UREA AND HEAT UNFOLDING OF COLD-ADAPTED ATLANTIC COD (GADUS-MORHUA) TRYPSIN AND BOVINE TRYPSIN

The reversible unfolding reactions for phenylmethylsulphonyl fluoride (PMSF)-modified trypins from Atlantic cod (cod PMS-trypsin) and cattle (bovine PMS-trypsin) were monitored by fluorescence spectrophotometry as a function of urea concentration and temperature. For urea unfoldi ng at 25 degrees C, the free energy change at zero concentration of ur ea (Delta G(H2O)) for cod PMS-trypsin was 11(+/-44) kJ mol(-1) compare d with 18(+/-1.14) kJ mol(-1) for bovine PMS-trypsin, while the mid-po int concentration for urea unfolding curve ([urea](1/2)) was 3.0(+/-0. 57)M and 4.1(+/-0.16) M, respectively. From studies of enzyme heat unf olding, the mid point temperature of the thermal unfolding curve (T-m) was 46(+/-1.4)degrees C for cod PMS-trypsin compared with 57(+/-2)deg rees C for bovine PMS-trypsin. The standard free energy change (Delta G degrees) for reversible thermal unfolding of cod PMS-trypsin was 9(/-1) kJ mol(-1) compared with 9(+/-1) kJ mol(-1) for bovine PMS-trypsi n. Values for the enthalpy (Delta H-m), entropy (Delta S-m) and heat c apacity (Delta C-p) for heat unfolding are compared. Results from urea and thermal unfolding studies show that cod PMS-trypsin has a signifi cantly lower conformational stability than bovine PMS-trypsin.