TY - JOUR
T1 - Reevaluation of the electrophoretic migration behavior of soluble globular proteins in the native and detergent-denatured states in polyacrylamide gels
AU - Westerhuis, Willem H.J.
AU - Sturgis, James N.
AU - Niederman, Robert A.
N1 - Funding Information: 1This work was supported by U.S. Department of Agriculture Grant 91-01640 and National Science Foundation Grant MCB90-19570 to R. Niederman. W. Westerhuis was the recipient of fellowships from the Charles and Johanna Busch Memorial Fund Award and the European Community Human Capital and Mobility Programme.
PY - 2000/8/15
Y1 - 2000/8/15
N2 - Although sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis is widely used for estimating molecular masses of proteins, considerable uncertainty 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 presence of SDS and their relative total molecular volume and net charge were estimated from Ferguson plots of electrophoretic mobility vs acrylamide concentration. Native globular protein served as standards for overall molecular size and effective radii. Results revealed at least two independent electrophoretic migration mechanisms for the SDS-protein complexes: (i) for proteins in the 14-65 kDa range at <15% acrylamide, linear Ferguson plots suggested that they migrated ideally and that their effective radii could be estimated in this manner: (ii) concave plots at higher gel concentrations, and 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 concentrations was not well described by either theory, representing intermediate behavior not described by these mechanisms. These data support models in which all but the smallest SDS-protein complexes adopt a necklace-like structure in which spherical micelles are distributed along the unfolded polypeptide chain. Possible relations to recent alternative models of gel electrophoresis are also discussed. (C) 2000 Academic Press.
AB - Although sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis is widely used for estimating molecular masses of proteins, considerable uncertainty 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 presence of SDS and their relative total molecular volume and net charge were estimated from Ferguson plots of electrophoretic mobility vs acrylamide concentration. Native globular protein served as standards for overall molecular size and effective radii. Results revealed at least two independent electrophoretic migration mechanisms for the SDS-protein complexes: (i) for proteins in the 14-65 kDa range at <15% acrylamide, linear Ferguson plots suggested that they migrated ideally and that their effective radii could be estimated in this manner: (ii) concave plots at higher gel concentrations, and 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 concentrations was not well described by either theory, representing intermediate behavior not described by these mechanisms. These data support models in which all but the smallest SDS-protein complexes adopt a necklace-like structure in which spherical micelles are distributed along the unfolded polypeptide chain. Possible relations to recent alternative models of gel electrophoresis are also discussed. (C) 2000 Academic Press.
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U2 - https://doi.org/10.1006/abio.2000.4684
DO - https://doi.org/10.1006/abio.2000.4684
M3 - Article
C2 - 10933867
SN - 0003-2697
VL - 284
SP - 143
EP - 152
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -