Removal of the Nonionic Detergent Emulgen 911 from Solubilized Microsomes by HPLC

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Abstract
The removal of a nonionic detergent, Emulgen 911, by HPLC from solubilized microsomes is described. Microsomal samples were dissolved in phosphate buffer containing sodium cholate and tritiated Emulgen 911. The HPLC column was eluted with phosphate buffer containing 20% (v/v) glycerol, sodium cholate and, for one set of conditions with Lubrol WX. The detergent, Emulgen 911, when eluted with sodium cholate, binds tightly to the column eluting after the inclusion volume. Elution in the presence of Lubrol WX resulted in decreased retention, with Emulgen 911 eluting within the inclusion volume. Microsomal samples chromatographed with sodium cholate are resolved into two peaks. The first eluting peak is associated with the cytochrome P-450 fraction. Effective detergent removal under these conditions was 88% of the eluted radioactivity. For purified cytochrome P-4501 detergent removal was 99% efficient and provided a sample with a ratio of 2.5 microgram detergent per nanomole of P-450. Elution with Lubrol WX did not provide a full quantitative picture because of overlapping protein and detergent peaks but it provided evidence for strong hydrophobic interactions between the bonded phase and Emulgen 911, and between the two nonionic detergents.