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The rate of binding of Asp-hemolysin to human erythrocyte ghosts was determined by the single radial immunodiffusion technique. This binding rate was found to be reduced by various chemical reagents, yielding inhibition rates in the order of: glyoxal>TNBS>4-PDS>DTNB>HgCl2. These findings suggest that sulfhydryl and arginine-guanidino group(s) in the toxin may play an important role as a site of binding to the erythrocyte membrane. On the other hand, the hemolytic activity of Asp-hemolysin for chicken erythrocytes was significantly inhibited by arginine, lysine, and ornithine, while it was not affected by the similar compounds citruline, betaine, and histidine. Arginine was shown to be the most effective competitive inhibitor. On the basis of these results, the presence of a binding site in the Asp-hemolysin molecule is suggested. Furthermore, we suggest that the inhibition caused by arginine and related compounds is competitive.

The fashion of binding of Asp-hemolysin to human erythrocytes and the isolation of Asp-hemolysin-binding proteins from erythrocyte membranes were investigated by the immunocytochemical technique and affinity chromatography. Asp-hemolysin bound best at a pH range from 5 to 7. The erythrocytes treated with Asp-hemolysin showed diffuse, ring-like or cap-like staining by the peroxidaselabeled antibody method under the light microscope. The distribution of Asphemolysin on the erythrocyte surface was clearly observed as patches or caps in the scanning electron microscope. The erythrocyte ghosts were extracted with 1% sodium deoxycholate-0.1 M Tris-HC1 buffer (pH 7.5) containing 0.2 M NaCl and 1 aim EDTA, and the extract was chromatographed on an affinity column consisting of Asp-hemolysin attached to activated thiol-Sepharose 4B. Four proteins present in the membrane extract were retained by activated thiol-Sepharose 4B and eluted with 50 mm cysteine as toxin-membrane components. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the polypeptides correspond to band 2.1, one protein of the 2 region, band 3 and band 7 in the Steck nomenclature system.

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