Effect of Heat Treatment on Bovine β-Lactoglobulin A, B, and C Explored Using Thiol Availability and Fluorescence

Abstract

Dilute solutions of beta-lactoglobulin (beta-Lg) A, B, and C were heated at temperatures between about 40 and 94 degrees C for 10 min, cooled, and analyzed using Trp fluorescence and extrinsic fluorescence spectra of the probe 1,8-anilinonaphthalene sulfonate (ANS). Thiol availabilities using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were determined using a separate set of samples. The normalized ANS fluorescence emission intensity and the thiol availability results showed a 1:1 relationship with the loss of nativelike but not SDS-monomeric protein, as determined by PAGE analysis. The normalized Trp emission intensity results did not show a comparable 1:1 relationship with the loss of nativelike protein, indicating that the Trp intensity arose from consequential disulfide bond reorganization and not the initial unfolding reaction. The results were also analyzed in terms of two-state models, and the midpoint temperatures (T(mid)) for the proteins were generally beta-Lg C > beta-Lg A > beta-Lg B, and the slopes at the midpoint temperatures for the A variant were generally less than those for the B and C variants indicating that beta-Lg A may denature by a different mechanism from that of beta-Lg B or beta-Lg C. The T(mid) parameters derived from the ANS fluorescence intensity results were similar to those for thiol availability and both were lower than the T(mid) values for Trp emission intensity showing that creation of an ANS binding site on a beta-Lg molecule was linked to the irreversible exposure of a thiol group and the loss of native beta-Lg but preceded the decrease in Trp(61) fluorescence quenching. These results for the differences between the behavior of the A and B or the C variants involved the creation of a destabilizing cavity by the Val(118)Ala (A --> B) substitution and the changed charge distribution within the CD loop caused by the Asp(64)Gly (A --> B) substitution.

Pulsed Electric Field-Induced Structural Modification of Whey Protein Isolate

“…This study confirmed that intrinsic fluorescence intensity of WPI increased and the red shifts in the emission spectra are apparently due to the unfolding of the whey protein resulting during PEF treatment. This result is consistent with the results observed by Manderson et al (1998) and Moro et al (2001) for thermal processing. Increases in the intrinsic fluorescence intensities and red shifts observed after PEF treatments indicated changes in the polarity of tryptophan residues microenvironment in whey proteins from a less polar to a more polar environment.…”

“…The intrinsic tryptophan fluorescence intensities of WPI increased significantly (P≤0.05) with the increase in electric field intensity ranging from 12, 16, and 20 kV cm −1 . Manderson et al (1998) established that the emission spectra of bovine β-lactoglobulin appeared as red shift when β-lactoglobulin was heated to 85°C. Moro et al (2001) used different techniques to study surface hydrophobicity of whey protein concentrate heated at 85°C.…”

pH-dependent structures and properties of casein micelles

“…Matulis and Lovrien [36] reported that electrostatic interactions are the predominant interactions between ANS and bovine serum albumin, and hydrophobic affinity is a minor interaction between them, which is in agreement with our results.…”

Purification and physicochemical characterization of ovine β‐lactoglobulin and α‐lactalbumin

“…The elution was carried out at a flow rate of 0.8 mL/min with 30 mM sodium phosphate, pH 6.7, or 30 mM glycine, pH 2.5. at 268 nm originating from its phenylalanyl residues and two characteristic deep minima at 287 and 297 nm arising from tryptophanyl residues, especially Trp19. At pH 6.7, the intensity of the two minima, slightly shifted at 286 and 294 nm, increased; a more significant increase of the intensity at 268 nm was observed, which agrees well with the results of Manderson et al [23]. The main b-lactoglobulin fluorescent residue is Trp 19 and is responsible for 80% of the protein fluorescence emission [24].…”