ALKALINE-INDUCED UNFOLDING AND SALT-INDUCED FOLDING OF PIG-HEART LACTATE-DEHYDROGENASE UNDER HIGH PH CONDITIONS

The alkaline-induced unfolding and the salt-induced folding of pig hea rt lactate dehydrogenase under high pH conditions have been followed b y fluorescence emission spectra and circular dichroism spectra. The re sults for alkaline-induced denaturation of lactate dehydrogenase show that at low ionic strength, increasing the pH value increased the exte nt of unfolding of the enzyme to the maximum ultimate unfolded conform ation at about pH 13.0. At pH 12.5, although the enzyme was completely inactivated, most of the ordered structure was retained. Even at pH 1 3.5, the apparently fully unfolded enzyme still retained some ordered secondary structure. Kinetic analysis showed that at high pH, the inac tivation rate constants of the enzyme are an order of magnitude faster than the unfolding rate constants at least. The above results are in accord with the suggestion by Tsou (Trends Biochem Sci 1986;11:427-429 and Science 1993;262:380-381) that the active site is usually more fl exible than the enzyme molecule. At pH 13.0, adding salt to the soluti on caused the relatively unfolded state of the denatured enzyme to cha nge into a compact conformational state by hydrophobic collapsing. The folded state induced by the salt bound ANS strongly, indicating the e xistence of an increased hydrophobic surface. The above results sugges t that the salt-induced folded state at high pH may be the folded inte rmediate which exists in the general protein folding and that the larg e residual ordered secondary structure might become folded during the salt-induced folding. (C) 1998 Elsevier Science B.V. All rights reserv ed.