INTERACTION OF GUANIDINE-HYDROCHLORIDE AND GUANIDINE THIOCYANATE WITH WHEAT-GERM LIPASE

Effect of two classical and potent denaturants, guanidine hydrochloride (GuHCl) and guanidine thiocyanate (GuHSCN) on purified wheat germ lipase has been studied. Lipase was found to be active only up to 5 M GuHCl and 1.5 M GuHSCN. The extent of interaction was determined by the measurement of apparent partial specific volume of the enzyme in presence of these two denaturants. While the preferential interaction parameter (xi(3)) has values of 0.08+/-0.02 and 0.14+/-0.03 g/g, the interaction parameter (delta m(3)/delta m(2))(Tau,mu 1,mu 3) has values of 35+/-9 and 50+/-10 mole/mole for GuHCl and GuHSCN, respectively. The number of denaturant molecules bound to the enzyme, A(3), obtained experimentally were 0.486+/-0.020 and 0.348+/-0.020 g/g and the calculated values were 0.459+/-0.023 and 0.567+/-0.030 g/g for 6 M GuHCl and 3 M GuHSCN, respectively. The volume change occurring upon denaturation results in -420+/-42 and -462+84 ml/mole in 6 M GuHCl and 3 M GuHSCN, respectively. The denaturation is accompanied by exposure of hydrophobic groups to the bulk solvent as confirmed by fluorescence emission measurements of the enzyme. The T-m measurements indicated a control value of 56+/- 1 degrees C. In presence of 6 M GuHCl/3 M GuHSCN, the value was 42+/-1 degrees C. These results explain the retention of lipase activity even at 5 M GuHCl from a mechanistic point of view.