The interactions of fatty acids with porcine and bovine beta-lactoglobulins were measured using tryptophan fluorescence enhancement. In the case of bovine beta-lactoglobulin, the apparent binding constants for most of the saturated and unsaturated fatty acids were in the range of 10(-7) M at neutral pH. Bovine beta-lactoglobulin displays only one high affinity binding site for palmitate with an apparent dissociation constant of 1 x 10(-7) M. The strength of the binding was decreasing in the following way: palmitate > stearate > myristate > arachidonate > laurate. Caprylic and capric acids are not bound at all. The affinity of beta-lactoglobulin for palmitate decreased as the pH of the incubation medium was lowered and BLG/palmitate complex was not observed at pH's lower than 4.5. Surprisingly, chemically modified bovine beta-lactoglobulin and porcine beta-lactoglobulin did not bind fatty acids in the applied conditions.

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It has been demonstrated using CD that ethanol induces important secondary structure changes of beta-lactoglobulin. CD spectra indicate that beta-lactoglobulin secondary structure, which is mainly composed of beta-strands, becomes mostly alpha-helical under the influence of the solvent polarity changes. The midpoint of beta-strand/alpha-helix transition in beta-lactoglobulin is observed at dielectric constant approximately 60 (35% ethanol; v/v). According to CD measurements, the ethanol-dependent secondary structure changes are reversible. The alkylation of lysines epsilon-NH2 in beta-lactoglobulin weakens the central beta-barrel structure, since the beta-strand/alpha-helix transition midpoint of alkylated beta-lactoglobulin is shifted to lower ethanol concentration (25% ethanol; v/v). beta-Lactoglobulin structural changes are triggering the dissociation of the beta-lactoglobulin-retinol complex as judged from complete quenching of its fluorescence in ethanol concentration greater than 30% (v/v). However, in 20% ethanol (v/v), beta-lactoglobulin still retains most of its native secondary structure as shown by CD and, in this condition, one beta-lactoglobulin molecule binds an additional second retinol molecule. This suggests that the highly populated species observed around 20% ethanol (v/v) might represent an intermediate state able to bind two molecules of retinol.

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Measurement of tryptophan fluorescence quenching and the excitation energy transfer from tryptophanyl residues to the bound ligand indicates that b-lactoglobulin binds tightly to hemin and protoporphyrin IX in a ligand-to-protein stoichiometric ratio. The apparent dissociation constants of hemin-b-lactoglobulin and protoporphyrin IX-/I-lactoglobulin complexes are 2.5 x lo-' M and 4 x lo-' M, respectively. The addition of /3-lactoglobulin (final concentration = 10 PM, phosphate buffer 50 mM, pH 7.1) to the solution containing retinol and protoporphyrin IX triggers an energy transfer between &lactoglobulin tryptophan and protoporphyrin IX as well as between retinol and protoporphyrin IX. The efficiency of energy transfer depends on the distance between the donor (retinol) and the acceptor (protoporphyrin IX). Using the Fiirster theory, a retinolprotoporphyrin IX distance of 25 A was calculated. These results indicate that retinol and protoporphyrin IX are bound to the p-lactoglobulin monomer at two different sites.p-Lactoglobulin; Protoporphyrin IX; Retinol; Binding site