Evidence for the spontaneous formation of disulfide crosslinked aggregates of tubulin during nondenaturing electrophoresis

Abstract

Phosphocellulose-purified tubulin has been shown to form a characteristic “ladder” of nonmicrotubular aggregates during nondenaturing gel electrophoresis ( J. J. Correia and R. C. Williams, Jr. (1985) Arch. Biochem. Biophys. 239, 120–129 ). In this paper we describe evidence that the intersubunit bonds responsible for formation of these oligomeric particles are disulfides. Two-dimensional nondenaturing-denaturing gel electrophoresis demonstrates that each aggregate zone is composed of α- and β-subunits of tubulin. Omission of β-mercaptoethanol during the sodium dodecyl sulfate (SDS)-electrophoresis step causes a pattern of aggregates to appear and implicates disulfide linkages in their stabilization. Molecular weights, estimated from mobilities in the second (SDS) dimension of two-dimensional gels, suggest that the aggregates are crosslinked in units of monomers, not heterodimers. Consistent with this conclusion, α- or β-subunits alone (isolated by isoelectric focusing) will form the same ladder of aggregates. The disulfide crosslinking of tubulin is also achievable in solution. It is favored by high concentrations of alcohol, the presence of oxidizing agents, high pH, and high temperature, conditions that denature tubulin and cause rapid noncovalent aggregation or precipitation. When aggregate formation was monitored as a function of time by SDS-gel electrophoresis in the absence of β-mercaptoethanol and by quantitative sulfhydryl and disulfide titrations, the most effective conditions for the crosslinking reaction included >75% alcohol, excess H 2O 2, or excess iodine. These results suggest that proximity of a hydrophobic gel matrix, high pH, the presence of oxidizing agents, high protein concentration, tubulin's propensity to aggregate nonspecifically, and the availability of as many as 20 sulfhydryls in αβ-tubulin contribute, during nondenaturing gel electrophoresis, to the spontaneous formation of disulfide-crosslinked tubulin aggregates.