Chapter 23
Infrared Spectroscopic Study of β-Lactoglobulin Interactions with Flavor Compounds Markus Lübke, Elisabeth Guichard, and Jean-Luc Le Quéré
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Fourier transform infrared spectroscopy of aqueous solutions of βlactoglobulin is used to study the incidence of binding of small organic ligands on the protein conformation at p H 2.0 and 7.5. Differential spectroscopy allows the comparison of conformational changes for different ligands, while curve fitting techniques are used to determine the influence of ligand binding on the relative proportions of the protein's secondary structural elements. Results are presented which suggest that retinol, fatty acids and β-ionone bind to the central cavity of β-lactoglobulin. Binding of α-ionone to the same site is also likely, although clear differences exist compared to the β isomer. A number of other flavor compounds did not induce any conformational changes to the protein. It is therefore assumed that these compounds bind to the protein surface.
It has long been recognised that the perception of food flavors not only depends on the quantitative composition of the volatile fraction, but that matrix effects also play a very important role. One cause for this matrix dependence is the interaction between the flavor active compounds and biopolymers present in most foodstuffs. Hydrophobicity of the flavors, acting as ligands, has a strong influence on these interactions, but sterical factors or the presence or absence of certain functional groups are also known to be important. Several methods have been developed to quantitatively study interactions either in model solutions of biopolymers or in more complex systems, among which are fluorescence spectroscopy (1,2), static headspace (3, 4), affinity chromatography on protein bonded stationary phases (5, 6) and equilibrium dialysis (7, 8). While all techniques allow the determination of association constants or at least of global affinities, usually no insight in the involved binding mechanisms can be gained. Spectroscopic techniques, on the other hand, can
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© 2000 American Chemical Society
Roberts and Taylor; Flavor Release ACS Symposium Series; American Chemical Society: Washington, DC, 2000.
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283 be used to study a broad range of systems, and in principle quantitative data about complexes can be obtained. More importantly, information at the molecular level is available: how exactly is the ligand bound to the macromolecule, and what implications does the binding have on the protein structure? In this study, we have focused on Fourier transform infrared spectroscopy to study complexation between the whey protein β-lactoglobulin (β-lg) and selected flavor compounds. Infrared spectroscopy is a very useful tool to study protein secondary structures (for a review, see e.g. (9)). It is in general the so called amide I band region between 1600 and 1700 cm" that reveals the most information, because it is highly conformation-sensitive. For example, the changes induced by varying the p H of aqueous solutions of β-lg can be clearly followed (10), and even the subtle structural differences between β-lg variants A and B, which differ by only two amino acids, can be detected (11). This amide I band is due mainly to C=0 stretching vibrations, and is a spectroscopically unresolvable complex of a certain number of overlapping individual bands. These individual bands represent the particular molecular environments of the protein backbone in secondary structures such as ahelices, β-sheets and turns (12). B y analysing this "fine structure" of the amide I band, the elements of the secondary structure can be quantified, and the obtained compositions usually correlate quite well with results computed from X-ray data. In order to avoid confusion the term "band" will be used in the following for those individual bands as opposed to the "amide I envelope" as a whole. 1
The protein β-lg, like other members of the lipocalin family, has a central calyxshaped cavity that is well established by X-ray studies (13-15). However,, there has been ongoing discussion on whether hydrophobic molecules bind inside that central cavity or to a putative second binding site, which has been proposed as being a groove on the protein surface (14). In the light of two very recent X-ray studies, it seems that the central cavity is indeed the favored binding site for fatty acids (16, 17). As far as retinol is concerned (a molecule which is of special interest in this context due to the close structural resemblance between β-lg and retinol binding protein), results are still contradictory (18-22), although one might conclude, in the absence of final evidence, that binding takes place in the central cavity, too. Ligand binding to βlg is further complicated by the fact that the protein undergoes a pH-driven structural change, known as the Tanford transition, which potentially restricts the access to the central cavity at low pH. It has been shown (21) that this transition involves the displacement of a flexible loop which links the strands Ε and F. Part of the contradictions in the literature may be due to this phenomenon. In principle, it is reasonable to assume that flavor compounds behave like other hydrophobic ligands such as the mentioned retinol and fatty acids. But the size and geometry of the molecule are obviously also important, and exclusion phenomena from the central cavity and weak or no binding due to an insufficient number of favorable interactions within the cavity can be expected in some cases. To our knowledge, no direct evidence has been reported for where flavor compounds bind on the β-lg molecule. Indirect clues comes from (1), who found competition between
Roberts and Taylor; Flavor Release ACS Symposium Series; American Chemical Society: Washington, DC, 2000.
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retinol and β-ionone, thus suggesting the central cavity as the binding site. Based on competition experiments between β-ionone and other flavor compounds (6), one can further conclude that (i) lactones have some (rather weak) affinity for the central cavity, and (ii) ct-ionone, β-damascenone, methyl benzoate and unsaturated aliphatic aldehydes and ketones bind elsewhere on the protein. It is our intention here to demonstrate the feasibility of the use of infrared spectroscopy to study interactions between proteins and flavor compounds in solution, to charcterize structural changes induced by the ligand binding and to correlate the findings with those obtained notably by X-ray and by ligand competition studies for a deeper understanding of where on the β-lg molecule binding takes place depending on the ligand structure. To this end, band shapes of amide I envelopes as well as the intensities of the underlying individual bands will be analyzed and discussed.
Materials and Method Solutions were prepared by dissolving 4.6 mg of β-lg variant A (Sigma) in 50 m M phosphate buffer (pH either 2.0 or 7.5) made up to 900 pL, then adding 100 p L of 1:1 ethanol/buffer mixture containing the appropriate amount of ligand. Final protein concentration was 250 μΜ. A l l ligands were from the in-house collection of volatile compounds, except alRr
