Acrylamide quenching of apo- and holo-alpha-lactalbumin in guanidine hydrochloride

We have examined the fluorescence properties and acrylamide quenching of ca lcium-loaded (holo) and calcium-depleted (apo) a-lactalbumin ((U-LA) as a f unction of guanidine hydrochloride (GDN/HCl) concentration. The spectral ch anges accompanying increasing GDN/HCl are consistent with protein unfolding and a release of internal fluorescence quenching, which occurs among the t hree tryptophan residues located in the region of the so-called "tertiary f old." Values for the intrinsic fluorescence emission, the wavelength maximu m of the emission, the Stern/Volmer dynamic quench constant, and the static quench constant are consistent with a significant stabilization effect by calcium against protein unfolding. The dynamic quench constant of apo-alpha -LA increases fourfold to its maximum, in the transition from the native st ate to protein in 1.5 M GDN/HCl, The dynamic quench constant for holo-alpha -LA remains unchanged until exposed to 2.5 M GDN/HCl, but increases by thre efold with addition denaturant to 4 M GDN/HCI. The static quench constant o f the ape-protein in the native solvent, approximately 0.2 M-1, declines to zero in 1 M denaturant, where the molten globule folding intermediate is m ost populated. A more protracted denaturant-dependent decline in the static quench constant occurs for the holoprotein. Sharp increase in the static q uenching occurs for ape-a-LA and holo-cu-LA above 1.5 M GDN/HCI and 3.5 M G DN/HCl, respectively. The results for apo-alpha-LA in dilute GDN/HCl sugges t that acrylamide can penetrate the protein molecule las judged by the coll ision quenching) but is unable to form a stable complex within the quenchin g domain for the tryptophans (as judged by the absence of the static quench constant). It seems reasonable to suggest that the protein folding interme diate which occurs in dilute denaturant represents a structure in which the tryptophans are, on average, more accessible to collisional quenching but sufficiently compact to prevent formation of a stable, dark equilibrium com plex with acrylamide. (C) 2000 Academic Press.