Microenvironmental contributions to the chromatographic behavior of subtilisin in hydrophobic-interaction and reversed-phase chromatography.

Genetically engineered variants were used to examine how microenvironmental changes in the S1 substrate binding subsite of subtilisin contribute to chromatographic behavior of proteins on hydrophobic-interaction chromatography (HIC) and reversed-phase chromatography (RPC) columns. Gradient elution studies over a wide pH range showed that conditions could be found where a HIC support could separate proteins varying by one amino acid. Although all single-site variants could not be separated by HIC, this chromatographic mode was found to be complementary to cation-exchange chromatography for the separation of such variants. RPC was found to be of much less utility in the resolution of variant proteins. Retention and resolution of subtilisin variants was found to vary on RPC with the concentration and type of mobile phase pairing agent.