Acrylamide Quenching of apo- and holo-α-Lactalbumin in Guanidine Hydrochloride

doi:10.1006/bbrc.2000.2346

Biochemical and Biophysical Research Communications

Volume 269, Issue 3, 24 March 2000, Pages 709-712

https://doi.org/10.1006/bbrc.2000.2346 Get rights and content

Abstract

We have examined the fluorescence properties and acrylamide quenching of calcium-loaded (holo) and calcium-depleted (apo) α-lactalbumin (α-LA) as a function of guanidine hydrochloride (GDN/HCl) concentration. The spectral changes accompanying increasing GDN/HCl are consistent with protein unfolding and a release of internal fluorescence quenching, which occurs among the three tryptophan residues located in the region of the so-called “tertiary fold.” Values for the intrinsic fluorescence emission, the wavelength maximum 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-α-LA increases fourfold to its maximum, in the transition from the native state to protein in 1.5 M GDN/HCl. The dynamic quench constant for holo-α-LA remains unchanged until exposed to 2.5 M GDN/HCl, but increases by threefold with addition denaturant to 4 M GDN/HCl. The static quench constant of the apo-protein in the native solvent, approximately 0.2 M⁻¹, declines to zero in 1 M denaturant, where the molten globule folding intermediate is most populated. A more protracted denaturant-dependent decline in the static quench constant occurs for the holo-protein. Sharp increase in the static quenching occurs for apo-α-LA and holo-α-LA above 1.5 M GDN/HCl and 3.5 M GDN/HCl, respectively. The results for apo-α-LA in dilute GDN/HCl suggest that acrylamide can penetrate the protein molecule (as judged by the collision quenching) but is unable to form a stable complex within the quenching domain for the tryptophans (as judged by the absence of the static quench constant). It seems reasonable to suggest that the protein folding intermediate 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 complex with acrylamide.

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Malate dehydrogenase enzyme plays an essential role in energy metabolism in all eukaryotes. It catalyzes the interconversion of malate to oxaloacetate, producing NAD⁺/NADH. In the current study, we monitored the urea-induced conformational changes in Fasciola gigantica malate dehydrogenase (FgMDH) structure using multiple spectroscopic techniques combined with all-atom molecular dynamics (MD) simulations. FgMDH showed a two-state cooperative unfolding revealed by intrinsic tryptophan (Trp) fluorescence and CD spectroscopy; however, their non-superimposable curves indicate a partially folded intermediate state formation. The study showed that the unfolding of tertiary structures preceded the secondary structure unfolding. The binding of the fluorescence probe 1-anilino-8-naphthalene sulfonate (ANS) at 1.5 M urea confirmed stabilization of the intermediate state. The free energy of stabilization determined from Trp fluorescence and CD spectroscopy were 11.4 and 13.2 kcal.mol⁻¹, respectively. The loss of enzymatic activity was observed before any significant loss in structural components. This suggested that low urea concentrations induce minor structural changes in the substrate or cofactor binding site leading to activity loss. Quenching of fluorescence intensity also provides information on unfolding and activity loss. MD simulations confirmed that FgMDH formed an intermediate state at 1.5 M urea at 300 K, which destabilized at higher urea concentration and temperature due to disrupted hydrogen-bond networks and hydrophobic interactions. We conclude that a partially folded metastable state is stabilized during the unfolding of cytosolic FgMDH. A strong correlation was observed between the data obtained from in-vitro and in-silico approaches.
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The altered fluorescence intensity in the presence of KI and acrylamide aids in determining the location of Trp residues in the protein as both have specific quenching behavior [53]. KI is anionic and provides information about the surface Trp residue of a protein, while acrylamide is neutral and can predict the sensitive changes in the conformation of buried Trp residues [54–58]. In order to elucidate the conformational changes due to R451A mutation, we studied the changes in the fluorescence intensity of both proteins with increasing concentrations of the quencher.
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When a Trp residue is buried in the folded protein core, the quencher has limited accessibility to Trp residues, as indicated by the maximum fluorescence emission intensity [41]. Because of unfolding, the buried Trp residues get exposed to the solvent, and thus, the encounter distance required by the quencher to deactivate the excited fluorophore is easily achieved [42]. A decrease in the Trp emission intensity was observed upon acrylamide binding to both proteins in the presence and absence of urea.
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This will be investigated in a future study. The emission maxima of ∼343–347 nm observed in areas 2 and 3 of Fig. 2A, detected under the studied conditions where the majority of the protein was converted to aggregates, are between those reported for α-LA molten globular states under acidic conditions (∼342 nm) (Kelkar, Chaudhuri, Haldar, & Chattopadhyay, 2010) and chemically denatured states (∼350–355 nm) (France & Grossman, 2000; Hanssens, Houthuys, Herreman, & Cauwelaert, 1980), respectively. This suggests that the tryptophan residues are largely exposed to the solvent in α-LA aggregates, and are likely to be more exposed in the aggregate structure than in the molten globular state (Kelkar et al., 2010; Lala & Kaul, 1992).
α-Lactalbumin (α-LA) is a key commercial whey protein for nutritional purposes. The holo protein (calcium saturated) is considered the most heat stable whey protein, capable of refolding from unfolded states under many conditions. This is due to the absence of free thiols (cysteine residues) that are typically involved in thermal aggregation and thiol–disulphide exchange reactions of other whey proteins. Heating (0–120 min at 90 °C, pH 7.0) holo α-LA generates free thiols through thermal cleavage of disulphide bonds, resulting in aggregates comprising unfolded α-LA species. The addition of free cysteine promotes the formation of soluble aggregates, effectively decreasing the holding time required to reach a particular aggregate size in a dose-dependent manner (0.35–1.4 mm cysteine). Excess cysteine (≥14 mm) causes a destabilisation of α-LA, shown by decreased denaturation temperature and gel formation. These data indicate that low doses of cysteine can be used to control α-LA aggregation.
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Acrylamide is a polar but uncharged quencher. It is assumed that acrylamide can penetrate into the interior of proteins through diffusive processes enabled by small fluctuations in the polypeptide conformation [34]. As a consequence, acrylamide has accessibility to Trp-214 which is buried in hydrophobic pocket.
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The aim of the present work was to estimate how non-enzymatic glycation of human serum albumin altered its tertiary structure using fluorescence technique. We compared glycated human serum albumin by glucose (gHSA_GLC) with HSA glycated by fructose (gHSA_FRC). We focused on presenting the differences between gHSA_FRC and nonglycated (HSA) albumin used acrylamide (Ac), potassium iodide (KI) and 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS). Changes of the microenvironment around the tryptophan residue (Trp-214) of non-glycated and glycated proteins was investigated by the red-edge excitation shift method. Effect of glycation on ligand binding was examined by the binding of phenylbutazone (PHB) and ketoprofen (KP), which a primary high affinity binding site in serum albumin is subdomain IIA and IIIA, respectively.
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Regular ArticleAcrylamide Quenching of apo- and holo-α-Lactalbumin in Guanidine Hydrochloride

Abstract

Biophys. Chem.

Biophys. Chem.

Biophys. Chem.

Anal. Biochem.

FASEB J.

J. Biochem.

Regular Article
Acrylamide Quenching of apo- and holo-α-Lactalbumin in Guanidine Hydrochloride