Agglutinins in the horseshoe crab hemolymph: purification of a potent agglutinin of horse erythrocytes from the hemolymph of Tachypleus tridentatus, the Japanese horseshoe crab.

Agglutinins from Tachypleus (Tachypleus tridentatus, the Japanese horseshoe crab) hemolymph were isolated by affinity chromatography on BSM-coupled Sepharose 4B. The agglutinins showed multiple species and were composed of eight heterogeneous subunits with molecular weights of 45,000, 42,000, 41,000, 39,000, 33,000, 29,000, 27,000, and 22,000 as determined by SDS-polyacrylamide gel electrophoresis. The affinity-isolated agglutinins were fractionated into four groups by gel filtration on a Fractogel TSK (Toyopearl) HW-65 column, and these were designated as Tachypleus tridentatus agglutinin (TTA)-I, -II, -III, and -IV in the order of elution. These agglutinins were demonstrated to be heterogeneous as judged by their specificity towards horse erythrocytes, subunit structures, and immunological properties. TTA-III showed a potent agglutination activity towards horse erythrocytes and was further purified by gel filtration on a Cellulofine GC-700 column. The purified TTA-III is a highly purified (46,000-fold) protein composed of homogeneous subunits (Mr, 42,000) as judged by SDS-polyacrylamide gel electrophoresis and immunological analysis.