The detection of kinetic intermediates during the unfolding of lipoxygenase-1 by urea or guanidine hydrochloride.

Sonati, Srinivasulu and Appu Rao, A. G. (1996) The detection of kinetic intermediates during the unfolding of lipoxygenase-1 by urea or guanidine hydrochloride. Biochimica et Biophysica Acta, 1296. pp. 115-120. ISSN 0167-4838

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Abstract

The unfolding of lipoxygenase-1 by urea and guanidine hydrochloride has been followed at the optimum pH of enzyme activity. The unfolding of lipoxygenase-I by urea or guanidine hydrochloride was characterized by equilibrium transition curves for different parameters like (i) enzyme activity, (ii) change in ellipticity values at 222 nm, and (iii) relative fluorescence intensity at 332 nm could not be superimposed. The transition curves displayed more than one plateau region suggesting the presence of stable intermediates during unfolding. At urea concentratJ~ons less than 1 M there was no significant loss in activity although loss in secondary structure was = 20%. At 4.0 M urea concentration there was complete loss of activity with a midpoint concentration of 2.5 M urea. The loss in secondary structure was biphasic. The first transition had a midpoint concentration of 1.2 M, while the second transition which was complete at 8.0 M urea had a midpoint concentration of 3.5 M urea. The changes in relative fluorescence intensity and shift in emission maximum were complete at 8.0 M urea. The Stem-Volmer constant for acrylamide and potassium iodide did not change at urea concentrations less than 4 M and then at higher concentrations increased. The reactivity of sulfhydryl groups to Ellman's reagent increased during the course of unfolding. The kinetics of unfolding supported the presence of stable intermediates during unfolding. The unfolding was irreversible and complex because of the multidomain nature. The apparent irreversibility could be related to aggregation during unfolding which precluded the determination of thermodynamic parameters.

Item Type:	Article
Additional Information:	Copyright of this article belongs to Elsevier Ltd.
Uncontrolled Keywords:	Kinetics; Irreversibilil:y; Aggregation; Stability; Unfolding
Subjects:	500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 29 Protein Chemistry
Divisions:	Protein Chemistry and Technology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	26 Jun 2007 06:12
Last Modified:	28 Dec 2011 09:28
URI:	http://ir.cftri.res.in/id/eprint/1262

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