KINETICS OF CYSTEINE OXIDATION IN IMMOBILIZED PH GRADIENT GELS

It was recently found that, during the gel polymerization step, persulphate could oxidize the alkaline Immobiline species (acryloyl weak bases used for producting immobilized pH gradients, IPG) by forming N-oxides. When focusing proteins in alkaline pH ranges, free SH groups were oxidized to SS bonds, generating artefactual, higher pI bands. The kinetics of this phenomenon were investigated in a model system consisting in incubating free Cys with a crushed IPG gel auto-buffered at pH 9.0 under aerobic or anaerobic conditions. After appropriate incubation periods, any excess free SH was blocked with acrylonitrile. After dansylation, Cys and its oxidized derivatives were separated and quantified by high-performance liquid chromatography in a C18 column. Under anaerobic conditions, control Cys (incubated in the absence of IPG gel) showed 30% oxidation after a period of 12 h, whereas Cys incubated with a pH 9.0 crushed IPG gel was 100% oxidized to cystine. When the same experiment was repeated under aerobic conditions, the oxidation process was accelerated by a factor of 3-4. Successful focusing of alkaline proteins can still be achieved if (a) the IPG gel is washed in 100 mM ascorbic acid, prior to the run, for elimination of N-oxides from the matrix, (b) the IPG gel is run anaerobically and (c) the focusing time is kept to a minimum (e.g., 4 h vs. the standard overnight run). © 1990.