p-Chloromercuribenzoate-induced inactivation and partial unfolding of porcine heart lactate dehydrogenase.
Purified porcine heart lactate dehydrogenase was inactivated and partially unfolded with p-chloromercuribenzoate (pCMB). With the increase of pCMB/enzyme ratio the enzyme was gradually inhibited till almost completely inactivated at the pCMB/enzyme ratio of 20 : 1. Native polyacrylamide gel electrophoresis showed that with the increase of pCMB/enzyme ratio the bands of native enzyme decreased till completely vanished. Meanwhile inactive multiple bands emerged and became thicker, which implied that lactate dehydrogenase became loose. The conformational changes of the enzyme molecule modified with pCMB were followed using fluorescence emission, ultraviolet difference, and circular dichroism (CD) spectra. Increasing pCMB concentration resulted in the decrease of fluorescence emission intensity. The ultraviolet difference spectra of the enzyme modified with pCMB exhibited an increasing absorbance in the vicinity of 240 nm with the increasing concentration of the inhibitor. The changes of the fluorescence and ultraviolet difference spectra reflected the conformational changes of the enzyme. The CD spectrum changes of the enzyme showed that its secondary structure changed as well. These results suggest that pCMB not only inhibits this enzyme but also influences its conformation (partial unfolding).
Zheng
Y-B
YB
Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, People's Republic of China.
Chen
B-Y
BY
Wang
X-C
XC
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology*; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D013057:Spectrum Analysis; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013552:Swine; D020245:p-Chloromercuribenzoic Acid / Q000494:pharmacology*
D013439:Sulfhydryl Reagents; D020245:p-Chloromercuribenzoic Acid; D007770:L-Lactate Dehydrogenase