POLYACRYLAMIDE-GEL POLYMERIZATION UNDER NONOXIDIZING CONDITIONS, AS MONITORED BY CAPILLARY ZONE ELECTROPHORESIS

It has been established that, during peroxodisulphate-catalysed polyacrylamide gel polymerization, there is a concomitant oxidation of all amino buffers present in the mixture, with the formation of N-oxides. This is deleterious in conventional isoelectric focusing and immobilized pH gradients because, even after discharging excess of peroxodisulphate at the anode, a residual oxidizing power remains in the matrix. When focusing proteins under mildly alkaline conditions, a redox reaction occurs during migration, and free SH groups are oxidized to -S-S- bridges, giving rise to spurious bands. The kinetics of riboflavin-catalysed gel polymerization have been reinvestigated and conditions were elucidated that allow > 95% conversion of monomers into the growing chains, full control of the lag phase and short polymerization times, as typical of peroxodisulphate catalysis. It was demonstrated, by capillary zone electrophoresis and direct NMR analysis, that there is an additional unique advantage in photopolymerization, namely that even in a large excess of catalyst, no traces of oxidizing power can be found. The gel matrix thus generated is unable to induce any oxidation of proteins during gel electrophoresis.