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.
1. Fully active ficin (EC 3.4.22.3) containing 1 mol of thiol with high reactivity towards 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) at pH4.5 per mol of protein was prepared from the dried latex of Ficus glabrata by covalent chromatography on a Sepharose-glutathione-2-pyridyl disulphide gel. 2. 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-S-S-2-Py. 3. Use of 2-Py-S-S-2-Py as a reactivity probe demonstrates (a) that in ficin, as in papain (EC 3.4.22.2), the active-centre thiol and imidazole groups interact to provide a nucleophilic state at pH values of approx. 6 additional to the uncomplicated thiolate ion that predominates at pH values over 9, and (b) a structural difference between ficin and papain that leads to a much higher rate of reaction of 2-Py-S-S-2-Py with ficin than with papain at pH values 3-4. This difference is suggested to include a lack in ficin of a carboxyl group conformationally equivalent to that of aspartic acid-158 in papain. 4. The high electrophilicity of the 2-Py-S-S-2PyH+ monocation allows directly the detection of the exposure of the buried thiol group of ficin at pH values below 4.
D015082:2,2'-Dipyridyl; D001120:Arginine / Q000031:analogs & derivatives; D001586:Benzoylarginine Nitroanilide; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D004220:Disulfides; D010450:Endopeptidases / Q000302:isolation & purification*; D005361:Ficain / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008623:Mercaptoethanol; D009596:Nitrophenols; D010944:Plants / Q000201:enzymology*; D011489:Protein Denaturation; D011725:Pyridines
D004220:Disulfides; D009596:Nitrophenols; D011725:Pyridines; D015082:2,2'-Dipyridyl; D008623:Mercaptoethanol; D001586:Benzoylarginine Nitroanilide; D001120:Arginine; D010450:Endopeptidases; D005361:Ficain