Covalent cross-linking of erythrocyte spectrin by carbon disulfide in vivo.
Covalent cross-linking of proteins by CS2 has been demonstrated in vitro and represents a potential mechanism for the toxicity of this compound. In the present investigation the ability of CS2 to cross-link proteins covalently in vivo is demonstrated using denaturing polyacrylamide gel electrophoresis. Intraperitoneal injection of CS2 in rats at 2 or 5 mmol/kg for 21 or 42 days produced several high-molecular-weight (approximately 410 kDa) proteins eluted from erythrocyte membranes which were not present in control animals. Limited proteolysis of the high-molecular-weight protein bands, monomeric alpha spectrin, and monomeric beta spectrin using endoproteinase glu-C, followed by peptide mapping on denaturing polyacrylamide gels, showed the high-molecular-weight proteins to be alpha,beta 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 CS2-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 CS2 and may provide a means to correlate pathological changes with existing methods of CS2 exposure monitoring. The ability of CS2 to covalently cross-link erythrocyte spectrin suggests that CS2 may also cross-link other proteins in vivo and supports covalent cross-linking of proteins as a possible molecular mechanism through which CS2 manifests toxicity. If so, then spectrin cross-linking may parallel cross-linking reactions in the axon and provide a sensitive, preneurotoxic biomarker of this molecular event.
Valentine
W M
WM
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710.
Graham
D G
DG
Anthony
D C
DC
D000818:Animals; D001345:Autoradiography; D002246:Carbon Disulfide / Q000633:toxicity*; D003432:Cross-Linking Reagents; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000187:drug effects*; D006868:Hydrolysis; D007274:Injections, Intraperitoneal; D008297:Male; D008970:Molecular Weight; D010449:Peptide Mapping; D011108:Polymers; D051381:Rats; D017207:Rats, Sprague-Dawley; D013049:Spectrin / Q000737:chemistry / Q000187:drug effects*; D013859:Thiocarbamates / Q000737:chemistry; D013890:Thiourea / Q000737:chemistry
D003432:Cross-Linking Reagents; D011108:Polymers; D013859:Thiocarbamates; D013049:Spectrin; D013890:Thiourea; D002246:Carbon Disulfide