INTERACTION OF GUANIDINE-HYDROCHLORIDE AND GUANIDINE THIOCYANATE WITHWHEAT-GERM LIPASE

Effect of two classical and potent denaturants, guanidine hydrochlorid e (GuHCl) and guanidine thiocyanate (GuHSCN) on purified wheat germ li pase has been studied. Lipase was found to be active only up to 5 M Gu HCl and 1.5 M GuHSCN. The extent of interaction was determined by the measurement of apparent partial specific volume of the enzyme in prese nce of these two denaturants. While the preferential interaction param eter (xi(3)) has values of 0.08+/-0.02 and 0.14+/-0.03 g/g, the intera ction parameter (delta m(3)/delta m(2))(Tau,mu 1,mu 3) has values of 3 5+/-9 and 50+/-10 mole/mole for GuHCl and GuHSCN, respectively. The nu mber of denaturant molecules bound to the enzyme, A(3), obtained exper imentally 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 re sults in -420+/-42 and -462+84 ml/mole in 6 M GuHCl and 3 M GuHSCN, re spectively. The denaturation is accompanied by exposure of hydrophobic groups to the bulk solvent as confirmed by fluorescence emission meas urements 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 ac tivity even at 5 M GuHCl from a mechanistic point of view.