Acylation of red blood cell membrane proteins studied with bio-orthogonal chemical probe analogs of fatty acids

Red blood cells (RBCs) have long been known to contain acylated proteins and to display an active palmitate turnover in selected membrane and membrane-skeletal proteins. Evidence of in vivo palmitoylation of RBC proteins was obtained after metabolic labeling of RBCs with tritiated palmitic acid, electrophoretic separation of RBC proteins followed by weeks-long exposure times for revealing the labeled proteins by fluorography. Considerable efforts are being made to pursue alternate routes for the study of protein fatty-acylation. One such alternative is the use of bioorthogonal chemical probe analogues of fatty acids, such as omega alkynyl analogues, for metabolically labeling the cells, followed by cell lysis and chemoselective ligation of the alkynyl group to azide-tagged biotin via a Cu+ catalyzed alkyne-azide (3+2) cycloaddition reaction. Subsequently, proteins are separated by SDS-PAGE, electrotransferred to PVDF membranes and visualized with streptavidin-HRP and chemiluminescence. Results obtained in this study show that the omega alkynyl analog of palmitic acid is indeed metabolically incorporated into a number of protein bands, the main of which is, as expected, p55. The method is sensitive (less than 20 microg total proteins loaded per lane) and rapid: in less than 4 days a complete result, including the hydroxylamine treatment, can be obtained, as opposed to the much longer time required by fluorography. White RBC ghosts, i.e. ghost membranes that have been carefully washed free of hemoglobin, must be used to carry out the alkyne-azide reaction: even the slightest contamination by hemoglobin interferes with the reaction. In the preparation of ghost membranes, chelating agents such as EDTA must be avoided, because they strongly interfere with the Cu+-catalyzed alkyne-azide reaction. Studies are in progress to investigate the state of palmitoylated proteins in RBCs of different age and their partition into different membrane subdomains.