Limited proteolysis as a tool to study the kinetics of protein folding: conformational rearrangements in acid-dissociated lactic dehydrogenase as determined by pepsin digestion

Archives of Biochemistry and Biophysics
G ZettlmeisslR Jaenicke

Abstract

Noncovalent aggregation as a side reaction competing with the reconstitution of oligomeric enzymes is enhanced by slow conformational changes within the partially unfolded subunits. This has been shown for lactic dehydrogenase from pig muscle after acid dissociation [G. Zettlmeissl, R. Rudolph, and R. Jaenicke (1981) Eur. J. Biochem. 121, 169-175]. The present experiments confirm previous spectroscopic evidence (from circular dichroism) applying pepsin digestion and subsequent analysis of the fragments on sodium dodecyl sulfate-polyacrylamide gradient gels. The susceptibility of certain fragmentation sites toward pepsin digestion changes with increasing incubation at acid pH, in accordance with a slow M1 leads to M2 transition of the acid-dissociated monomers. Constant pulses of pepsin at varying times after transferring native enzyme to pH 2.3 yield distinct changes in the fragmentation pattern consisting of undigested monomers (Mr = 35,000) plus 12 fragments ranging from 31,000 to 5000. Short digestion of the M2 species at low concentrations of pepsin preferentially yields 25,000 and 10,500 fragments (molar ratio pepsin:lactic dehydrogenase = 1:24). The time-dependent decrease of monomers upon incubation in 0.1 M sodium phosp...Continue Reading

References

Apr 1, 1976·European Journal of Biochemistry·R Rudolph, R Jaenicke
May 1, 1975·Archives of Biochemistry and Biophysics·F Widmer, C Widmer
Jul 1, 1977·Proceedings of the National Academy of Sciences of the United States of America·W EventoffH H Kiltz
Jan 1, 1981·Advances in Protein Chemistry·D B Wetlaufer
Jul 1, 1982·European Journal of Biochemistry·U LillH Eggerer

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