Fluorescent photochemical surface labeling of intact human erythrocytes

A photolabile nitrene precursor, 3-azido-(2,7)-naphthalene disulfonate (ANDS), has been synthesized and used as a membrane-impermeable probe. The aryl azide was nonfluorescent. When activated by light, a highly reactive nitrene was generated which was capable of nonspecific covalent modifications of hydrophilic regions of cell surfaces. The products of the photolysis were highly fluorescent and modified proteins could be identified by their characteristic fluorescence after electrophoresis on sodium dodecyl sulfate polyacrylamide gels. When intact human erythrocytes were labeled with ANDS, Protein 3, the major membrane protein, and the sialoglycoproteins were modified. No proteins of apparent molecular weight greater than Protein 3 were labeled by ANDS, suggesting that none of these membrane components was exposed to the hydrophilic external surface of the red blood cell. When open erythrocyte stroma were labeled with ANDS, virtually all protein bands detectable by Coomassie blue staining could be shown to contain some fluorescence label. The significance of these findings are discussed with relation to the use of various aryl azides as surface labels of membranes.