Differential solubilization of lipids along with membrane proteins by different classes of detergents.
Membrane proteins are typically extracted by detergent concentrations of 0.5-2.0%, using detergent/protein ratios of 1:1 to 3:1. We have compared the ability of 14 different detergents from seven different structural and ionic classes, at a concentration of 2.0% and a detergent/protein ratio of 2:1, to extract an integral membrane protein (the serotonin 5-HT1A receptor) in active form and have observed profound differences in both lipids and proteins. All extracts were freed from detergents and dialyzed to form vesicles containing 95-100% of the extracted lipids, prior to [3H]8-hydroxy-2-(N,N-di-n-propylamino)tetralin ([3H]8-OH-DPAT) binding. The most efficient detergents in extracting active 5-HT1A receptor protein were the zwitterionic 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 3-[(cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), followed by the neutral n-dodecyl-beta-D-maltoside. Zwitterionic detergents also produced the highest solubilized lipid/protein ratio (3.0 and 2.5, respectively) and in general the relative amounts of extracted lipids and proteins followed inverse profiles. Thus, hydrophobic detergents such as Tritons (with critical micelle concentrations similar to CHAPS) and Thesit (structurally similar to Lubrol) extracted the most protein, but relatively little lipid (ratios of less than 0.2) and very little active 5-HT receptor. Dramatic differences were also observed in the ratios of individual lipids extracted by the same concentrations of different detergents and resolved by high-performance thin-layer chromatography. For example, galactosylceramide (GalCer) content ranged from 2.7% (CHAPSO) to 13.4% (sodium cholate) of the total lipid extract and cholesterol ranged from 0% (digitonin) to 17.9% (Triton X-100). The detergent-extractability profile for phosphatidylethanolamine (PE) (range 15-40% of total lipid) paralleled that of phosphatidylinositol (PI) (range 4-10%), but was inverse to that for GalCer and cholesterol. Detergent-extractability profiles for phosphatidylcholine (PC) and phosphatidylserine (PS) also followed inverse profiles, with zwitterionic detergents giving high PS/PC and high PE/PC ratios (approximately 2:1), whereas the Tritons and digitonin gave ratios of 1:2. We believe that differential solubilization of lipids, as well as proteins, by detergents is important for the biological activity of the extracted proteins, and lipid extractability should be taken into account when purifying membrane proteins.
Banerjee
P
P
Department of Chemistry, City University of New York, College of Staten Island 10314, USA.
Joo
J B
JB
Buse
J T
JT
Dawson
G
G
D017371:8-Hydroxy-2-(di-n-propylamino)tetralin / Q000378:metabolism; D000818:Animals; D000838:Anions / Q000737:chemistry; D001923:Brain Chemistry; D002412:Cations / Q000737:chemistry; D002518:Ceramides / Q000737:chemistry; D002784:Cholesterol / Q000737:chemistry; D002855:Chromatography, Thin Layer; D003505:Cyclodextrins / Q000737:chemistry; D003902:Detergents / Q000737:chemistry*; D006860:Hydrogen Bonding; D008055:Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D015394:Molecular Structure; D010743:Phospholipids / Q000737:chemistry; D011985:Receptors, Serotonin / Q000737:chemistry; D012756:Sheep; D012995:Solubility; D013012:Sorbitol / Q000031:analogs & derivatives / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry
D000838:Anions; D002412:Cations; D002518:Ceramides; D003505:Cyclodextrins; D003902:Detergents; D008055:Lipids; D008565:Membrane Proteins; D010743:Phospholipids; D011985:Receptors, Serotonin; D013501:Surface-Active Agents; D013012:Sorbitol; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; C063161:MEGA 8; D002784:Cholesterol