Studies on Perivitelline Fluid of Horseshoe Crab Embryo. I. Purification and Properties of Agglutinin from the Perivitelline Fluid of Tachypleus gigas Embryo

Agglutinin in the perivitelline fluid (PVF) of Tachypleus gigas, horseshoe crab, embryo was isolated and purified by a combination of affinity column chromatography on Sepharose 4B coupled with bovine submaxillary gland mucin and gelfiltration of Fractogel TSK (Toyopearl) HW-60 in Tris-NaCl-CaCl2, (0.05 M Tris HCl, pH 7.5, containing 0.5 M NaCl and 0.1 M CaCl2 buffer, containing 1 M urea. The specific activity of the purified protein was increased about 1,300 times in comparison with that of the starting material. The active protein was present in highly polymerized forms which were multimers of an identical subunit with a molecular weight of approximately 40,000 as measured by sodium dodecyl sulfate-polyacryl amide gel electrophoresis. This agglutinin was shown to have muttimeric activity towards different kinds of erythrocytes and its hemagglutinating activity was in hibited by N-acetylamino sugars and bovine submaxillary gland mucin containing sialic acid. Urea and guanidine-HCl inhibited the agglutinating activity but the activity recovered after dilution or dialysis, whereas the effect of HCl, NaOH, or 2-mercaptoethanol was irreversible.