Properties of the hatching enzyme from frog embryos

The stage 18 Rana chensinensis embryos were deprived of their envelopes, and were reared until stage 21. The culture medium concentrated by lyophilization contains the enzyme (hatching enzyme; HE) which digests the whole egg‐envelopes of the homologous species. The enzyme also digests the outer egg‐jelly layers of a closely related species, but does not affect its inner layers and the whole envelopes of the less‐related anuran species. HE is a protease, as defined by its activity towards casein. Successive fractionation by (NH 4 ) 2 SO 4 precipitation, Sephadex gel‐filtration, and DEAE‐cellulose chromatography yielded a 100‐fold increase of proteolytic activity in the original culture medium. The molecular weight estimation by gel‐filtration gave a value of 55,000–60,000 for the HE. The pH optimum for the enzyme is 7.4–7.8. The enzyme is easily inactivated by preincubation at temperatures higher than 50°C, but is quite stable to autodigestion at pH 7.4. Its activity is unaffected by Na + , K + , and the soybean trypsin inhibitor, but is strongly inhibited by Ca ++ , Mg ++ , ethylenediaminetetraacetic acid, and diisopropyl‐fluorophosphate. The properties of the HE demonstrated here provide a basis for the view that this enzyme can be regarded as a unique protein functioning at the particular stage of embryogenesis.