The influence of site-specificity of single amino acid substitutions on electrophoretic separation of yeast iso-1-cytochrome c.

This study dealt with the ability of non-denaturing gel electrophoresis to separate iso-1-cytochrome c with single amino acid replacements isolated from revertants of various cyc1 nonsense mutants of the yeast Saccharomyces cerevisiae. A total of 28 different iso-1-cytochromes c with single amino acid substitutions of one of seven amino acids at six positions were examined on nondenaturing polyacrylamide gels at pH 4.8. Each of these iso-1-cytochromes c exhibited 1 of 16 distinct electrophoretic mobilities. We could distinguish the majority of iso-1-cytochromes c, even those having the same replacement at different sites and those having different replacements that resulted in the same net charge. These results provide confirmation of the importance of site-specific effects on the electrophoretic mobility, and presumably other properties, of proteins differing in sequence by as little as one amino acid. They demonstrate that nondenaturing electrophoresis is able to separate the majority of, but not all, proteins differing by single amino acids.