Agglutinin from Limulus polyphemus. Purification with formalinized horse erythrocytes as the affinity adsorbent.
We have purified an agglutinin from the hemolymph of Limulus polyphemus about 1500-3000-fold by adsorption to formalinized horse erythrocytes, elution with N-acetylneuraminic acid and subsequent fractionation on Sephadex G-200. Recovery was in the range of 50 percent. On ultracentrifugation the agglutinin behaves as an homogenous protein with a molecular weight of about 460 000. On polyacrylamide gel electrophoresis of the dissociated protein in sodium dodecylsulfate we found a single prominent diffuse band with an apparent molecular weight of 22 000 plus or minus 2000. This band contained carbohydrate as determined by periodic acid-Schiff staining. The intensity of staining compared with standards suggested a carbohydrate content of less than 4 percent. The protein contains a preponderance of acidic amino acids and has an isoelectric point of 4.83.5 residues per 1000 of glucosamine were detected on amino acid analysis. Agglutination of formalinized horse erythrocytes by the purified protein is inhibited not only by N-acetylneuraminic acid but also by D-glucuronic acid; but not by a number of other monosaccharides. D-Glucuronic acid may be used in place of N-acetylneuraminic acid as the eluting sugar in the purification procedure.
D000373:Agglutinins / Q000302:isolation & purification; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003386:Brachyura / Q000032:analysis*; D002850:Chromatography, Gel; D004912:Erythrocytes / Q000502:physiology*; D005557:Formaldehyde; D005688:Galactosamine / Q000032:analysis; D005944:Glucosamine / Q000032:analysis; D006386:Hemagglutination Tests; D006458:Hemolymph / Q000032:analysis*; D006736:Horses; D046911:Macromolecular Substances; D008970:Molecular Weight; D011485:Protein Binding; D012794:Sialic Acids
D000373:Agglutinins; D000596:Amino Acids; D046911:Macromolecular Substances; D012794:Sialic Acids; D005557:Formaldehyde; D005688:Galactosamine; D005944:Glucosamine