Chemo-mechanical leak formation in human erythrocytes upon exposure to a water-soluble carbodiimide followed by very mild shear stress. II. Chemical modifications involved.

The chemical reactions underlying the chemo-mechanical leak formation in human erythrocytes upon treatment with the carboxyl-modifying reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and subsequent minimal shearing, described in the preceding paper (Thelen, P. and Deuticke, B. (1988) Biochim. Biophys. Acta 944, 285-296), are here characterized in more detail. The capacity to form leaks under minimal shearing results from the transformation of certain membrane carboxyl groups into an activated state, i.e., the O-acylisourea derivative of the original COO- group. This activated state re-disappears, i.e., the cell become shear-resistant again, when the O-acylisourea derivative under goes spontaneous hydrolysis upon removal of excess EDC in the suspension by addition of a cation-exchange resin. The activated state can be stabilized by addition of N-hydroxysuccinimide or N-hydroxysulfosuccinimide, which both form activated esters. The additional presence of nucleophilic amines, e.g., glycine methyl ester or aminomethane sulfonate, during the pretreatment with EDC strongly suppresses leak formation during subsequent shearing, which substantiates the involvement of COO- groups. EDC reactive side groups other than COO- (e.g., tyrosyl-OH or sulfhydryls) can be discarded as candidates for the underlying chemical reaction. The formation of the chemo-mechanical leaks most likely results from the cross-linking between a subpopulation of activated carboxyl groups and endogenous amino groups. This cross-linking, however, seems only to occur when the two reacting groups are brought into contact by the shear-induced cell--cell interactions required for leak formation. Besides information on chemo-mechanical leak formation the study provides new data helpful for future work with carbodiimides.