High-Capacity Adsorbents for Affinity Chromatography

One simple structural change in compound (I), the destruction of its symmetry by its conversion into n-propyl-2-pyridyl disulphide [III, R = C3H7(propyl)-S-S-2-Py], has permitted the demonstration of an important property of the thiol proteinases, the existence of nucleophilic character in the uninterrupted thiol-imidazole interactive systems of their active centres. The reactions of 2-Py-S-S-2-Py (I) with papain and ficin are characterized by striking rate maxima at p H values 3-4 (see Malthouse & Brocklehurst, 1976), and the three reactive protonic states of the pH-k profile are designated X-XH,. Although the existence of reactive XH, and X H states of these reactions appears t o demonstrate nucleophilic character of the interactive systems, the symmetry of the probe reagent does not permit resolution of one important ambiguity. Jolley & Yankeelov (1972) suggested that it may be necessary for the ion-pair components of such active-centre systems t o be interrupted by a n electrophilic reagent t o release the nucleophilic character of the system. We here report that the reaction of ficin with propyl-S-S-2-Py occurs at the sulphur atom distal from the pyridyl ring, and that this reaction also is characterized by three reactive protonic states, including a striking rate maximum a t p H 3.4. Since the asymmetrical probe reagent possesses only one pyridyl nitrogen atom, it is not possible for both a pyridyl-imidazolium interaction and a n additional activating protonation t o exist simultaneously. High reactivity in the XH2 state of the reaction of ficin with propyl-S-S-2-Py therefore provides compelling evidence for nucleophilic character in the interactive cysteine-histidine system (pK, approx. 4) without the necessity of interruption by a pyridyl-imidazolium interaction. A similar conclusion was reached also for papain (M. Shipton & K. Brocklehurst, 1978).