Steady-state and dynamic fluorescence titrations show that: (a) the complex between β-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~2) to the native state (pH~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.

D'Alfonso, L., Collini, M., Baldini, G. (1999). Evidence of heterogeneous 1-anilinonaphthalene-8-sulfonate binding to β-lactoglobulin from fluorescence spectroscopy. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 1432(2), 194-202 [10.1016/S0167-4838(99)00105-3].

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

D'ALFONSO, LAURA;COLLINI, MADDALENA;BALDINI, GIANCARLO
1999

Abstract

Steady-state and dynamic fluorescence titrations show that: (a) the complex between β-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~2) to the native state (pH~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.
Articolo in rivista - Articolo scientifico
β-lactoglobulin; ANS; Fluorescence lifetime; Heterogeneous binding;
English
1999
1432
2
194
202
none
D'Alfonso, L., Collini, M., Baldini, G. (1999). Evidence of heterogeneous 1-anilinonaphthalene-8-sulfonate binding to β-lactoglobulin from fluorescence spectroscopy. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 1432(2), 194-202 [10.1016/S0167-4838(99)00105-3].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/15725
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