Properties of the hatching enzyme from frog embryos

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Abstract
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 (NH4)2SO4 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.