Reversed-phase high-performance liquid chromatographic purification of subunits of oligomeric membrane proteins. The nuclear coded subunits of yeast cytochrome c oxidase.
Reversed-phase chromatography of the subunits of an oligomeric membrane protein such as yeast cytochrome c oxidase requires additional sample handling techniques which are not necessary for soluble proteins. This paper considers these and discusses (1) methods for the removal of ballast material by preliminary batchwise extraction with solvent mixtures similar to those used for reversed-phase elution; (2) the chromatographic heterogeneity induced by partial cysteine oxidation; (3) the removal of tightly bound proteins from the stationary phase; and (4) the generation of an elution system with continuously variable selectivity based on acetonitrile-1-propanol ratios (0.05% triethylamine, 0.05% trifluoroacetic acid). These methods are designed to simplify complex mixtures of hydrophobic proteins prior to chromatography and to purify them chromatographically in high yield.
D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D003576:Electron Transport Complex IV / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000302:isolation & purification*; D010455:Peptides / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D000596:Amino Acids; D008565:Membrane Proteins; D010455:Peptides; D003576:Electron Transport Complex IV