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The chorionase (hatching enzyme) of Fundulus heteroclitus digests the chorion by producing water soluble peptide fragments. The chorion may be labeled with 14C-iodoacetamide so that the soluble products of digestion are also labeled. We used this labeled substrate as the basis for a quantitative assay for chorionase. Chorionase was found to be quite stable at temperatures below 30°C and had a Ql0 of 2.2 between 15 and 30°C. The pH optimum for enzymatic activity was between pH 8.0 and pH 8.5, and an ionic strength optimum was found between 0.1 M and 0.2M.The enzyme was inhibited at ionic strengths similar to those found in sea water. Chorionase was also inhibited by divalent cations, EDTA, and PMSF. These results suggest that divalent cations are necessary for activity even though they are inhibitory at concentrations above 10mM.During the hatching process teleosts secrete an enzyme, chorionase, that digests the chorion surrounding the embryo. It has long been assumed that the enzyme digests the chorion by a proteolytic action (Wintrebert, '12; Bourdin, '26; Ishida, '44). Kaighn (' 64) was able to demonstrate that the chorionase of Fundulus heteroclitus is capable of cleaving the beta chain of insulin. More recently, Iuchi and Yamagami (' 76) have shown that peptide fragments are produced when chorionase acts on its natural substrate.Several assays based on the proteolytic action of chorionase have been reported. Kaighn ('64) observed the digestion of a s m d fragment of chorion by chorionase on a glass slide to obtain a qualitative estimate of chorionase activity.Yamagami ('70) developed an assay based on the increased transmittance of light as chorionase digested a turbid suspension of chorionase fragments. He used this assay to characterize the action of chorionase on its natural substrate in Oryzias latipes. Hagemaier ('74) also used the casein assay to characterize the chorionase of Salmo gairdneri.Our purpose in this study was to extend the characterization of Fundulus heteroclitus chorionase, initiated by Kaighn ('64), using Fundulus chorions as the substrate. We developed an assay based on formation of aradioactive, water-soluble product. This assay is presented here together with a scheme for the partial purification of the chorionase of Fundulus heteroclitus. MATERIALS AND METHODSFertilized eggs were obtained by stripping gametes from gravid males and females into a beaker of sea water. The eggs were washed several times with tap water and placed in a 33-liter aquarium equipped with an undergravel filter. The aquarium was maintained at 15°ioo salinity, pH 7.0, 20"C, 13mg02/liter, and LD14:lO. Eggs held under these conditions develop normally, but hatching is prevented by the high oxygen concentrations (DiMichele and Taylor, '81). EnzymeTo obtain a crude preparation of chorionase, 500-3000 eggs were removed from the stock aquarium on day 16 of development and placed in a beaker of nitrogen-saturated HEPES buffer (1mM Na' HEPES, pH 8.1). Hatching began within 10 minutes after immersion of the ...

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Cytochrome C3 was purified from Desulfovibrio africanus Benghazi by extraction with alkaline deoxyribonuclease, fractionation with ammonium sulfate, batch elution from carboxymethyl Sephadex followed by chromatography on the same resin, and gel filtration on Sephadex G-75. The preparation was judged homogeneous by a variety of criteria. The moleculer weight was determined in an analytical ultracentrifuge, and values between 14,400 and 15,490 were obtained, depending upon the presumed value of partial specific volume. Gel filtration on a calibrated column of Sephadex G-75 gave a value of 14,900 daltons. The amino acid composition was very similar to that observed for the cytochrome from other species of Desulfovibrio, with the exception of increased levels of ThR and PhE. S-Carboxymethylation of the protein before and after heme removal by HgCl2 demonstrated eight Cys molecules involved in heme binding or four heme sites per molecule. Titration with sodium dithionite under N2 gave an electrochemical potential (E'o) of-276 mV relative to the normal hydrogen electrode. Electrochemical titration of the cytochrome gave a Nernst plot with two linear regions with E'o values of-0.376 and-0.534 V. The spectra produced at various potentials exhibited shifts in isosbestic points upon reduction, suggesting changes in conformation during the reaction.