EFFECT OF 2 POLAR ORGANIC-AQUEOUS SOLVENT SYSTEMS ON THE STRUCTURE-FUNCTION RELATIONSHIP OF PROTEASES .1. PEPSIN

The effect of various organic-aqueous solvent systems on the kinetic p arameters, intrinsic spectral properties, thermal stability, and prote olytic patterns of porcine pepsin were studied. Two substrates, Z-His- Phe (-NO2)-Phe-OMe and sodium (Na-) caseinate, were chosen. Three diff erent buffer compositions were used in the investigation: (1) the stan dard buffers (20 mM formate buffers, pH 2. 1 and 10 mM phosphate buffe r, pH 5. 7); (2) 5 % (v/v) ethanol (EtOH) in the standard buffers; (3) 5 % (v/v) acetonitrile (ACN) in the standard buffer. Relative to peps in in formate buffer (pH 2.1), the K(m) for Z-His-Phe (-NO2)-Phe-OMe i n 5 % EtOH increased from 0.57 mM to 1.03 mM (p less-than-or-equal-to 0. 05), while no significant difference was observed in 5 % ACN (p > 0 . 05). The solvent-induced decrease in V(max) was much larger in 5 % A CN than in 5 % EtOH, from 48. 0 nmoles/min. mg to 12.3 nmoles/min. mg (p less-than-or-equal-to 0. 05) and 35. 0 nmoles/min. mg (p > 0. 05), respectively, as compared to standard buffer. Relative to pepsin in 10 mM phosphate buffer (pH 5.7), the K(m) for Na-caseinate in both 5 % E tOH and 5 % ACN increased from 4.1 mg/ml to 7.8 mg/ml and 6 2 mg/ml (p less-than-or-equal-to 0. 05), respectively while only 5 % ACN caused a significant decrease in V(max), compared with standard buffer, from 11.8 mumoles/min. mg to 7.6 mumoles/min. mg (p less-than-or-equal-to 0 . 05). Changes in kinetic parameters generally corresponded with solve nt-induced structural changes as evidenced by circular dichroism (CD) spectroscopy, a low K(m) corresponding to low ellipticity in the near- UV CD spectra (240-320 nm) possibly indicative of a greater protein fl exibility. The above results were attributed to differences in propert ies other than polarity of the solvent systems since the polarity of b oth 5 % EtOH and 5 % ACN solutions, as measured by E(T)(30) scale, was the same. Differential scanning calorimetric studies of pepsin in the different solvent systems showed destabilization of the enzyme in the organic solutions relative to standard buffer, i. e., lowered tempera ture of denaturation. Altered specificity of pepsin for Na-caseinate h ydrolysis in the presence of the various organic solvents was demonstr ated in SDS-PAGE peptide maps as differences in the banding patterns.