Oxidation of alkaline immobiline buffers for isoelectric focusing in immobilized pH gradients.
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 -SH groups in human alpha-globin chains, which produced an intense band with higher isoelectric point.
Righetti
P G
PG
Department of Biomedical Sciences and Technologies, University of Milano, Italy.
Chiari
M
M
Casale
E
E
Chiesa
C
C
D000178:Acrylamides / Q000138:chemical synthesis / Q000378:metabolism*; D002021:Buffers; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007525:Isoelectric Focusing; D010087:Oxides / Q000378:metabolism; D013431:Sulfates
D000178:Acrylamides; D002021:Buffers; D010087:Oxides; D013431:Sulfates; C059413:Immobiline