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Evidence for internal and external binding sites on human tear lipocalin
Authors:Gasymov Oktay K  Abduragimov Adil R  Glasgow Ben J
Affiliation:Departments of Pathology and Ophthalmology, UCLA School of Medicine, Jules Stein Eye Institute, 100 Stein Plaza, Rm# B269, Los Angeles, CA 90095, USA
Abstract:
8-Anilino-1-naphthalenesulfonic acid (ANS) is widely used as a probe for locating binding sites of proteins. To characterize the binding sites of tear lipocalin (TL), we studied ANS binding to apoTL by steady-state and time-resolved fluorescence. Deconvolution of ANS binding revealed that two lifetime components, 16.99 ns and 2.76 ns at pH 7.3, have dissociation constants of 0.58 μM and 5.7 μM, respectively. At pH 3.0, the lifetime components show decreased affinities with dissociation constants of 2.42 μM and ∼21 μM, respectively. Selective displacement of ANS molecules from the ANS-apoTL complex by stearic acid discriminates the internal and external binding sites. Dependence of the binding affinity on ionic strength under various conditions provides strong evidence that an electrostatic interaction is involved. Time-resolved fluorescence is a promising tool to segregate multiple binding sites of proteins.
Keywords:Tear lipocalin   Time-resolved fluorescence   Binding sites   ANS   Heterogeneous binding   Lipocalin-1   von Ebner&rsquo  s gland protein   Human tear protein
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