Further Characterization of the Anti-Encephalitogenic Protein (SCP): Isolation from Bovine Spinal Cord and Spinal Roots

Abstract

The three molecular forms of the anti-encephalitogenic protein, beta-SCP, gamma-SCP, and SCP-peptide were isolated in higher yield by a shortened procedure, which involved 1) extraction of bovine spinal cord (BSC) or bovine spinal roots (BSR) with 0.05 M sodium acetate buffer, pH 4.5, 2) batch absorption on CM-52 cellulose, 3) stepwise elution with sodium acetate buffers, pH 5.8, containing increasing concentrations of sodium chloride and finally, 4) removal of trace contaminants by gel-exclusion chromatography on Sephadex G-50 superfine. The m.w. of the purified proteins determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was 13,200 daltons. The same value for the molecular sizes was obtained by gel-exclusion chromatography by using 0.1% SDS in 0.05M sodium chloride as eluant. In the absence of SDS the molecular sizes estimated by gel exclusion chromatography ranged from 14,000 to 18,500. The amino acid compositions of the beta-SCP and gamma-SCP from BSC and BSR were similar except that beta-SCP from BSR lacked half-cystine whereas gamma-SCP from BSR contained three times as much half-cystine as the SCP forms prepared from BSC. All forms of SCP showed reactions of identity when compared by immunodiffusion analyses with a rabbit anti-bovine SCP serum; none formed precipitin lines with a rabbit anti-bovine myelin basic protein (MyBP) serum.