MANNITOL PREVENTS METHIONINE SULFOXIDATION MEDIATED ELECTROPHORETIC HETEROGENEITY OF APOLIPOPROTEIN-A-I

Hybrid isoelectic focusing of apolipoprotein A-I in polyacrylamide gel s with immobilized pH-gradients under non-denaturing conditions result ed in the occurrence of additional bands which could prevent the speci fic and sensitive detection of genetic variants. Hybrid isoelectric fo cusing of two chromatographically distinguishable apolipoprotein A-I i soforms that differ by sulphoxidaton of methionine residues, apo A-I(M et) and apo A-I(MetSO), revealed that the additional bands were caused by this post-translational modification. Several antioxidative additi ves and conditions were compared for their ability to prevent methioni ne sulphoxidation in apolipoprotein A-I. In the presence of 200 g/L ma nnitol in the gel, apolipoprotein A/I focused as a single band. Since methionine sulphoxidation in proteins is a general phenomenon either t aking place in vivo or in vitro by isoelectric focusing, we conclude t hat isoelectric focusing in the presence of mannitol will improve the quality of resolution of many proteins in gels with immobilized pH-gra dients.