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 heart lactate dehydrogenase under high pH conditions have been followed by fluorescence emission spectra and circular dichroism spectra. The results for alkaline-induced denaturation of lactate dehydrogenase show that at low ionic strength, increasing the pH value increased the extent of unfolding of the enzyme to the maximum ultimate unfolded conformation 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 13.5, the apparently fully unfolded enzyme still retained some ordered secondary structure. Kinetic analysis showed that at high pH, the inactivation 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 flexible than the enzyme molecule. At pH 13.0, adding salt to the solution caused the relatively unfolded state of the denatured enzyme to change into a compact conformational state by hydrophobic collapsing. The folded state induced by the salt bound ANS strongly, indicating the existence of an increased hydrophobic surface. The above results suggest that the salt-induced folded state at high pH may be the folded intermediate which exists in the general protein folding and that the large residual ordered secondary structure might become folded during the salt-induced folding.
Bai
J H
JH
Department of Biological Science and Biotechnology, Tsinghua University, Beijing, China.
Wang
H J
HJ
Zhou
H M
HM
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology*; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013552:Swine
D000817:Anilino Naphthalenesulfonates; C027132:1-anilino-8-naphthalenesulfonate; D011189:Potassium Chloride; D007770:L-Lactate Dehydrogenase