International Journal of Biological Macromolecules 35 (2005) 231–242 The binding of novel two-color fluorescence probe FA to serum albumins of different species Sebnem Ercelen a , Andrey S. Klymchenko a,b , Yves M´ ely b , Alexander P. Demchenko a,c,∗ a TUBITAK Research Institute for Genetic Engineering and Biotechnology, 41470 Gebze-Kocaeli, Turkey b Laboratoire de Pharmacologie et Physicochimie, UMR 7034 du CNRS, Facult´ e de Pharmacie, Universit´ e Louis Pasteur BP 24, 67401 Illkirch, France c A.V. Palladin Institute of Biochemistry, Kiev 01030, Ukraine Received 3 January 2005; received in revised form 13 February 2005; accepted 15 February 2005 Abstract The novel two-color ratiometric fluorescence probe FA belonging to a class of 3-hydroxychromone dyes was applied for characterization of binding sites in serum albumins obtained from seven species (bovine, dog, horse, human, pig, rabbit and sheep). On strong and highly specific FA binding to the same location in protein structure, the species-dependent differences were observed in positions of absorption maxima, positions of two fluorescence emission bands and the intensity ratios between them. They were analyzed by multiparametric algorithm that allowed a detailed characterization of probe-binding sites and were characterized by very low polarity, high electronic polarizability and different extent of intermolecular hydrogen bonding. The species-dependent differences were also observed in time-resolved fluorescence emission decays. Fluorescence competition experiments with the drug warfarin, suggested the location of FA binding site within or in proximity to Domain IIA. © 2005 Published by Elsevier B.V. Keywords: Fluorescence probe; Ligand binding; Polarity sensing; Two-color ratiometric dye; 3-Hydroxychromones; Serum albumin species 1. Introduction Serum albumin is the most abundant of all proteins in blood plasma of many species, where it reaches a concen- tration of about 40 mg/ml (0.6 mM). It accounts for about 60% of its total protein content and provides about 80% of the blood osmotic pressure. Biological and pharmacokinetic functions of albumin are also very important. It is the major transporter in the blood of non-esterified fatty acids as well as of many other low-polar metabolites and drugs [1,2]. Albu- Abbreviations: ANS, 1-anilinonaphthalene-8-sulfonic acid; BSA, bovine serum albumins; DSA, dog serum albumins; HoSA, horse serum albumins; HAS, human serum albumins; PSA, pig serum albumins; RSA, rabbit serum albumins; SSA, sheep serum albumins; ESIPT, excited-state intramolecular proton transfer; FA, 2-(6-diethylaminobenzo[b]furan-2-yl)- 3-hydroxychromone; warfarin, 3-(-acetonylbenzyl)-4-hydroxycoumarin; MEM, maximum entropy method ∗ Corresponding author. Tel.: +90 262 641 2300/4011; fax: +90 262 646 3929. E-mail address: dem@rigeb.gov.tr (A.P. Demchenko). min is able to bind an enormous variety of these compounds, most of them are hydrophobic and low-soluble in aqueous media. The binding affinity of any drug to serum albumin is one of the major factors that determine the pharmacokinet- ics of these drugs, their lifetimes and availabilities in various tissues. Serum albumin is also one of the most extensively studied of all proteins. As it was clearly shown, it is far from being a universal carrier of all hydrophobic compounds. Its ligand affinities are determined not only by their hydrophobicities, but also by a number of other factors including steric ones [3]. These affinities can vary over broad ranges, from 10 3 to 10 7 M -1 [4]. This is because of existence in this molecule of discrete number of binding sites possessing different speci- ficity, the most important of which are the so-called sites I and II [5]. These sites were first assigned based on fluores- cence probing and then identified in crystal structures of HSA and its complexes with several ligands [2,6]. Site I located in subdomain IIA is called the warfarin-binding site, and site II called the indole/benzodiazepine site is located in subdomain 0141-8130/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.ijbiomac.2005.02.002