ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 206, No. 2, February, pp. 424-431, 1981 Isolation and Characterization of the Hatching Enzyme from the Amphibian, Xenopus laevisl UMBERT A. URCH AND JERRY L. HEDRICK Department of Biochemistry and Biophysics, University of California, Davis, California 95616 Received July 9, 1980 The proteolytic activity released at the time of Xenopus laevis embryo hatching, termed the hatching enzyme, was purified and characterized in terms of its physical and enzymatic properties. Using predominantly isoelectric focusing and preparative ultracentrifugation, the enzyme was purified 2200-fold over the starting crude hatching media. From disc gel electrophoretic experiments, the most highly purified form of the enzyme had two enzymatically active charge isomers present with molecular weights of 62,500. With time, the purified enzyme gave rise to a family of enzymatically active charge isomeric proteins. The enzymatic activity of hatching enzyme toward its lz51- labeled natural substrate, the fertilization envelope, was optimal at pH 7.7 and was ionic strength dependent. The enzyme was inhibited by ZnZ+ and by EDTA. From inhibition by the site-specific reagents diisopropylfluorophosphate and phenylmethylsulfonylfluoride, we concluded that the enzyme was of the serine protease type, although its inhibition by Zn*+ and EDTA prevents a clear and unequivocal classification of the protease. This enzyme is different from the hatching enzymes reported in fish and echinoderms, on the basis of size, but it is similar to that described in Rana chensinensis on the basis of size and specificity. The escape of an embryo from protective integuments (e.g., jelly coats and fertiliza- tion envelopes of amphibians and echino- derms, the zona pellucida of mammals) is a fundamental process in animals. This escape, defined as hatching, is accomplished mechanistically in a limited number of ways. The hatching of an embryo may involve: (i) release of a hatching enzyme by the embryo to digest part or the whole of the egg jelly coats or envelopes; (ii) increases in perivitelline space volume due to os- motically active agents secreted by the embyro resulting in rupture of the envelope; (iii) mechanical hatching with either special- ized embryonic structures that puncture the egg envelope or by muscular or ciliary movement (l-3). There are additional examples of hatching by increases in em- bryo volume causing envelope rupture and by parental aid (3). Many species ’ This work was supported in part by a grant from the United States Public Health Service, Grant HD 04906. employ a combination of these mechanisms to first weaken the integuments and then rupture them. The hatching process in the African clawed toad, Xenopus laevis, was ini- tially described by Bles (4) and the in- vestigation was extended by Carroll and Hedrick (5). Two phases of hatching have been described. The first phase involves rupture of the outer jelly layers, JZ and J3, by water imbibition of the innermost jelly layer, J1, and by changes in perivitelline space volume. The second phase involves muscular movement and enzymatic action to hatch from the fertilization envelope (FE).2 In a previous publication (5), hatching enzyme (HE) was defined as the proteolytic/ arylamidase activity associated with the culture media of hatching embryos. The present paper further defines the hatching ’ Abbreviations used: J, jelly layer; FE, fertilization envelope; HE, hatching enzyme; Tris, tris(hydroxy- methylkuninomethane; TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate; DFP, diisopropyl fluorophos- phate; TEAE-cellulose, O-(triethylaminoethyl)-cellulose. 0003-9861/81/020424-08$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. 424