Urea and heat unfolding of cold-adapted Atlantic cod (Gadus morhua) trypsin and bovine trypsin

The reversible unfolding reactions for phenylmethylsulphonyl fluoride (PMSF)-modified trypsins 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 unfolding at 25 degrees C, the free energy change at zero concentration of urea (delta(G)(H2O)) for cod PMS-trypsin was 11(+/- 4.4) kJ mol-1 compared with 18(+/- 1.14) kJ mol-1 for bovine PMS-trypsin, while the mid-point 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 unfolding, the mid point temperature of the thermal unfolding curve (Tm) was 46(+/-1.4) degrees C for cod PMS-trypsin compared with 57(+/-2) degrees 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 19(+/-1) kJ mole-1 for bovine PMS-trypsin. Values for the enthalpy (delta(H)m), entropy (delta(S)m) and heat capacity (delta(C)p) for heat unfolding are compared. Results from urea and thermal unfolding studies show that cod PMS-trypsin has a significantly lower conformational stability than bovine PMS-trypsin.