Reevaluation of the electrophoretic migration behavior of soluble globularproteins in the native and detergent-denatured states in polyacrylamide gels

Although sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis is widely used for estimating molecular masses of proteins, considerable unce rtainty still exists both about the structure of SDS-protein complexes and about their mechanism of electrophoretic migration, In this study, soluble globular proteins, with masses of 14-200 kDa, were heat-denatured in the pr esence of SDS and their relative total molecular volume and net charge were estimated from Ferguson plots of electrophoretic mobility vs acrylamide co ncentration. Native globular protein served as standards for overall molecu lar size and effective radii. Results revealed at least two independent ele ctrophoretic migration mechanisms for the SDS-protein complexes: (i) for pr oteins in the 14-65 kDa range at <15% acrylamide, linear Ferguson plots sug gested that they migrated ideally and that their effective radii could be e stimated in this manner: (ii) concave plots at higher gel concentrations, a nd for complexes derived from larger proteins, indicated that migration in these cases could be described by reptation theory. Migration of the large proteins at lower gel concentrations and small proteins at higher gel conce ntrations was not well described by either theory, representing intermediat e behavior not described by these mechanisms. These data support models in which all but the smallest SDS-protein complexes adopt a necklace-like stru cture in which spherical micelles are distributed along the unfolded polype ptide chain, Possible relations to recent alternative models of gel electro phoresis are also discussed. (C) 2000 Academic Press.