Bead reagents for membrane proteins

The synthesis of long-arm thiolated beads from beaded polyacrylamide ( $400 mesh, 10-37^ M diameter, 2,000 Dalton inclusion limit) is described. The long spacer arm, polyethylene glycol di(3-mercapto-propionate)(PEG-DMP) was i introduced via thioether formation with haloacetamidoethyl beads. The latter were prepared via the N-hydroxysuccinimide ester (NHS) after surveying a number of peptide coupling methods. Iodide was chosen as the leaving group following a survey of the corresponding chloro-, bromoand iodobead derivatives. The thiol group densities in the range 3-7 ^ e q / g of dry beads were achieved in the final derivative as determined by spectrophotometric titration. Radiochemical titration with (1-^cJ-iodoacetamide indicated sulfhydryl group density about 40# of the spectrophotometric one. Thiol beads were characterized as the mixed disulfides with thionitrobenzoic acid and 2-thiopyridone. Characterization of long-arm thiolated beads in thiol-disulfide interchange reactions was made by the use of bead-S-S-2-Py coupling with 2-mercaptoethanol, PEG-DMP, papain, BSA-SH and cell (ghost). In papain experiments, with bead-S-S-2-Py in large excess (40-50-fold) over protein thiol, thiopyridone release corresponding to £ of protein thiol groups occurred rapidly and this exceeded amounts of