THE DETECTION OF KINETIC INTERMEDIATES DURING THE UNFOLDING OF LIPOXYGENASE-1 BY UREA OR GUANIDINE-HYDROCHLORIDE

The unfolding of lipoxygenase-l by urea and guanidine hydrochloride ha s been followed at the optimum pH of enzyme activity. The unfolding of lipoxygenase-l by urea or guanidine hydrochloride was characterized b y equilibrium transition curves for different parameters like (i) enzy me activity, (ii) change in ellipticity values at 222 nm, and (iii) re lative fluorescence intensity at 332 nm could not be superimposed. The transition curves displayed more than one plateau region suggesting t he presence of stable intermediates during unfolding. At urea concentr ations less than 1 M there was no significant loss in activity althoug h loss in secondary structure was approximate to 20%. At 4.0 M urea co ncentration there was complete loss of activity with a midpoint concen tration of 2.5 M urea. The loss in secondary structure was biphasic. T he first transition had a midpoint concentration of 1.2 M, while the s econd transition which was complete at 8.0 M urea had a midpoint conce ntration of 3.5 M urea. The changes in relative fluorescence intensity and shift in emission maximum were complete at 8.0 M urea. The Stern- Volmer constant for acrylamide and potassium iodide did not change at urea concentrations less than 4 M and then at higher concentrations in creased. The reactivity of sulfhydryl groups to Ellman's reagent incre ased during the course of unfolding. The kinetics of unfolding support ed the presence of stable intermediates during unfolding. The unfoldin g was irreversible and complex because of the multidomain nature. The apparent irreversibility could be related to aggregation during unfold ing which precluded the determination of thermodynamic parameters.