Detergent Mediated Effects on the High-Performance Liquid Chromatography of Proteins

Abstract

Present chromatographic systems for the high-performance liquid chromatography (HPLC) of hydrophobic-proteins are generally limited to size-exclusion or ion-exchange chromatography. A major stumbling block to the successful chromatography of membrane-proteins is their limited solubility. Detergent is usally required to solublize these proteins. This detergent causes some problems in size-exclusion chromatography, but does not always interfere with the separation. It is more deleterious in anion-exchange chromatography, where ionic detergents can poison the column, and reversed-phase chromatography, where strong interactions can occur between the stationary phase and detergent. Successful chromatography of membrane-proteins requires favorable detergent/stationary-phase interactions that enhance, rather than interfere with, the separation.

To study these “detergent-mediated effects” a series of protein standards were chromatographed by reversed-phase HPLC. The column was then saturated with detergent and the standards rechromatographed. To evaluate any irreversible effects (caused by detergent/stationary-phase interactions) the column was washed extensively and re-evaluated. Following this procedure a variety of stationary-phases and detergents were tested.

The results of these studies showed that resolution was enhanced by detergent. Retention time was generally uneffected, but peak width was noticeably decreassed. Proteins were separated by fast gradients and recovered in high yields (95–99%). A C-18 stationary-phase gave better resolution than a C-8 stationary-phase. In all cases studied the column was irreversibly modified.

A final test of the “detergent-modified” columns was the chromatography of membrane-proteins. Prior attempts at the reversed-phase HPLC of these proteins had resulted in either no sample recovery, or of very low yields of purified protein. An acetylcholinesterase containing sample chromatographed as series of fused peaks, two of which were found to contain cholinesterase activity. Human lymophocyte function-antigen chromatographed as a single peak and was recoved with a 95% yield.