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Volume 17, Issue 2-3, Pages 183-193

Folding mechanism of canine milk lysozyme studied by circular dichroism and fluorescence spectroscopy

Masaharu Nakao,1 Munehito Arai,1,3 Takumi Koshiba,2,4 Katsutoshi Nitta,2 and Kunihiro Kuwajima1

1Department of Physics, Graduate School of Science, University of Tokyo, 7‒3‒1 Hongo, Bunkyo-ku, Tokyo -113-0033, Japan
2Division of Biological Sciences, Graduate School of Science, Hokkaido University, N10 W8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan
3Institute of Molecular and Cell Biology, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki-305-8566, Japan
4Division of Biology, MC 156-29, California Institute of Technology, 1200 E. California Blvd., Pasadena, CA 91125, USA

Copyright © 2003 Hindawi Publishing Corporation. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


We have studied the guanidine hydrochloride‒induced equilibrium unfolding and the kinetics of refolding of canine milk lysozyme by circular dichroism and fluorescence spectroscopy. The thermodynamic analysis of the equilibrium unfolding measured by circular dichroism and fluorescence has shown that unfolding is represented by a three‒state mechanism and that the intermediate state of canine milk lysozyme is remarkably more stable than the intermediates observed in other lysozyme and α-lactalbumin. In the kinetic refolding of this protein, there are at least two kinetic intermediates; a burst=phase intermediate accumulated within the dead time (4 ms) of the measurement and an intermediate that has been observed during the kinetics with a rate constant of 10–20 s–1 after the burst phase. This result is apparently in contrast with those previously observed in the kinetic refolding of α‒lactalbumin and equine lysozyme that show only the burst‒phase intermediate. The relationship between the extraordinarily stable equilibrium molten globule and the kinetic folding intermediates will be discussed.