Inactivation and conformational changes of lactate dehydrogenase from porcine heart in sodium dodecyl sulfate solutions.
The inactivation and conformational changes of porcine heart lactate dehydrogenase (LDH) have been studied in sodium dodecyl sulfate (SDS) solutions. Increasing SDS concentration led to a quick and concentration-dependent inhibition of the enzyme, with complete inactivation within 5 min in the presence of 1.0 mM SDS. Meanwhile, fluorescence emission and circular dichroism spectra were used to follow the conformational changes of the enzyme during this process, concurrently showing that SDS less than 1.0 mM induced only limited conformational changes to LDH. The above results are in accordance with the suggestion by Tsou (Trends Biochem. Sci. 11 (1986) 427; Science 262 (1993) 380) that the active site usually be more flexible than the enzyme molecule as a whole. Furthermore, the results of polyacrylamide gel electrophoresis (PAGE) implied that unfolding intermediates were presented in the above process. When the SDS concentration used to treat LDH was increased, the bands of native enzyme on native PAGE faded and finally almost disappeared. Meanwhile, multiple bands with lower mobility but no activity emerged behind and enhanced correspondingly. Fast protein liquid chromatography indicated that dissociation occurred during the course of denaturation. The reasons for the above phenomena have been discussed. It was suggested that SDS, binding to LDH to form different LDH-SDS complexes, conferred an array of different unfolding states over the enzyme, and in turn resulted in the formation of the multiple bands on the native PAGE.
Zheng
Yan-bin
YB
Department of Biological Science and Biotechnology, Tsinghua University, 100084, Beijing, China.
Meng
Fan-guo
FG
Chen
Bao-yu
BY
Wang
Xi-cheng
XC
D000818:Animals; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D009206:Myocardium / Q000201:enzymology*; D011487:Protein Conformation; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry* / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013552:Swine; D013997:Time Factors
D012967:Sodium Dodecyl Sulfate; D007770:L-Lactate Dehydrogenase