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Preincubation with cysteine prevents modification of sulfhydryl groups in proteins by unreacted acrylamide in a gel
Author(s) -
Chiari Marcella,
Righetti Pier Giorgio,
Negri Armando,
Ceciliani Fabrizio,
Ronchi Severino
Publication year - 1992
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.11501301193
Subject(s) - cysteine , acrylamide , chemistry , polyacrylamide gel electrophoresis , chromatography , biochemistry , combinatorial chemistry , organic chemistry , polymer , enzyme , copolymer
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 β‐lactoglobulin was incubated at concentration levels lower than those of free acrylamide, left after polymerizing a 5% T, 4% C gel (barely 12 m M ), 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 lle‐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 ‐NH 2 terminus and free ‐NH 2 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 m M free Cys.