Nondisulfide Crosslinking and Chemical Cleavage of Tubulin Subunits: pH and Temperature Dependence

Tubulin is known to be extremely unstable. The denaturation process partially involves irreversible aggregation, mediated by disulfide crosslinking. In addition, tubulin is known to undergo chemical cleavage during boiling in sodium dodecyl sulfate (SDS), a process that generates small peptides that have been mistaken for low molecular weight MAPs. Similar peptide cleavage has now been observed during two-dimensional denaturing isoelectric focusing-SDS-polyacrylamide gel electrophoresis. This phenomenon has complicated interpretation of limited proteolysis studies of tubulin by subtilisin. In an effort to avoid this problem we have undertaken a detailed study of the solution conditions that promote chemical cleavage of tubulin. The cleavage reaction is found to be strongly pH, time, and temperature dependent. Nondenatured and denatured tubulin is susceptible to peptide cleavage, suggesting that primary structure is more important than secondary structure in selection of susceptible bonds. After transfer of cleavage products from an SDS gel to a polyvinylidene difluoride membrane, amino acid sequencing has confirmed cleavage at Asp-Pro bonds, at position 306 in α-tubulin and at position 304 in β-tubulin. We also infer cleavage at the only additional Asp-Pro peptide bond located at position 31 in β-tubulin. Heat-induced cleavage at Asp-Pro accounts for 5 of the 13 bands observed on SDS gels. In addition, a minor α-tubulin band has been sequenced from a two-dimensional gel, corresponds to cleavage at Asp-Cys located at α-tubulin position 200, and accounts for two additional bands observed on SDS gels. Under non-denaturing and nonpolymerizing conditions tubulin undergoes extensive intermolecular, disulfide crosslinking. At elevated temperatures and high pH, a small fraction of the crosslinking is not reduced by β-mercaptoethanol. Disulfide-crosslinked aggregates are not suspected because carboxymethylation of tubulin does not prevent their formation. Lysinoalanine has been found by amino acid analysis and thus covalent lysine-dehydroalanine crosslinks are suspected. Dehydroalanine is formed by β-elimination at serine and thus the presence of lysinoalanine is consistent with cleavage at Gly-Ser peptides, the most unstable serine peptide bond, and accounts for most of the remaining cleavage data.