PURIFICATION AND CHARACTERIZATION OF A BLOOD-GROUP A REACTIVE HEMAGGLUTININ FROM SNAIL HELIX POMATIA AND A STUDY OF ITS COMBINING SITE

A protein specifically agglutinating human A erythrocytes has been isolated from the albumin gland of the snail Helix pomatia. The hemagglutinin, obtained in high yield (85.5%) by adsorption to insoluble polyleucyl blood-group A substance and subsequent elution with 0.005 m D-GalNAc, was homogeneous by gel filtration and ultracentrifugation and gave one precipitin band in double diffusion and immunoelectrophoresis against rabbit antisera to the crude extract. It was completely precipitated by human blood-group A substance. On polyacrylamide gel at alkaline pH, several bands with closely similar mobilities were observed, probably ascribable to heterogeneity of hemagglutinin molecules. The amino acid composition has been determined. It contains both halfcystine and methionine and has about 7% carbohydrate of which 4.0% is galactose and 3.3 % is mannose. The molecular weight is 100,000. Precipitation between blood-group A substance and purified hemagglutinin is best inhibited with Me-α- D-GalNAc and with twice the molar amount of D-GalNAc. No inhibition was obtained with Et-β-D-GalNAc even at very high concentrations. Me-α-D-GNAc and Me-β-D-GNAc also inhibited precipitation although four and ten times higher concentrations than for Me-α-D-GalNAc were needed. The size of the combining site may be no larger than that of an α-linked monosaccharide since about the same degree of inhibition was obtained by the A-active di-, tri-, and pentasaccharides, isolated from human or hog blood-group A substances, as with Me-α-D-GalNAc. The hemagglutinin not only precipitates with human blood-group A substance but reacts to a lesser extent with B, H, or Lea blood-group substances and increased reactivity is observed with a P-1 fraction or with a cyst material of low fucose content. The structure responsible for this reaction is not known. Precipitin analyses with teichoic acids from different strains of Staphylococcus aureus containing d-GNAc linked either α or β to the ribitol phosphate backbone or with mixtures of the α- and β-D-GNAc polymers revealed that only the α-D-GNAc polymer precipitates; using the 100% α-linked teichoic acid as a standard, the amount of α-linked polymer in mixtures could be estimated quantitatively. By comparing the relative capacities of Me-α-D-GalNAc, D-GalNAc, Me-α-D-GNAc, and d-GNAc in inhibiting precipitation between blood-group A substance and various fractions of snail hemagglutinin, homogeneity of the combining site was indicated. © 1969, American Chemical Society. All rights reserved.