Abstract: Lysozyme was coupled on Sepharose 4FF media activated with 1,4-butanediol diglycidyl ether. HPLC-MS and enzymatic digestion were used to identify the coupling sites of lysozyme on the media. The effects of epoxy group density and the coupling reaction condition on the coupling sites were investigated. It was found that K96 was the main coupling site on the media when the epoxy group density was 11.34 mmol/g and pH 9.5. The coupling reaction time did not affect the coupling site. Epoxy group density and pH value during the coupling reaction are the main factors affecting the coupling sites of lysozyme on the media. More lysozyme molecules were coupled on the media via K33 and K97 when pH value was above 10.5.

Key words: affinity chromatographic media, protein ligand, coupling site, MS analysis, enzymatic digestion

摘要： 以溶菌酶为模型蛋白，将其偶联在环氧活化的琼脂糖介质上，采用液质联用技术结合蛋白质酶切技术对琼脂糖介质表面配基蛋白的偶联位点进行了识别，考察了活化时间、偶联时间和溶液pH值对偶联位点的影响. 结果表明，环氧基密度为11.34 mmol/g、反应pH 9.5条件下，溶菌酶偶联位点发生在K96，延长偶联反应时间蛋白配基偶联量增加，对偶联位点种类无显著影响；增加环氧基密度或提高偶联反应pH值使溶菌酶通过多种位点偶联，在pH≥10.5条件下偶联位点主要发生在K33, K96和K97.

关键词: 亲和层析介质, 蛋白质配基, 偶联位点, 质谱分析, 蛋白质酶解