PMID: 5380Jan 1, 1976

Electron paramagnetic resonance studies on spin-labelling of pepsin: effects of temperature, pH and urea on its conformation

International Journal of Peptide and Protein Research
H AoshimaH Hatano

Abstract

Pepsin was spin-labelled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidyl) bromoacetamide, possibly at the active site, at a beta-catboxyl group of a reactive aspartic acid. The spectrum of the spin-labelled pepsin showed that the spin probe was strongly immobilized (correlation time is greater than or equal to 10(-8) sec). Spin-labelled pepsin was thermally denatured at various temperatures and electron paramagnetic resonance (e.p.r.) spectra were taken at various times. Rates of denaturation estimated from the e.p.r. spectra at various temperatures showed that the enthalpy and entropy of thermal denaturation of spin-labelled pepsin at pH 3.5 were 48.0+/-4.9 kcal/mole and 214.7+/-14.5 e.u. respectively. Addition of conc. NaOH or 1 M acetate buffer at pH 6.0 sharpened e.p.r. spectra of the spin-labelled pepsin, indicating that the spin probe became mobilized by alkaline denaturation. Addition of urea caused unfolding of the protein which increased with the urea concentration, although only slight transition of conformational changes was observed in the e.p.r. spectra.

References

Oct 1, 1965·Proceedings of the National Academy of Sciences of the United States of America·T J StoneH M MCCONNELL
May 3, 1966·Biochemical and Biophysical Research Communications·B F ErlangerA G Cooper
May 20, 1965·Journal of the American Chemical Society·S I OHNISHI, H M MCCONNELL
Sep 20, 1938·The Journal of General Physiology·M L Anson

Citations

Related Concepts

Hydrogen-Ion Concentration
Pepsin 3
Protein Conformation
Spin Labels
Carmol

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