β-lactoglobulin binding properties during its folding changes studied by fluorescence spectroscopy

doi:10.1016/0167-4838(94)90098-1

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

Volume 1205, Issue 1, 16 March 1994, Pages 105-112

Biochimica et Biophy...

https://doi.org/10.1016/0167-4838(94)90098-1 Get rights and content

Abstract

The milk protein, β-lactoglobulin (BLG) exhibits structural and binding properties which vary widely, depending on the medium. These properties of BLG are reflected in fluorescence intensities, steady-state anisotropies and phase lifetimes of BLG tryptophan residues and of retinol and diphenyl hexatriene (DPH) bound to BLG, as functions of pH, ethanol concentration and protein modifications (22% ethylated, 90% methylated and 85% acetylated BLGs). Tryptophan quenching experiments show that retinol and DPH bind to BLG in 1:1 molar ratios with apparent dissociation constants around 10⁻⁷–10⁻⁸M. The strength of retinol binding is pH-dependent in the range 3–8, whereas that of DPH binding is not. Two different binding sites for these two ligands coexist on the protein. Modified BLGs exhibit higher affinities for DPH than the unmodified protein. At all pH values investigated, the fluorescence emission at 480 nm of retinol/BLG mixtures and retinol, DPH and tryptophan anisotropies and lifetimes change dramatically with midpoint at 27% ethanol for the first parameter and 35% for the others, suggesting simultaneous β-strand to α-helix transition and the dissociation of BLG complexes at 35% ethanol. An intermediate state, possibly ‘molten globular’, occurs around 20% ethanol, as deduced from anisotropy and lifetime measurements.

References (42)

H.L. Monaco et al.
J. Mol. Biol.
(1987)
R. Huber et al.
J. Mol. Biol.
(1987)
C. Tanford et al.
J. Biol. Chem.
(1961)
R. Townend et al.
J. Biol. Chem.
(1967)
S. Futterman et al.
J. Biol. Chem.
(1972)
M. Coke et al.
J. Colloid Interface Sci.
(1990)
E. Dufour et al.
Biochim. Biophys. Acta
(1991)
R.D. Fugate et al.
Biochim. Biophys. Acta
(1980)
E. Dufour et al.
FEBS Lett.
(1990)
S. Georghiou et al.
J. Biol. Chem.
(1975)

M. Shinitzky et al.

J. Biol. Chem.

(1974)

B. Mély-Goubert et al.

Biochim. Biophys. Acta

(1980)

J.C. Cabacungan et al.

Anal. Biochem.

(1982)

H. Fraenkel-Conrat et al.

J. Biol. Chem.

(1945)

J. Yguerabide et al.

J. Mol. Biol.

(1970)

M.Z. Papiz et al.

Nature

(1986)

M.E. Newcomer et al.

EMBO J.

(1984)

S.N. Timasheff et al.

Nature

(1964)

K.D. Zimmerman et al.

Arch. Biochem. Biophys.

(1970)

K. Takeda et al.

J. Protein Chem.

(1989)

C. Tanford et al.

J. Am. Chem. Soc.

(1960)

Cited by (118)

Probing the binding sites of bioactives with β-Lactoglobulin at different gastrointestinal pHs
2024, Food Hydrocolloids
Encapsulation is an efficient strategy to preserve the nutritional value of bioactive compounds in functional foods. The whey protein, β-lactoglobulin (βLg), is a potential carrier for bioactives due to its stability over a range of pHs and in the presence of the gastric enzyme, pepsin. In this study, curcumin and folic acid are used as representative hydrophobic and hydrophilic bioactives compounds respectively to investigate the suitability of βLg to encapsulate at different pH conditions of the gastrointestinal tract (2.0, 4.0, and 7.4). Unexpectedly, we found through intrinsic fluorescence spectroscopy that βLg binds both curcumin and folic acid with similar binding affinity at all three pHs. Fourier-transform infrared experiments showed, that with an exception at pH 2.0, H-bonding play a role in the interaction of both bioactives with βLg. Circular dichroism spectroscopy and small angle X-ray scattering showed that the interactions does not alter the structure of the protein. Further, 2D-protein nuclear magnetic resonance spectroscopy demonstrated, that curcumin binds to an external groove at pH 2.65. Whereas at pH 4.0, curcumin was found to bind at the groove and contrary to expectations, also at the calyx (the EF loop was previously thought to close the entrance to the calyx at this pH). At pH 6.5, both curcumin and folic acid bind to βLg at the calyx and the groove. Overall, our study shows that βLg binds curcumin and folic acid at gastrointestinal pHs, suggesting its potential ability to encapsulate both hydrophobic and hydrophilic bioactive molecules in dairy functional foods.
Insights into the supramolecular self-assembly of sodium caseinate and calixarene
2023, Food Hydrocolloids
Casein is widely used in the creation of biocompatible and functional particles, which are of interest as a delivery system based on the use of colloidal protein aggregates for the encapsulation of nutritional supplements and drugs. A very promising method for the functionalization and ordering of casein molecules is supramolecular self-assembly, which makes it possible to create nanosized materials using bottom-up approach. In this work, three-dimensional calixarene, namely the amphiphilic viologen calix[4]resorcinol, was used for the first time for spontaneous co-aggregation with sodium caseinate in an aqueous medium. A wide range of physicochemical methods were used to study the mechanisms of interaction between sodium caseinate and viologen calix[4]resorcinol, which lead to the formation of functional nanoparticles. Such nanoparticles, obtained spontaneously and without the use of organic solvents, are able to solubilize both hydrophobic (quercetin and oleic acid) and hydrophilic molecules (doxorubicin hydrochloride). In addition to encapsulating and stimulus-sensitive properties, the cytotoxic and penetrating properties of the formed nanoparticles were determined, as well as their distribution in cells.
Influence of pH and low/high- methoxy pectin complexation on the hydrophobic binding sites of β-lactoglobulin studied by a fluorescent probe method
2022, Food Hydrocolloids
Citation Excerpt :
The Kd at pH 7.0 determined in this study is comparable to some previous values (i.e., between 0.02 and 10 μM), despite the fact that these values vary considerably depending on experimental parameters, such as temperature, selection of fitting models, and the correction of the inner filter effect (Keppler, Sönnichsen, Lorenzen, & Schwarz, 2014). Likewise, a fluorescence quenching method observed the pH-dependent Kd of β-Lg-retinol complexes (Dufour, Genot, & Haertlé, 1994). However, Fugate and Song (1980) claimed that retinol binding to β-Lg was pH-independent in pH 2–7.5.
β-lactoglobulin(β-Lg)-polysaccharide soluble complexes formed in a specific pH range through electrostatic attraction have attracted a growing interest in the design of food-grade encapsulation systems for hydrophobic compounds, which is mainly ascribed to the ligand-binding properties of β-Lg. However, it remains unclear whether pH-induced conformational changes in β-Lg and its electrostatic complexation with anionic pectin affect their ability to bind hydrophobic compounds. Here, a fluorescent probe method was employed to provide useful insights into the field. Three solvatochromic fluorescent probes (i.e. Nile red, retinol and curcumin) were selected as representative models of hydrophobic compounds that were bound to the inner cavity or/and the outer surface of β-Lg. Binding with β-Lg or β-Lg/pectin complexes largely enhanced the fluorescence of the hydrophobic probes. Especially, the polarity difference of different binding sites on β-Lg was revealed by the fluorescence spectra of β-Lg-Nile red as a function of pH. Both Nile red and retinol were bound more favorably to β-Lg at neutral pH than at acidic pH, possibly due to the accessibility of the inner cavity in the former case. Upon acidification, the gradual reduction in fluorescence intensity of the pre-formed protein-ligands complexes (i.e. at pH 7.0) was ascribed to the dissociation between Nile red (or retinol) and the inner cavity of β-Lg. β-Lg-curcumin interactions were less affected by pH variations, suggesting curcumin mainly binding to the outer surface of β-Lg. For the three tested hydrophobic compounds, the formation of soluble complexes between β-Lg with pectin had no adverse effects on their interactions with the protein. These results may provide useful insights into the binding of hydrophobic compounds to β-Lg or β-Lg/pectin complexes. The methodology may also be extended to study the encapsulation performance of other biopolymers or particles.
Investigating the effect of sugar-terminated nanoparticles on amyloid fibrillogenesis of β-lactoglobulin
2020, International Journal of Biological Macromolecules
In vivo tissue deposition of fibrillar protein aggregates is the cause of several degenerative diseases. Evidence suggests that interfering with the pathology-associated amyloid fibrillogenesis by inhibitory molecules is envisaged as the primary therapeutic strategy. Amyloid fibril formation of proteins has been demonstrated to be influenced by nanoparticles/nanomaterials. As compared with their molecular form counterpart, this work examined the effect of sucrose-terminated nanoparticles on the in vitro amyloid fibrillogenesis and structural properties of β-lactoglobulin at pH 2.0 and 80 °C. ThT binding and electron microscopy results demonstrated that sucrose-terminated nanoparticles were able to suppress β-lactoglobulin fibrillogenesis in a concentration-dependent fashion. Importantly, sucrose-terminated nanoparticles showed better β-lactoglobulin fibril-inhibiting ability than sucrose molecules. ANS fluorescence and right-angle light scattering results showed reduced solvent exposure and decreased aggregation, respectively, in the β-lactoglobulin samples upon treatment with sucrose-terminated nanoparticles. Moreover, fluorescence quenching analyses revealed that the static quenching mechanism and formation of a non-fluorescent fluorophore–nanoparticle complex are involved in the nanoparticle-β-lactoglobulin interaction. We believe that the results from this study may suggest that the nanoparticle form of biocompatible sugar-related osmolytes may serve as effective inhibiting/suppressing agents toward protein fibrillogenesis.
Norbixin binding to whey protein isolate - alginate electrostatic complexes increases its solubility and stability
2020, Food Hydrocolloids
Norbixin is a carotenoid extracted from the Annatto plant (Bixa orellana) and widely used as a natural colorant in pharmaceuticals, cosmetics and food. It is water-soluble at neutral and alkaline pH, but below neutral pH, norbixin starts to precipitate limiting the use in acidic food products. As a new approach to enhance the solubility and stability of norbixin in aqueous solution under acidic conditions, the colorant was applied in the presence of whey protein isolate (WPI) and alginate (ALG). Addition of WPI prevented norbixin precipitation between pH 2 and pH 7 except at the pI of WPI, pH 5. The visual appearance, colorimetric measurements and a blue shift in the absorbance spectrum indicated that norbixin formed H-aggregates at pH < 7, but the aggregates were stabilized by WPI addition. Fluorescence spectroscopy measurements indicated interactions between both soluble norbixin and WPI at pH 7 and aggregated norbixin and WPI at pH 3. Particle size measurements indicated formation of complexes between norbixin aggregates and WPI. Additionally to preventing the precipitation of norbixin, the WPI-norbixin interaction also increased the color stability of norbixin during storage at both neutral and acidic pH. At pH 5, isoelectric precipitation of WPI was prevented by the inclusion of ALG, likely due to electrostatic interactions between WPI and ALG leading to the formation of norbixin, WPI and ALG complexes. The use of WPI and ALG complexation of norbixin can therefore be a new way to expand the usage of norbixin coloring of foods at acidic pH conditions.
Structure and stability of whey proteins
2019, Milk Proteins: From Expression to Food
The chemical and physical stabilities of the more common proteins of bovine and, where available, ovine, caprine, and equine whey (β-lactoglobulin, α-lactalbumin, serum albumin, immunoglobulins, and lactoferrin) are reviewed with regard to their molecular structures and dynamics. The behavior of the proteins separately and in combination with respect to temperature, pressure, pH, denaturants (such as guanidinium chloride and urea), and stabilizers (such as fatty acids and metal ions) is considered. The combination of high temperature and low pH in the hydrolysis and subsequent formation of fibrils constitutes a new body of study and knowledge since the first edition of this chapter in 2009. Particular emphasis is placed on studies that have utilized x-ray, nuclear magnetic resonance, fluorescence, and circular dichroism techniques. Attention is directed to the role of cysteines and disulfide bridges with regard to chemical stability. Whereas there is considerable knowledge of the structure-function relationships of individual proteins, there remains a dearth of three-dimensional structural knowledge of combinations of proteins, despite the clear importance of such knowledge to functionality, especially with regard to food processes.

View all citing articles on Scopus

View full text

β-lactoglobulin binding properties during its folding changes studied by fluorescence spectroscopy

Abstract

J. Mol. Biol.

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Colloid Interface Sci.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

Anal. Biochem.

J. Biol. Chem.

J. Mol. Biol.

Nature

EMBO J.

Nature

Arch. Biochem. Biophys.

J. Protein Chem.

J. Am. Chem. Soc.