琼脂糖4B的对-汞苯甲酰氨基乙基衍生物中的+HgC6H4CO基团之间的反应，用含有半胱氨酸的小牛胸腺组蛋白粗制品对亲和色谱法进行了研究。发现在含有5M尿素的0.1M柠檬酸盐缓冲液（pH 5.5）中，通过特异性偶联将组蛋白吸附到柱上是最佳的，以防止组蛋白聚集及其在柱上的非特异性吸附，并且通过用含有0.05M 2-巯基乙醇的尿素缓冲溶液裂解所得的S-Hg键成功地从柱上进行洗脱。在这些条件下，吸附和洗脱都是定量的；当使用SH封闭的组蛋白或未取代的琼脂糖时，未观察到吸附。通过Bio-Gel P-60色谱进一步纯化以去除一些污染起始材料的含半胱氨酸的非组蛋白后，回收的含半胱氨酸的组蛋白在聚丙烯酰胺凝胶电泳上显示出一条带，并且氨基酸组成与该组蛋白的已知序列一致。
