Preparation of fully active ficin from Ficus glabrata by covalent chromatography and characterization of its active centre by using 2,2'-depyridyl disulphide as a reactivity probe

Abstract

Fully active ficin (EC 3.4.22.3) containing 1 mol of thiol with high reactivity towards 2,2''-dipyridyl disulfide (2-Py.sbd.S.sbd.S.sbd.2-Py) at pH 4.5/mol of protein was prepared from the dried latex of F. glabrata by covalent chromatography on a Sepharose-glutathione-2-pyridyl disulfide gel. Ficin thus prepared is a mixture of ficins I-IV and ficin G, in which ficins II and III predominate. The various ficins exhibit similar reactivity characteristics towards 2-Py.sbd.S.sbd.S.sbd.2-Py. Use of 2-Py.sbd.S.sbd.S.sbd.2-Py as a reactivity probe demonstrates that in ficin, as in papain (EC 3.4.22.2), the active-center thiol and imidazole groups interact to provide a nucleophilic state at pH values of approximately 6 in addition to the uncomplicated thiolate ion that predominates at pH values over 9, and a structural difference between ficin and papain that leads to a much higher rate of reaction of 2-Py.sbd.S.sbd.S.sbd.2-Py with ficin than with papain at pH values 3-4. This difference probably includes a lack in ficin of a carboxyl group conformationally equivalent to that of aspartic acid-158 in papain. The high electrophilicity of the 2-Py.sbd.S.sbd.S.sbd.2-PyH+ monocation allows directly the detection of the exposure of the buried thiol group of ficin at pH values below 4.