Covalent Cross-Linking of Erythrocyte Spectrin by Carbon Disulfide in Vivo

Covalent cross-linking of proteins by CS 2 has been demonstrated in vitro and represents a potential mechanism for the toxicity of this compound. In the present investigation the ability of CS 2 to cross-link proteins covalently in vivo is demonstrated using denaturing polyacrylamide gel electrophoresis. Intraperitoneal injection of CS 2 in rats at 2 or 5 mmol/kg for 21 or 42 days produced several high-molecular-weight (∼410 kDa) proteins eluted from erythrocyte membranes which were not present in control animals. Limited proteolysis of the high-molecular-weight protein bands, monomeric α spectrin, and monomeric β spectrin using endoproteinase glu-C, followed by peptide mapping on denaturing polyacrylamide gels, showed the high-molecular-weight proteins to be αβ heterodimers. The production of multiple heterodimers exhibiting different distances of migration was consistent with the existence of several preferred sites for cross-linking. Evidence for the presence of dithiocarbamate ester and thiourea cross-linking structures in spectrin dimers was obtained using selective base hydrolysis. No spectrin dimer was detected in control animals, and dimer formation demonstrated a cumulative dose response in CS 2-treated rats. The longevity of red blood cells, the cumulative dose response, and the stability of the cross-linking structures endows spectrin cross-linking with the potential to serve as a biomarker of chronic low-level exposures to CS 2 and may provide a means to correlate pathological changes with existing methods of CS 2 exposure monitoring. The ability of CS 2 to covalently cross-link erythrocyte spectrin suggests that CS 2 may also cross-link other proteins in vivo and supports covalent cross-linking of proteins as a possible molecular mechanism through which CS 2 manifests toxicity. If so, then spectrin cross-linking may parallel cross-linking reactions in the axon and provide a sensitive, pre-neurotoxic biomarker of this molecular event.