Studies on the Redox State in Polyacrylamide Gels

Abstract

The oxidation-reduction properties of polyacrylamide gels were examined to test the possibility that persulfate-or other free radical donors-alter proteins during polyacrylamide gel electrophoresis. Polyacrylamide gels formed either by use of persulfate or riboflavin and light as catalysts were shown to oxidize sulfhydryl compounds. Electrophoresis of thioglycolate into the gel in amounts equivalent to persulfate resulted in reducing conditions (titratable SH groups) in the gel. Acrylamide monomer and, therefore, presumably unreacted monomer in polyacrylamide react with the α-amino group of amino acids: the reaction with glycine proceeded slowly at pH 9 and at acrylamide concentrations comparable to that of amino acid. Acrylamide at high concentrations (0.5 M) also reacted with yeast enolase and, like the thiol groups of reduring agents, prevented the appearance of characteristic changes in the pattern of enolase in polyacrylamide gel electrophoresis, previously attributed to “persulfate damage” and now designated the “enolase band shift”. The enolase band shift was demonstrated in gels photopolymerized by riboflavin in the absence of persulfate. Pattern changes resembling those of the enolase band shift were observed after direct incubation of enolase with persulfate at the high level used for polymerization (4 mM), although pattern changes indicative of drastic alteration of the protein result from such incubation. The enolase band shift was obtained whether or not gels were purified by pre-electrophoresis which should remove all persulfate. The pattern ascribed previously to persulfate damage only occurred in gels containing urea and was abolished by reaction of the protein with thioglycolate, hydroquinone, dithiothreitol, or acrylamide prior to the migration of the protein into the gel. Under the conditions of the riboflavin-catalyzed photopolymerization of acrylamide the cyanmethemoglobin molecule was altered; illumination by itself had no such effect.