Separation of the major proteins of central and peripheral nervous system myelin using reversed-phase high-performance liquid chromatography

Abstract

A general method to separate the major proteins of rat central and peripheral nervous system myelin has been developed. The key step is the initial quantitative removal of the lipids under conditions where the proteins retain their solubility in HPLC solvents. Lipids are removed by a combination of solvent extraction and column chromatography on Sephadex LH-60 in 2-chloroethanol:10 m m HCl (9:1). Proteins are then separated by reversed-phase (RP) HPLC. Samples are applied to a wide pore reversed-phase C-3 column and eluted with a linear gradient of 10–70% 1-propanol in 0.1% trifluoroacetic acid (0–100% B) over a 60-min period. Myelin basic proteins elute between 25 and 30% B, Wolfgram and other high molecular weight proteins at 35–50% B, proteolipid protein at 65–80% B, and P0 glycoprotein at 55–65% B. This elution pattern is consistent with the known relative hydrophobicity of these proteins. Protein recovery for the entire procedure is greater than 74%. Proteolipid and P0 proteins isolated by HPLC contain 2.3 and 1.1 mol of covalently bound fatty acids, respectively. This fatty acid composition is similar to that previously reported using different isolation procedures. The analysis of central and peripheral nervous system myelin proteins by RP-HPLC permits the isolation of purified proteins for structural and metabolic experiments.