Amidination of the outer and inner surfaces of the human erythrocyte membrane

We have synthesized a novel imidoester, isethionyl acetimidate, which is unable to penetrate the membrane of the human erythrocyte. It has the same specificity for amino groups as ethyl acetimidate, which penetrates the membrane. Either reagent can be labeled with ³H or ¹⁴C and, thus, be used to convert amines to radioactive amidines. An erythrocyte membrane saturated with either compound functions nearly normally. Therefore, the membrane can be double labeled if the amino groups on the outer surface of a cell are saturated with isethionyl acetimidate (e.g. labeled with ¹⁴C) and the remaining active sites are saturated with ethyl acetimidate (labeled with ³H). Alternatively, the membrane can be isolated after saturation with [¹⁴C]isethionyl acetimidate and treated with [³H]isethionyl acetimidate. From quantitative experiments of this kind we conclude that there are more than ten times as many reactive amino groups in protein on the inner surface than on the outer surface of the membrane. Nearly all of the reactive amino groups in lipid are on the inner surface. The localization of individual polypeptides confirms and extends assignments made previously by other techniques; as many as four major components may span the membrane. The proteins and lipids react to the same extent with ethyl acetimidate in the intact cell as they do in isolated membranes; this implies that the isolation does not load to major structural rearrangements.