PMID: 2488590Jan 1, 1989Paper

Oxidation of alkaline immobiline buffers for isoelectric focusing in immobilized pH gradients

Applied and Theoretical Electrophoresis : the Official Journal of the International Electrophoresis Society
P G RighettiC Chiesa

Abstract

Two degradation pathways had been previously reported for the four alkaline Immobilines: (a) hydrolysis of the amido bond (Pietta et al., 1985) and (b) auto-polymerization (Righetti et al., 1987a). Both these degradation pathways are fully abolished by dissolving the alkaline buffers in n-propanol (Immobiline II; Gåveby et al., 1988). We report here a third, previously undetected degradation pathway: pH-dependent oxidation of the four alkaline Immobilines by persulphate during the polymerization process, with formation of N-oxides. These R3N+O species are in turn strongly oxidizing and attack Cys residues in proteins by transforming them into -S-S-bridges. In extreme cases, -S-S- bridges are transformed into cysteic acid. For alkaline proteins, this often results in spurious, additional bands existing in three possible equilibria: -SH, -S-S- and -SO3-. A remedy has been found for this phenomenon: polymerization is still carried out around pH 7 as recommended (Righetti, 1984) (at this pH the pK 8.5 and 9.3 Immobilines remain unaltered) and the two oxidized morpholino derivatives (pK 6.2 and 7.0 species) reduced by a short washing step in 100 mM ascorbic acid at pH 4.5. This procedure is seen to completely prevent oxidation of -S...Continue Reading

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