Phenyl-Sepharose-mediated detergent-exchange chromatography: its application to exchange of detergents bound to membrane proteins

Abstract

Detergent-saturated phenyl-Sepharose was used to exchange detergents for one another in the presence of membrane proteins. The alkyl detergents lauryl maltoside, octyl glucoside, and dodecyl sulfate were each successfully exchanged for Triton X-100, Triton N-101, or Nonidet P-40 present in a solution of either cytochrome c oxidase, a mixture of inner mitochondrial membrane proteins, or a mixture of erythrocyte membrane proteins. The method involves (1) saturating a small column of phenyl-Sepharose (1-2 mL) with one of the alkyl detergents at a pH of 8 or 9 and an ionic strength of 0.01, (2) applying a detergent-solubilized membrane protein sample containing as much as 20 mg/mL of Triton X-100, Triton N-101, or Nonidet P-40, and (3) eluting the protein with buffer containing the detergent with which the resin had been saturated. With this approach, 90-99% of the detergent in the initial protein sample was exchanged for the second detergent with an 80-100% recovery of protein. The advantages of this method over previous approaches for exchanging detergents include the rapidity of the technique and the apparent general applicability of the method to a wide variety of detergents and membrane proteins.

Quantitative Determination of Cardiolipin in Mitochondrial Electron Transferring Complexes by Silicic Acid High-Performance Liquid Chromatography

“…3B). To eliminate this problem, most of the Triton X-100 was removed by exchanging for another detergent by phenyl-Sepharose CL 4B chromatography (25). Triton X-100 can be exchanged for either Tween 20 or dodecyl maltoside, neither of which causes any detectable silicic acid HPLC elution peaks when monitored at 208 nm.…”

“…Triton X-100 interferes with the determination of cardiolipin by silicic acid HPLC; therefore, residual Triton X-100 in native cytochrome bc 1 was exchanged for dodecyl maltoside or Tween 20 prior to the HPLC analysis of cardiolipin using phenyl-Sepharose CL 4B chromatography (25). Two milligrams of cytochrome bc 1 was applied to a 1-ml column equilibrated with 20 mM Tris-HCl, pH 9.0, I ϭ 0.01, containing 2 mg/ml dodecyl maltoside.…”

Purification and characterization of two forms of hydrogenase isoenzyme 1 from Escherichia coli

“…However, the hydrogenase-deoxycholate complex did bind to this weak anion exchanger. This necessitated exchange of the Triton X-114 with deoxycholate (26).…”

Selective Depletion and Enrichment Methods for the Analysis of Protein and Peptide Pools

“…Covalently bound lipids that allow the protein to be attached to a lipid bilayer membrane could also provide a convenient selection tool for combinatorial depletion or enrichment strategies (both on protein and peptide levels). Lipoproteins can be isolated with hydrophobic interaction chromatography using alkyl agaroses (most commonly having the length of the alkyl chains [(CH 2 ) n -H], n = 8) or phenyl agarose (the latter being a more frequently used sorbent) [77][78][79]. Lipid adsorption reagents that are suitable for use in a solid-phase mode include Cleanascite HC TM -a nonionic lipid adsorption reagent for the selective collection of lipoproteins or lipid-modified peptides (available from Biotech Support Group, www.biotechsupportgroup.com).…”