Perfluoroalkylphosphocholines are poor protein-solubilizing surfactants, as tested with neutrophil plasma membranes.
We have tested the membrane-protein solubilizing properties of two perfluoroalkylphosphocholines. These compounds belong to a series of fluorinated amphiphiles which are being investigated as potential stabilizing agents for a variety of fluorocarbon-based systems. We are particularly interested in cytochrome b558 from phagocytes, the redox component of NADPH oxidase. Its heavy subunit is believed to carry binding sites for NADPH and FAD. Nevertheless, when the cytochrome is purified in the presence of classical detergents, it carries no FAD. This could be due to a delipidating, denaturing effect of these detergents (octyl glucoside, Triton, etc). The first perfluoroalkyphosphocholine, C8F17(CH2)2O-P(O2-)-O(CH2)2N+(CH3)3(F8C2PC), extracted about as much protein from neutrophil plasma membranes into a 100,000 g supernatant as octyl glucoside. The second compound, C8F17(CH2)11O-P(O2-)-O(CH2)2N+(CH3)3(F8C11PC), was less efficient. We found that flavin was still protein-bound in the crude F8C2PC extract at a FAD to heme ratio of about 1, and a good NADPH oxidase activity was obtained without addition of exogenous FAD, even after dialysis or gel filtration, whereas dialysis eliminated most of the FAD from the octyl glucoside extracts. These experiments appeared to make F8C2PC an interesting membrane-solubilizing agent. Nevertheless, no protein in the F8C2PC extract could be adsorbed on the chromatographic supports normally used for purification. After dilution of the extract and addition of 15 mM octyl glucoside, some of the proteins, such as myeloperoxidase, could be adsorbed (and eluted), but not cytochrome b558. Freeze-fracture electron microscopy showed that the F8C2PC extracts contained numerous vesicles and aggregates of small shapeless particles. Higher centrifugal forces sedimented most proteins of the 100,000 g supernatant. As a check, the effect of F8C2PC was tested on sarcoplasmic reticulum vesicles, the behavior of which with respect to the usual non-denaturating detergents has been well studied. There was little, if any, solubilization. We conclude that, although supernatants of F8C2PC extracts of neutrophil membranes are optically clear, proteins are not really solubilized. This result is in keeping with the absence of lytic effects of F8C2PC on erythrocyte membranes.
Der Mardirossian
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Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, France.
Krafft
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Gulik-Krzywicki
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T
le Maire
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M
Lederer
F
F
D002462:Cell Membrane / Q000737:chemistry*; D020285:Cryoelectron Microscopy / Q000379:methods; D003573:Cytochrome b Group / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D005415:Flavins / Q000032:analysis / Q000737:chemistry; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008823:Micelles; D009249:NADP / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000737:chemistry*; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000737:chemistry*; D012519:Sarcoplasmic Reticulum / Q000737:chemistry; D012995:Solubility; D013501:Surface-Active Agents / Q000737:chemistry*; D005466:Fluorocarbons
D003573:Cytochrome b Group; D003902:Detergents; D005415:Flavins; D008565:Membrane Proteins; D008823:Micelles; D009249:NADP; D019255:NADPH Oxidases; D010767:Phosphorylcholine; D013501:Surface-Active Agents; C099918:3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecylphosphocholine; D005466:Fluorocarbons; C115690:F8C11PC, phosphocholine; C036888:cytochrome b558