Effects of proteolytic enzymes and neuraminidase on the I and i erythrocyte antigen sites. Quantitative and thermodynamic studies.

The effects of neuraminidase and proteolytic enzymes on I and i reactivities was studied with I and i adult red cells, using radioimmunological methods. An enhanced reactivity after enzyme treatment is not exclusively due to a membrane charge reduction. The increase in site numbers and association constants bring about the gain of the cold agglutinin fixation. The release of N-acetylneuraminic acid residues gradually increases the I antigen site density of I red cells and the i site density of i red cells. Similar behaviour was observed after proteolytic enzyme treatment with papain, bromelin or ficin. The proteolytic treatment of I erythrocyte reveals underlying i receptors on these cells. Following membrane glycoprotein chain removal, anti-i antibodies are specifically fixed on I erythrocytes. The accessibility to antibodies of the determinants responsible for I and i erythrocyte activities was influenced significantly by steric hindrance factors. While N-acetylneuraminic acid release increased antibody affinities for the antigenic receptor, the removal of glycopeptide chains greatly diminished steric hindrance and brought about higher affinity constants. After enzyme treatment, the antigenic structures become more homogeneous in their reaction with antibodies. The heterogeneity of binding constants observed with antigenic determinants of non-treated erythrocytes is probably due to the wide range of spatial distribution of these receptors within the membrane.