Chiari M, Righetti P G, Negri A, Ceciliani F, Ronchi S
Department of Biomedical Sciences and Technologies, University of Milano, Italy.
Electrophoresis. 1992 Nov;13(11):882-4. doi: 10.1002/elps.11501301193.
It has been found that the double bond of free, unreacted acrylamide in a gel can react with a free -SH group of proteins, forming a cysteinyl-S-propionamide adduct. When beta-lactoglobulin was incubated at concentration levels lower than those of free acrylamide, left after polymerizing a 5% T, 4% C gel (barely 12 mM), under anaerobic conditions in 0.1 M borate, pH 9.5, for 1 h and then the tryptic digests analyzed by high performance liquid chromatography (HPLC), two new peptides were detected. The two new peaks were recovered and sequenced by the Edman degradation procedure. They correspond to the sequence from Leu-149 to Ile-162. Residue No. 160 was found to be a cysteinyl-S-propionamide reaction product. Interestingly, only this residue, out of a total of 5 Cys residues, had reacted. No other amino acids (including -NH2 terminus and free -NH2 in Lys) reacted with free acrylamide. The addition of free acrylamide to the -SH group could be completely inhibited if: (i) the gel was extensively washed prior to sample application, or (ii) the gel was incubated for 1 h in 100 mM free Cys.
已发现凝胶中游离的、未反应的丙烯酰胺双键可与蛋白质的游离巯基反应，形成半胱氨酰 - S - 丙酰胺加合物。当β - 乳球蛋白在低于聚合5% T、4% C凝胶（仅12 mM）后残留的游离丙烯酰胺浓度水平下孵育时，在0.1 M硼酸盐（pH 9.5）的厌氧条件下孵育1小时，然后通过高效液相色谱（HPLC）分析胰蛋白酶消化产物，检测到了两种新的肽段。回收这两个新峰并通过埃德曼降解程序进行测序。它们对应于从Leu - 149到Ile - 162的序列。发现第160位残基是半胱氨酰 - S - 丙酰胺反应产物。有趣的是，在总共5个半胱氨酸残基中，只有这个残基发生了反应。没有其他氨基酸（包括 - NH2末端和赖氨酸中的游离 - NH2）与游离丙烯酰胺反应。如果：（i）在加样前对凝胶进行充分洗涤，或（ii）将凝胶在100 mM游离半胱氨酸中孵育1小时，则游离丙烯酰胺与巯基的加成可被完全抑制。
