On the Dissociation of Bovine ,b-Lactoglobulins A, B, and C Near Ph 7

McKenzie, HA; Sawyer, W. M.

doi:10.1071/bi9720949

Cited by 10 publications

(6 citation statements)

References 7 publications

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“…The higher intensity of the 1636 cm -1 band in the spectra of the B variant could potentially suggest that this variant had a higher dimeric content than the A variant. b-lg was earlier reported to form dimers with dissociation constants in the micromolar range at pH y7 and stability constants in the order b-lg CAb-lg B > b-lg A (Timasheff & Townend, 1961;McKenzie & Sawyer, 1972;Thresher & Hill, 1997). The results reported here would agree with these findings.…”

Section: Discussionmentioning

confidence: 99%

Thermal stability of β-lactoglobulins A and B: effect of SDS, urea, cysteine and N-ethylmaleimide

Boye

Ching

Ismail

2004

Journal of Dairy Research

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Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) were used to monitor changes in the secondary structure and thermal stability of β-lactoglobulin A and B in the presence of sodium dodecyl sulphate (SDS), N-ethylmaleimide (NEM), urea and cysteine. An increase in the thermal stabilities of both proteins was noted in the presence of 10 mM-SDS. In the presence of 50 mM-SDS, there was extensive denaturation of both variants. In general, the β-strand/β-sheet regions in the secondary structure of both variants were very susceptible to denaturation by SDS and cysteine, suggesting that these regions may be held by hydrophobic and disulphide bonds. At ambient temperature and physiological pH, a notable difference was observed in the 1636 and 1627 cm−1 regions of the FTIR spectra of the two β-lg variants. The results suggest possible differences in the nature of the β-sheet/β-strand distribution/content of the two proteins. Urea and NEM at a concentration of 50 mM, had little effect on the secondary structure and denaturation of both variants. New findings are presented which further indicate that although the β-lg B variant showed greater thermal stability than the A variant in all the cases studied, its denaturation temperature and secondary structure were affected to a greater extent by the protein perturbants than β-lg A.

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Section: Discussionmentioning

confidence: 99%

Thermal stability of β-lactoglobulins A and B: effect of SDS, urea, cysteine and N-ethylmaleimide

Boye

Ching

Ismail

2004

Journal of Dairy Research

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“…Investigators have determined the dimer dissociation constant (K D 2-1 ) using a variety of techniques, including analytical ultracentrifugation (AUC) (15,16,37,(39)(40)(41)(42)(43)(44)(45)(46)(47), isothermal titration calorimetry (48,49), small-angle x-ray scattering (SAXS) (50), and light scattering (16,31,50,51). For bLg A and B, the experimentally determined K D 2-1 values fall within the 1-300 mM range at an ionic strength of~100 mM and over a wide pH range either side of the isoelectric point of~5.2 (16,17,52,53) (see Table S1 in the Supporting Material).…”

Section: Introductionmentioning

confidence: 99%

Bovine β-Lactoglobulin Is Dimeric Under Imitative Physiological Conditions: Dissociation Equilibrium and Rate Constants over the pH Range of 2.5–7.5

Mercadante

Melton

Norris

et al. 2012

Biophysical Journal

144

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The oligomerization of β-lactoglobulin (βLg) has been studied extensively, but with somewhat contradictory results. Using analytical ultracentrifugation in both sedimentation equilibrium and sedimentation velocity modes, we studied the oligomerization of βLg variants A and B over a pH range of 2.5-7.5 in 100 mM NaCl at 25°C. For the first time, to our knowledge, we were able to estimate rate constants (k(off)) for βLg dimer dissociation. At pH 2.5 k(off) is low (0.008 and 0.009 s(-1)), but at higher pH (6.5 and 7.5) k(off) is considerably greater (>0.1 s(-1)). We analyzed the sedimentation velocity data using the van Holde-Weischet method, and the results were consistent with a monomer-dimer reversible self-association at pH 2.5, 3.5, 6.5, and 7.5. Dimer dissociation constants K(D)(2-1) fell close to or within the protein concentration range of ∼5 to ∼45 μM, and at ∼45 μM the dimer predominated. No species larger than the dimer could be detected. The K(D)(2-1) increased as |pH-pI| increased, indicating that the hydrophobic effect is the major factor stabilizing the dimer, and suggesting that, especially at low pH, electrostatic repulsion destabilizes the dimer. Therefore, through Poisson-Boltzmann calculations, we determined the electrostatic dimerization energy and the ionic charge distribution as a function of ionic strength at pH above (pH 7.5) and below (pH 2.5) the isoelectric point (pI∼5.3). We propose a mechanism for dimer stabilization whereby the added ionic species screen and neutralize charges in the vicinity of the dimer interface. The electrostatic forces of the ion cloud surrounding βLg play a key role in the thermodynamics and kinetics of dimer association/dissociation.

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“…These variants of bovine BLG have different self-association properties~Hill et al, 1996!. Variant A forms dimers and then octamers under increasingly acidic conditions, whereas variants B and C do not form octamers~Timasheff & Townend, 1961;Timasheff, 1964;Pessen et al, 1985!, but do form dimers, with dissociation constants in the micromolar range at pH ; 7 and stability constants in the order C Ͼ Ͼ B Ͼ A~Timasheff & Townend, 1961;McKenzie, 1971;McKenzie & Sawyer, 1972;Thresher et al, 1994;Hill et al, 1996;Thresher & Hill, 1997!. Both bovine BLG variants A and B undergo pH-dependent conformational changes in the range pH 6.5 to pH 7.5, the so called N ? R or Tanford transition~Tanford et al, 1959;Hambling et al, 1994!.…”

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confidence: 99%

Functional implications of structural differences between variants A and B of bovine β‐lactoglobulin

Qin

Bewley

Creamer³

et al. 1999

Protein Science

134

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The structure of the trigonal crystal form of bovine b-lactoglobulin variant B at pH 7.1 has been determined by X-ray diffraction methods at a resolution of 2.22 Å and refined to values for R and R free of 0.239 and 0.286, respectively. By comparison with the structure of the trigonal crystal form of bovine b-lactoglobulin variant A at pH 7.1, which was determined previously @Qin BY et al., 1998, Biochemistry 37:14014-14023#, the structural consequences of the sequence differences D64G and V118A of variants A and B, respectively, have been investigated. Only minor differences in the core calyx structure occur. In the vicinity of the mutation site D64G on loop CD~residues 61-67!, there are small changes in main-chain conformation, whereas the substitution V118A on b-strand H is unaccompanied by changes in the surrounding structure, thereby creating a void volume and weakened hydrophobic interactions with a consequent loss of thermal stability relative to variant A. A conformational difference is found for the loop EF, implicated in the pH-dependent conformational change known as the Tanford transition, but it is not clear whether this reflects differences intrinsic to the variants in solution or differences in crystallization.Keywords: bovine b-lactoglobulin; crystal structure; genetic variants; hydrophobic stabilization Bovine b-lactoglobulin~BLG!, the major whey protein of cow's milk at a concentration of 0.3 g0100 mL~Bell & McKenzie, 1964!, was first isolated by Palmer~1934!. Mature bovine b-lactoglobulin has 162 residues, and is a member of the lipocalin protein superfamily~Flower, 1994, 1996!. Members of this family have a distinctive eight-stranded b-barrel structure, the central cavity of which binds a variety of hydrophobic molecules~Banaszak et al., 1994!. In the case of BLG, the primary site for fatty-acid binding has recently been established crystallographically to be inside this cavity~Qin et al., 1998b; Wu et al., 1999!. The structure of BLG is now reliably known following two independent redeterminations of the structure in the triclinic~lattice X!~Brownlow et al., 1997! and orthorhombic~lattice Y! forms~Bewley et al., 1997!. The latter structure has led to our redetermination of the structure in the trigonal~lattice Z! form~Qin et al., 1998a!. In lattice Z, in contrast to lattices X and Y, the entire molecule is well defined from electron density maps.Three variants of BLG, labeled as A, B, and C, commonly occur in cow's milk. Variants A and B~BLGA and BLGB! differ at two sites: Asp64 in A is changed to Gly in B, and Val118 in A is changed to Ala in B. Variants B and C differ at one site: Gln59 in B is changed to His in C. Thus, the isoelectric points for variants A, B, and C differ slightly: pI ϭ 5.26, 5.34, and 5.33, respectively, in 0.1 M KCl at room temperature~McKenzie, 1971!. Of technological significance, bovine BLG variants have different effects on the industrial processing of milk and on the characteristics of milk products~Hill et al., 1996!. Thermal stability is in the order B ...

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On the Dissociation of Bovine ,b-Lactoglobulins A, B, and C Near Ph 7

Cited by 10 publications

References 7 publications

Thermal stability of β-lactoglobulins A and B: effect of SDS, urea, cysteine and N-ethylmaleimide

Thermal stability of β-lactoglobulins A and B: effect of SDS, urea, cysteine and N-ethylmaleimide

Bovine β-Lactoglobulin Is Dimeric Under Imitative Physiological Conditions: Dissociation Equilibrium and Rate Constants over the pH Range of 2.5–7.5

Functional implications of structural differences between variants A and B of bovine β‐lactoglobulin

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