Purification and Characterization of Hatching Enzyme of Strongylocentrotus intermedius

Hatching enzyme of the sea urchin, Strongylocentrotus intermedius, was purified 220-fold from the initial hatching supernatant by the following procedures: (a) gel filtration on Sephadex G-100; (b) hydrophobic chromatography on 4-phenylbutylamine-Sepharose; (c) ion-exchange chromatography on DEAE-cellulose; (d) gel filtration on Sephadex G-200; (e) gel filtration on Sephadex G-100. The purified enzyme gave a single band following electrophoresis on sodium dodecylsulfate/urea/polyacrylamide gel. The molecular weight of the enzyme was estimated as 44000 by electrophoresis on dodecylsulfate/urea/polyacrylamide gel, and as 45000 by gel filtration on both Sephadex G-200 and Sephadex G-100. It was free from β-1,3-glucanase, esterase activity toward synthetic substrates, and most of the proteinases, all of which were found in the initial hatching supernatant. The purified enzyme required Ca2+ ion for its activity, and this requirement was not replaced with Mg2+ ion. It was fairly resistant to chemical and physical treatments such as urea, freezing-thawing and lyophilization. The purified enzyme dissolved the fertilization envelope only in diluted saline but not in sea water, whereas the crude preparation dissolved the fertilization envelope in both sea water and diluted saline. This capacity to dissolve the fertilization envelope in sea water was eliminated by chromatography on DEAE-cellulose, and was not restored by the addition of either boiled hatching supernatant or other fractions eluted from the chromatography on DEAE-cellulose. The presence of two unknown factors was demonstrated in the initial hatching supernatant; one causes crinkling of the fertilization envelope, whereas the other results in the envelope becoming thin. These factors were separated from the hatching enzyme by 4-phenylbutylamine-Sepharose and DEAE-cellulose chromatography, respectively. However, hatching enzyme activity was not augmented by the addition of these factors.