Selective radiolabeling of cell surface proteins to a high specific activity.
A procedure was developed for selective radiolabeling of membrane proteins on cells to higher specific activities than possible with available techniques. Cell surface amino groups were derivatized with 125I-(hydroxyphenyl)propionyl groups via 125I-sulfosuccinimidyl (hydroxyphenyl)propionate (125I-sulfo-SHPP). This reagent preferentially labeled membrane proteins exposed at the cell surface of erythrocytes as assessed by the degree of radiolabel incorporation into erythrocyte ghost proteins and hemoglobin. Comparison with the lactoperoxidase-[125I]iodide labeling technique revealed that 125I-sulfo-SHPP labeled cell surface proteins to a much higher specific activity and hemoglobin to a much lower specific activity. Additionally, this reagent was used for selective radiolabeling of membrane proteins on the cytoplasmic face of the plasma membrane by blocking exofacial amino groups with uniodinated sulfo-SHPP, lysing the cells, and then incubating them with 125I-sulfo-SHPP. Exclusive labeling of either side of the plasma membrane was demonstrated by the labeling of some marker proteins with well-defined spatial orientations on erythrocytes. Transmembrane proteins such as the epidermal growth factor receptor on cultured cells could also be labeled differentially from either side of the plasma membrane.
D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000032:analysis; D006801:Humans; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000032:analysis* / Q000097:blood; D008970:Molecular Weight; D011865:Radioisotope Dilution Technique; D013388:Succinimides
D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D013388:Succinimides