Vanadate is known to cleave proteins in a near-uv-dependent manner. We have found that vanadate will cleave α- and β-tubulin upon photoirradiation (419 nm emission maxima) under conditions when tetravanadate, pentavanadate, and decavanadate are in solution. The reaction is independent of GTPMg or GDPMg, and cleavage occurs at two or more sites per chain. Cleavage was studied at pH 6.0 (2(N-morpholino)ethanesulfonic acid (Mes) and phosphate), pH 6.9 (piperaxine-N,N'-bis(2-ethanesulfonic acid) (Pipes)), pH 7.0 (phosphate), and pH 8.0 (N-(2-hydroxyethyl)piperazine-N'-bis(2-ethanesulfonic acid) (Hepes) and phosphate). The concentration of vanadate oligomer species, as determined by 51V NMR, was correlated with the extent of cutting. In organic buffers, low pH and high vanadate concentration favored oligomer formation, especially tetra and decavanadate. In phosphate buffer at pH 7 and 8, decamer is more prevalent, and at pH 6, phosphate buffer appears to favor a different oligomer form, V', appearing at -582 ppm. Cleavage is best correlated with the presence of cyclic tetravanadate at pH 6.9 in Pipes buffer and the V' species at pH 6.0 in phosphate buffer. Cleavage efficiency is also affected by interactions of photoactivated vanadate species with organic buffer components. In phosphate buffer no photochemical degradation of vanadate species occurs. Analysis using sodium dodecyl sulfate (SDS) gel electrophoresis and western blotting showed that vanadate produced cleavage patterns and nonenzymatic cleavage patterns resulting from boiling tubulin in SDS sample buffer (J.J. Correia, L.D. Lipscomb, and S. Lobert, 1993, Arch. Biochem. Biophys. 300, 105-114) are not the same. Attempts to identify the locations of the vanadate cleavage sites on the protein through N-terminal sequencing was unsuccessful, apparently due to the presence of blocked amino groups. We conclude that tetravanadate cleaves tubulin upon photoirradiation, that organic buffers can interact with vanadate oligomers upon photoirradiation, and that in phosphate buffer photocleavage is enhanced by an absence of photochemical degradation and a preference for forming photoactive vanadate oligomers. These results have general application to photoirradiation studies of any protein in the presence of vanadate.
ASJC Scopus subject areas
- Molecular Biology