Site-specific recognition of fluorescein by human serum albumin: A steady-state and time-resolved spectroscopic study Osama K. Abou-Zied * , Saba A.J. Sulaiman Department of Chemistry, Faculty of Science, Sultan Qaboos University, P.O. Box 36, 123 Muscat, Oman article info Article history: Available online 14 May 2014 Keywords: Fluorescein Human serum albumin Warfarin Fluorescence Lifetime Förster’s resonance energy transfer abstract The increased interest in using fluorescein as a fluorescent probe in biology and medicine is associated with its distinct absorption and fluorescence signals that are transparent to biological samples. Herein, we characterize the binding mechanism of fluorescein inside human serum albumin which is used as a carrier and protector for fluorescein in medical applications. Binding of fluorescein in human serum albumin causes partial fluorescence quenching of the sole tryptophan residue in the protein (W214). The estimated W214-fluorescein distance (2.42 nm) and the calculated quenching rate constant (k q ¼ 5.13  10 12 M 1 s 1 ), both indicate binding of fluorescein in subdomain IIA. A site-competitive experiment shows that fluorescein is located in the warfarin binding pocket. The estimated binding constant (K ¼ 10,000 M 1 ) points to a moderate binding strength of the fluoresceinehuman serum al- bumin complex that should not affect the fluorescein release to the target when used as a probe. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Fluorescein (FL) is widely used as an extrinsic label in proteins due to its distinct long absorption maximum near 490 with a high molar extinction coefficient, and emission wavelength from 480 to 600 nm with a high quantum yield [1,2]. These properties are sig- nificant in biochemical and biological applications which require a probe that possesses a fluorescence signal well removed from the intrinsic fluorescence of biological samples. One common use of FL is for labelling of antibodies. A wide variety of FL-labelled immu- noglobulins are commercially available which are frequently used as proteins in fluorescence microscopy and in immunoassays [3e5]. The spectral properties of FL in the visible region make this mole- cule a suitable probe for such study and eliminate the need for quartz optics. FL is also used as a biological pH sensor to determine intracellular pH values. The molecule displays a complex pH- dependent equilibrium as shown in Scheme 1 [1,2]. The lactone form is usually found in organic solvents and is absent in aqueous solutions above pH 5.0. Only the monoanionic and the dianionic forms are fluorescent. The absorption spectrum shifts to longer wavelengths as the pH increases. These spectral changes allow wavelength-ratiometric pH measurements with two excitation wavelengths near 460 nm (monoanion) and 490 nm (dianion). The intensity ratio (Int 490 /Int 460 ) increases with increasing pH. Self-quenching of FL fluorescence has been reported even at low concentrations [6]. This is attributed to the very small Stokes shift which results in resonance energy transfer between nearby FL molecules (homo-transfer). As a result, the fluorescence intensity of a labelled protein decreases with the extent of labelling [7]. FL has been used as a fluorescent tracer in medical applications due to its high quantum yield in physiological conditions. It is used as a probe to measure the permeability of the human blood-ocular barriers [8,9]. However a rapid metabolic mechanism to a weakly fluorescent conjugate (FL-monoglucuronide) was reported [8,9]. In order to minimize the metabolic effect on FL, the molecule was used as a tracer after binding to human serum albumin (HSA) [10,11]. But when FL is administered systemically to study its rate of penetration through the blood-ocular barrier, the degree of its binding to albumin may affect the observed results. In this regard, a detailed characterization of the binding between FL and HSA is beneficial. In the present work, we characterize the binding prop- erties of the HSAeFL complex using steady-state and time-resolved fluorescence measurements. The HSA protein is one of the major carrier proteins in the body and constitutes approximately half of the protein found in human blood [12]. This protein of 585 residues is composed of a single polypeptide chain, with three a-helical domains IeIII, each con- taining two subdomains A and B (Fig. 1) [13]. The protein is stabi- lized by 17 disulfide bridges. The crystal structure analyses indicate * Corresponding author. Tel.: þ968 2414 1468; fax: þ968 2414 1469. E-mail address: abouzied@squ.edu.om (O.K. Abou-Zied). Contents lists available at ScienceDirect Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig http://dx.doi.org/10.1016/j.dyepig.2014.05.005 0143-7208/Ó 2014 Elsevier Ltd. All rights reserved. Dyes and Pigments 110 (2014) 89e96