Preincubation with cysteine prevents modification of sulfhydryl groups in proteins by unreacted acrylamide in a gel.
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.
Chiari
M
M
Department of Biomedical Sciences and Technologies, University of Milano, Italy.
Righetti
P G
PG
Negri
A
A
Ceciliani
F
F
Ronchi
S
S
D020106:Acrylamide; D000178:Acrylamides / Q000737:chemistry*; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D005782:Gels; D007782:Lactoglobulins / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013997:Time Factors
D000178:Acrylamides; D005782:Gels; D007782:Lactoglobulins; D013438:Sulfhydryl Compounds; D020106:Acrylamide; D003545:Cysteine