Article pubs.acs.org/JAFC
Structural and Thermal Stability of β‑Lactoglobulin as a Result of Interacting with Sugar Beet Pectin Phoebe X. Qi,*,† Edward D. Wickham,† and Rafael A. Garcia‡ †
Dairy and Functional Foods Research Unit and ‡Biobased and Other Animal Co-products Research Unit, Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Wyndmoor, Pennsylvania 19038, United States ABSTRACT: Changes in the structural and thermal stability of β-lactoglobulin (β-LG) induced by interacting with sugar beet pectin (SBP) have been studied by circular dichroism (CD), Fourier transform infrared, and steady-state as well as time-resolved fluorescence spectroscopic techniques. It has been demonstrated that SBP not only is capable of binding to native β-LG but also causes a significant loss in antiparallel β-sheet, ∼10%, accompanied by an increase in either random coil or turn structures. In addition, the interaction also disrupted the environments of all aromatic residues including Trp, Phe, and Tyr of β-LG as evidenced by near-UV CD and fluorescence. When preheated β-LG was combined with SBP, the secondary structure of β-LG was partially recovered, ∼4% gain in antiparallel β-sheet, and Trp19 fluorescence was recovered slightly. Although forming complexes with SBP did not significantly impact the thermal stability of individual secondary structural elements of β-LG, the environment of Trp19 was protected considerably. KEYWORDS: β-lactoglobulin, sugar beet pectin, circular dichroism, FTIR, fluorescence spectroscopy, fluorescence lifetime decay
1. INTRODUCTION
The molecular structure of pectin contains predominately the “smooth” homogalaturonan (HG) regions, α-(1→ 4)-linked Dgalacturonate and their methyl esters, and the “hairy” regions, which are composed of rhamnogalacturonan (RG) units.21 Pectin from sugar beets often differs from other sources of pectin in that it tends to have a higher degree of acetylation and higher neutral sugar side chains (rich in hairy regions), and also contains feruloyl groups in these regions. These feruloyl moieties proved to be advantageous for spectroscopic studies such as those employed in this work because of the high quantum yield that these fluorescent groups exhibit (based on our experiment using free ferulic acid), making them an effective indicator when interacting with β-LG. In addition to reported approaches to creating conjugates between proteins and polysaccharides via covalent bonding,5,22−25 a great deal of effort2,4,26 also has been paid to complexes formed through noncovalent interactions including electrostatic, hydrogen bonding, hydrophobic, and steric interactions. It is assumed therefore, these interactions depend greatly on the type, molecular weight, charge density, and concentration of the protein and polysaccharide used and are therefore particularly sensitive to the ionic strength, pH, and temperature of the solution conditions.26,27 The effect of interaction with polysaccharide on the structure and stability of the protein, which in turns undermines the functional properties of the products, remains unexplored thus far. In a recent publication,28 we demonstrated that mild preheat-treatment of β-LG facilitated interactions with sugar beet pectin (SBP) through mainly hydrophobic and local charge− charge interactions at neutral pH when β-LG and SBP are
Recent years have seen growing successful attempts in preparing complexes formed between proteins and polysaccharides, particularly by the food, cosmetics, and pharmaceutical industries.1−5 Among many, the interacting system formed between β-lactoglobulin (β-LG) and pectin is of particular interest because β-LG is easily available, its structural features are intrinsically interesting, and it has broad-ranging uses.6−9 A subject of intense research for the past several decades,10 the biological function of β-LG still remains elusive. Its molecular structural features, however, are characterized extensively by both X-ray crystallography11,12 and NMR techniques.13 It contains two tryptophan residues, first one (Trp19), buried in the hydrophobic core, is mainly responsible for intrinsic fluorescence properties of β-LG, whereas the second one (Trp61) is fully solvent exposed.14 At room temperature, β-LG exists as a monomer at acidic (below pH 3.0) and alkaline pH (above pH 8.0); it is predominately present as a dimer at pH 3.0−8.0. The dimer is stabilized by hydrogen bonds distributed between the surface AB loop and the antiparallel β-sheet between the β-strands, and by tight packing of the residues in the interface. The tendency of β-LG to bind to a host of hydrophobic ligands, including palmitate,15 fatty acids, and retinol,16 is well established, suggesting its possible role as a lipid transport protein. Most of these ligands are believed to bind deeply into the central calyx that is held together by eight antiparallel β-strands and a α-helix located at the outer surface of the β-barrel17 and lead to the disruption and rearrangement of the calyx upon binding and releasing the ligands.15,17 The binding of phenolic compounds such as ferulic acid, a naturally occurring plant antioxidant,18,19 to β-LG, however, has attracted far less attention,20 thus warranting further investigation. This article not subject to U.S. Copyright. Published 2014 by the American Chemical Society
Received: Revised: Accepted: Published: 7567
June 5, 2014 July 7, 2014 July 8, 2014 July 8, 2014 dx.doi.org/10.1021/jf502699g | J. Agric. Food Chem. 2014, 62, 7567−7576
Journal of Agricultural and Food Chemistry
Article
mixture of β-LG and SBP was prepared by either mixing the two untreated or preheated solutions, denoted as β-LG-h and SBP-h respectively, to obtain the final mixture containing 0.3 mg/mL of β-LG and 0.1 mg/mL of SBP for the CD and fluorescence experiments unless otherwise noted. All solutions were made fresh prior to each use. 2.4. Circular Dichroism Spectroscopy. CD spectroscopy was performed at 20 °C with an Aviv Model 420 CD spectrometer (Aviv Biomedical, Inc., Lakewood, NJ, USA). Spectra in the far-UV region (190−260 nm) were measured at the concentration of 0.3 mg/mL for β-LG and/or 0.1 mg/mL for SBP in a 0.05 cm path length quartz cell. The CD spectra in the near-UV region (250−320 nm) were acquired using β-LG concentration at 1.0 mg/mL and/or SBP at 0.33 mg/mL in a path length of 1 cm rectangular cuvette. All spectra were recorded with 0.5 nm step and 5 s average time. Spectra were corrected for buffer (33 mM sodium phosphate, pH 6.75) contribution and for SBP signal when present. The mean residue ellipticity, [θ] (degree cm2 dmol−1), was calculated from the formula [θ] = (Mwθ)/(10Cln), where θ is the measured ellipticity (millidegrees), Mw is the molecular weight (Mw = 18320 Da, an average for a mixture of β-LG A and B variants), l is the path length of the cuvette (cm), C is the protein concentration (mg/mL), and n is the number of amino acid residues in β-LG (n = 162). Analysis of the secondary structure of β-LG (untreated and heated) in the presence and absence of SBP (untreated and heated) in phosphate buffer (33 mM, pH 6.75) from the far-UV CD spectra was carried out using the software CDNN (v.2.1, Applied Photophysics, Ltd., Leatherhead, United Kingdom) developed by Dr. Gerald Böhm.33 A database consisting of 33 reference proteins was used in the deconvolution analysis. The standard deviation (SD) was ±0.4% based on a set of triplicate CD experiments and followed by CDNN analysis. Temperature dependence of far-UV CD (190−260 nm) spectra was carried out on solutions containing 0.3 mg/mL β-LG or/and 0.1 mg/ mL SBP in the buffer containing 33 mM PBS (pH 6.75) in a 0.05 cm path length quartz cell. Temperature was adjusted using a solid-state Pelletier element, and a minimum of 30 min equilibration time was used between each temperature point. Each spectrum at each temperature was corrected for buffer and SBP (when present) contributions before being subjected to deconvolution using CDNN program. 2.5. FTIR Spectroscopic Measurements and Analysis. Infrared spectra were collected with a FTIR spectrometer (Nexus 670, Thermo Electron, Madison, WI, USA) equipped with a DTGS KBr detector and a KBr beam splitter. PBS buffer at reduced concentration, 20 mM, was used due to its IR contribution at the higher concentration (33 mM) used in all other experiments. β-LG and SBP samples were dissolved at 15 and 5 mg/mL respectively, and followed by subsequent mixing or/and preheating and then mixing. Aliquots of 200 μL each of the solutions investigated in this work were placed on a CaF2 plate, allowed to air-dry overnight, and then vacuum-dried for 30 min prior to measurement. The thin film was scanned from 4000 to 1000 cm−1 at 20 °C with a nominal resolution of 2 cm−1 and 128 accumulative scans. Analysis of the secondary structure of β-LG (untreated and heated) in the presence and absence of SBP (untreated and heated) from the FTIR spectra was carried out as previously reported.34 The baselines were corrected automatically using the built-in software of the spectrophotometer (OMNIC v.7.3). By means of the second derivative in the amide I region, 1600−1700 cm−1, eight and nine major peaks for β-LG in the absence and presence of SBP and β-LG-h in the absence and presence of SBP-h were resolved, respectively, and fitted using Peak Resolve (OMNIC v.7.3). A Gaussian function was used during the fitting routine for all peaks corresponding to α-helix (∼1660 cm−1), unordered (∼1550 cm−1), β-sheet (1610−1620 cm−1), β-turn (1670−1690 cm−1), and β-antiparallel sheet (∼1690 and 1630−1640 cm−1) according to the assignments by Barth,35 Dong et al.,36 and Hasni et al.37 A separate Fourier self-deconvolution procedure (OMNIC v.7.3)38 was also used to independently confirm the positions of overlapping peaks (±2 cm−1). The areas of all of the
incompatible for strong electrostatic interaction. In this work, we attempt to investigate and understand in detail the structural changes and thermal stability of β-LG as a result of interacting with SBP using circular dichroism (CD), Fourier transform infrared (FTIR), and steady-state and time-resolved fluorescence spectroscopic techniques.
2. MATERIALS AND METHODS 2.1. Materials. Purified β-lactoglobulin (β-LG) powder (Lot no. JE001-0-415) was kindly donated by Davisco Foods International (Le Sueur, MN, USA). The reported composition (expressed as dry weight percent unless otherwise indicated) of the powder was as follows: >95% protein (of which 90% was β-lactoglobulin);
