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, 16.99ns and 2.76ns at pH 7.3, have dissociation constants of 0.58μM and 5.7μM, respectively. At pH 3.0, the lifetime components show decreased affinities with dissociation constants of 2.42μM and ∼21μM, 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.
Accepted Version (
Free)
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call