The influence of fatty acids on determination of human serum albumin thiol group Vesna B. Jovanovic ´ a , Ivan D. Pavic ´ evic ´ a , Marija M. Takic ´ b , Ana Z. Penezic ´ -Romanjuk a , Jelena M. Ac ´ imovic ´ a , Ljuba M. Mandic ´ a,⇑ a Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade 11158, Serbia b Institute for Medical Research, Laboratory for Nutrition and Metabolism, University of Belgrade, Belgrade, Serbia article info Article history: Received 18 September 2013 Received in revised form 13 November 2013 Accepted 25 November 2013 Available online 4 December 2013 Keywords: HSA-thiol group determination Fatty acid Albumin isolation Affinity chromatography Diabetes abstract During investigation of the changes of the Cys34 thiol group of human serum albumin (HSA) (isolated by affinity chromatography with Cibacron Blue (CB)) in diabetes, we found that the HSA-SH content was higher (11–33%) than the total serum thiol content. The influence of fatty acids (FA) binding to HSA on this discrepancy was investigated in vitro (using fluorescence and CD spectroscopy and GC) and with HSA samples from diabetic (n=20) and control groups (n=17). HSA-bound FA determine the selection of HSA molecules by CB and enhance reactivity and/or accessibility of the SH group. A high content of polyunsaturated FA (35.6%) leads to weaker binding of HSA molecules to CB. Rate constants of DTNB reac- tion with the SH group of HSA applied to a CB column, bound-HSA and unbound-HSA fractions, were 4.810 -3 , 21.610 -3 , and 11.210 -3 s -1 , respectively. The HSA-SH group of diabetics is more reactive com- pared with control individuals (rate constants 20.910 -3 ±4.410 -3 vs 12.910 -3 ±2.610 -3 s -1 , P<0.05). Recovery values of the SH group obtained after chromatography of HSA with bound stearic acid ranged from 110 to 140%, while those for defatted HSA were from 98.5 to 101.7%. Thus, HSA-bound FA leads to an increase of HSA-SH content and a contribution to total serum thiols, which make the determination of the thiol group unreliable. Ó 2013 Elsevier Inc. All rights reserved. Human serum albumin (HSA) 1 is the most abundant plasma pro- tein (0.6 mM). It is a 66.5-kDa protein organized into three homol- ogous domains (labeled I–III) and each domain comprises two subdomains (A and B) that share common structural elements [1,2]. A total of 17 disulfide bridges located exclusively within sub- domains contribute toward HSA’s stability [3]. HSA transports many endogenous ligands such as long chain (C13–C21) fatty acids (FA), hemin, bilirubin, and thyroxin, all of which bind HSA with high affinity [3]. Although most ligands for HSA are hydrophobic anions, heavy metals are also known to bind to this protein [1,3–5]. Moreover, HSA has the ability to bind a wide variety of drug molecules and alter their pharmacokinetic param- eters [6]. This binding occurs via hydrophobic cavities in subdo- mains IIA and IIIA, known as Sudlow I and Sudlow II, respectively [7,8], with the sole tryptophan residue in HSA located in Sudlow I (Trp-214) [9]. HSA is the primary transporter for delivering FA to the tissues and possesses at least seven binding sites of varying affinities for this ligand (Fig. 1) [10–12]. Although none of the FA-binding sites are completely identical, each comprises a hydrophobic pocket that interacts with the hydrocarbon chain, while five binding sites cap FA at one end with basic or polar residues that interact closely with the carboxyl group of bound FA [13]. Under normal physio- logical conditions, between 0.1 and 2 mol of FA are bound to HSA, but the molar ratio of FA/HSA can rise above 6:1 in the peripheral vasculature during fasting or extreme exercise [5,14] or under pathological conditions such as diabetes, liver disease, and cardiovascular disease [3,15]. In order to completely under- stand the role of HSA in vivo, it is crucial to obtain detailed infor- mation about the variety of ligands that HSA binds, as well as how these ligands interact and influence each other during binding to HSA. Besides its role in transport, HSA is one of the most important extracellular antioxidants [3]. The one free cysteine-derived thiol (-SH) group (Cys34) (located in subdomain I), which can exist in both reduced and oxidized forms, provides a part of the antioxi- dant property of HSA. As HSA is the most abundant plasma protein 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.11.030 ⇑ Corresponding author. Address: Department of Biochemistry, Faculty of Chem- istry, University of Belgrade, P.O. Box 51, Studentski trg 16, 11158 Belgrade, Serbia. Fax: +381 11 2184 330. E-mail address: ljmandic@chem.bg.ac.rs (L.M. Mandic ´). 1 Abbreviations used: BCG, bromocresol green; CB, Cibacron Blue; CD, circular dichroism; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; DTNB, 5,5 0 - dithiobis-(2-nitrobenzoic acid); FA, fatty acids; GC, gas chromatography; HSA, human serum albumin; HSA-SH, human serum albumin thiol content; PUFA, polyunsaturated fatty acids; RSD, relative standard deviation; TG, triglyceride; UV, ultraviolet. Analytical Biochemistry 448 (2014) 50–57 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio