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Isoelectric focusing of basic proteins: The problem of oxidation of cysteines
Author(s) -
Altland Klaus,
Becher Pia,
Rossmann Ute,
Bjellqvist Bengt
Publication year - 1988
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.1150090906
Subject(s) - dithiothreitol , chemistry , iodoacetamide , iodoacetic acid , isoelectric focusing , polyacrylamide gel electrophoresis , isoelectric point , polyacrylamide , reducing agent , cysteine , gel electrophoresis , chromatography , inorganic chemistry , organic chemistry , biochemistry , polymer chemistry , enzyme
Isoelectric focusing of human globin chains in polyacrylamide gels dried in the ambient atmosphere and rehydrated in the presence of 8 mol/L urea produces artefactual doublets of zones as a result of oxidation by the gel. This oxidation can be avoided in separations of short duration by adding a reducing agent ( e. g. 2‐mercaptoethanol or dithiothreitol to the rehydration solution (Altland, K. and Rossmann, U., Electrophoresis 1985, 6 , 314–325)). We now demonstrate that the observed zone doublets can be explained by assuming neutralization of the contribution of dissociated sulfhydryl group of cysteine to p I by partial and reversible formation of globin dimers held together by disulfide bridges. Long time separations, requiring e. g. more than 4 h at > 500 V/cm, in pH gradients exceeding pH 7.5, are accompanied by artefactual oxidation from both the atmosphere and the gel matrix. Oxidation from the atmosphere as well as the effect of carbon dioxide can be eliminated by overlayering the gel with paraffin oil. Oxidation from the gel matrix can only partially be inhibited by rehydration of gels in the presence of 2‐mercaptoethanol or dithiothreitol. Nearly complete protection against oxidation by the gel matrix was achieved by adding a permanent supply of 2‐ME to the gel or by adding DTT to the cathodic wick towards the end of the experiment. Alkylation with iodoacetamide or iodoacetic acid resulted in stable globin patterns, which, however, displayed additional artefactual zones. Our experimental data indicate that the polyacrylamide gels function as an electron acceptor for dissociated sulfhydryl groups in proteins, even after pretreatment with strong reducing agents for proteins.