Spectroscopic characterization of beta-lactoglobulin-retinol complex.

1. The absorption spectrum of retinol when bound to beta-lactoglobulin is vibrationally resolved. The circular dichroism spectrum exhibits the same structure, as does the fluorescence excitation spectrum. 2. Two molecules of retinol are bound per protein dimer, with a binding constant (Kd) of 2 x 10(-8) M. Also, by fluorescence titration it was found that the monomer binds one molecule of retinol with essentially the same Kd. 2. Energy transfer occurs from tryptophan (donor) to retinol (acceptor) with a rate constant, k, of 4.4 x 10(8) s-1. The distance between the centers of mass of the transition is 34 A, corresponding to the energy transfer efficiency of 44%. 4. The fluoresence lifetime of retinol increases dramatically on binding to beta-lactoglobulin, from approx. 2 to approx. 10 ns, as does the fluorescence quantum yield. 5. The retinol binding to beta-lactoglobulin does not show a pH dependence and the binding site is hydrophobic. 6. On the Sephadex G-100 column, retinol is chemically modified to a retro derivative which binds even more strongly to beta-lactoglobulin than does retinol. 7. The beta-lactoglobulin-retinol complex rotates anisotropically in solution with a fast (3 ns) and a slower (12 ns) component. This may be attributed to retinol being found at a flexible region of the protein, where only segmental flexibility is observed, weighted by its proximity to one of the major axis rotational times.