EFFECT OF 2 POLAR ORGANIC-AQUEOUS SOLVENT SYSTEMS ON THE STRUCTURE-FUNCTION-RELATIONSHIPS OF PROTEASES .3. PAPAIN AND TRYPSIN

Studies of the structure-function relationships of papain and trypsin in relation to their proteolytic kinetic parameters, intrinsic CD spec tral properties, thermal stability and proteolytic patterns were perfo rmed in three different buffer systems: (1) standard buffer (20 mM bor ate, pH 8.1); (2) 5 % (v/v) ethanol (EtOH) in standard buffer, and (3) 5 % (v/v) acetonitrile (ACN) in standard buffer. Kinetic studies of t he hydrolysis of sodium caseinate by papain demonstrated a similar inc rease in K. (p > 0. 05) in both 5 % EtOH in standard buffer and 5 % AC N in standard buffer relative to standard buffer (from 0.68 mg/ml to 1 . 0 mg/ml and 1.1 mg/ml, respectively, p less-than-or-equal-to 0. 05). Relative to trypsin in standard buffer, the K(m) in organic solvents increased significantly (p less-than-or-equal-to 0.05) from 3. 7 mg/ml (standard buffer) to 5.1 mg/ml (5 % EtOH in standard buffer) and 4.5 mg/ml (5 % A CN in standard buffer). Despite its high K(m) values, the catalytic efficiencies (V(max)/K(m)) of papain (6.8, 5.9 and 5.7, res pectively in standard buffer, 5 % EtOH in standard buffer and 5 % ACN in standard buffer) were several orders of magnitude lower than those of trypsin (102, 68 and 74 in standard buffer, 5 % EtOH in standard bu ffer, and 5 % ACN in standard buffer, respectively). For both enzymes, changes in kinetic parameters generally corresponded with solvent-ind uced enzyme structural changes as evidenced by circular dichroism (CD) spectroscopy, with low K(m) corresponding to low ellipticity in the a romatic region of the near-UV CD spectra (240 320 nm) which may be ind icative of greater protein flexibility. Because the polarity of both 5 % EtOH and 5 % A CN solutions, as measured by E(T)(30), was the same, only a polarity effect of the me&a (i. e. , lack of sensitivity to th e nature of the solvent) on reaction kinetics was exhibited by papain; whereas, effects on reaction kinetics of trypsin arising from the int rinsic properties of the organic solvent systems (solvent effect) were also noted. Different hydrolytic patterns of Na-caseinate between pap ain and trypsin were demonstrated in SDS-PAGE peptide maps as differen ces in ban&ng patterns; however, no differences were observed in the p resence of the organic solvents. Differential scanning calorimetric st udies of papain and trypsin in the organic solvent systems showed dest abilization of papain in both 5 % EtOH in standard buffer and 5 % ACN in standard buffer relative to standard buffer, i. e. , lowered temper ature of denaturation (T(d)), while no destabilizing effects were obse rved for trypsin.