Urea and Heat Unfolding of Cold-Adapted Atlantic Cod(Gadusmorhua) Trypsin and Bovine Trypsin

Abstract

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 unfolding at 25°C, the free energy change at zero concentration of urea (ΔG(H 2 O)) 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 (T m ) was 46(±1.4)°C for cod PMS-trypsin compared with 57(±2)°C for bovine PMS-trypsin. The standard free energy change (ΔG°) for reversible thermal unfolding of cod PMS-trypsin was 9(±1) kJ mol -1 compared with 19(±1) kJ mol -1 for bovine PMS-trypsin. Values for the enthalpy (ΔH m ), entropy (ΔS m ) and heat capacity (ΔC 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.