A cationic hydroxysuccinimide ester A reagent for labeling exterior membrane proteins

Abstract

3H-labeled N,N,N, trimethylamino-β-alanyl-N- hydroxysuccinimido ester ([ 3H]TMAS), a new cationic membrane reagent, was synthesized. TMAS was shown to be impermeant through human erythrocyte membranes. Under mild physiological conditions TMAS reacted primarily with amino groups of the membrane proteins and lipids. The pattern of erythrocyte proteins labeled with [ 3H]TMAS was examined by polyacrylamide gel electrophoresis under denaturing conditions. Externally oriented labeling of intact erythrocytes revealed a major radioactive protein with an apparent molecular weight of 90 000. By labeling ghost preparations with [ 3H]TMAS the radioactivity incorporated into all the major Coomassie Brilliant Blue bands resolved by gel electrophoresis. The agreement between the results obtained with anionic and cationic amino reactive probes indicates that the ionic character of the reagent has a minor effect on the pattern of labeled exterior polypeptides observed in erythrocytes.