Evidence of heterogeneous 1-anilinonaphthalene-8-sulfonate binding to β-lactoglobulin from fluorescence spectroscopy

Steady-state and dynamic fluorescence titrations show that:-(a) the complex between beta-lactoglobulin (BLG) and 1-anilinonaphthalene-8-sulfonate (ANS) displays a heterogeneous equilibrium with large changes in the binding strength vs. pH and ion concentration; and (b) the fluorescence response of bound ANS reveals two separate lifetimes that suggest two different sites (or binding modes). While steady-state fluorescence titrations yield effective values of the binding constant and of the bound ANS quantum efficiency, it is shown that, by combining steady-state fluorescence and lifetime decay of ANS, it is possible to give quantitative estimates of the association constants for each site. When heading from the acid (pH similar to 2) to the native state (pH similar to 6) the main result is a very large reduction of the effective binding constant. This and the results of titrations vs. ionic strength suggest that electrostatic interactions are. a major contribution to ANS binding to BLG. (C) 1999 Elsevier Science B.V. All rights reserved.