Urea-induced binding between diclofenac sodium and bovine serum albumin: a spectroscopic insight Neeraj Dohare, Abbul Bashar Khan, Fareeda Athar, Sonu Chand Thakur and Rajan Patel* ABSTRACT: We investigated the interaction of diclofenac sodium (Dic.Na) with bovine serum albumin (BSA) in the absence and presence of urea using different spectroscopic techniques. A fluorescence quenching study revealed that the Stern–Volmer quenching constant decreases in the presence of urea, decreasing further at higher urea concentrations. The binding constant and number of binding sites were also evaluated for the BSA–Dic.Na interaction system in the absence and presence of urea using a modified Stern–Volmer equation. The binding constant is greater at high urea concentrations, as shown by the fluorescence results. In addition, for the BSA–Dic.Na interaction system, a static quenching mechanism was observed, which was further confirmed using time-resolved fluorescence spectroscopy. UV–vis spectroscopy provided information about the formation of a complex between BSA and Dic.Na. Circular dichroism was carried out to explain the conformational changes in BSA induced by Dic.Na in the absence and presence of urea. The presence of urea reduced the α-helical content of BSA as the Dic.Na concen- tration varied. The distance r between the donor (BSA) and acceptor (Dic.Na) was also obtained in the absence and presence of urea, using fluorescence resonance energy transfer. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: diclofenac sodium; bovine serum albumin; quenching; FRET Introduction The most commonly studied globular protein in the blood plasma and circulatory system is serum albumin (1,2), which has a major role in the transportation of fatty acids, amino acids, steroids and metal ions, on binding with these ligands. Serum albumin contrib- utes to 80% of the osmotic blood pressure (3), and is also respon- sible for the maintenance of blood pH (4). Bovine serum albumin (BSA) consists of a single polypeptide chain folded into a tertiary globular conformation forming three domains (5,6), with a micro- heterogeneous population of amino acids in its native form (7,8). It is basically made up of a single polypeptide chain with a well- known sequence of amino acids, and contains three homologous α-helix domains (I–III); each domain includes 10 helixes that are di- vided into six anti-parallel helixes and four subdomains. In total, 582 amino acid residues contribute to the formation of a BSA molecule, with the tryptophan at position 134 buried in a hydro- phobic pocket of domain IIA and that at position 212 forming an internal part of domain IIIA. The structural homology of BSA shows 76% similarity with human serum albumin (HSA) (9), but there are differences in the distance between subdomains IIA and IIB, the number of amino acids (582 in BSA and 585 in HSA) and the number of tryptophan molecules (two in BSA and one in HSA) (10). Diclofenac sodium (Dic.Na), a nonsteroidal anti-inflammatory drug, is used as an active compound in analgesic, anti-pyretic and anti- rheumatic medication. It is a derivative of phenylacetic acid and its chemical name is 2-(2,6-dichloroanilino)phenylacetic acid. It is a light-sensitive, weak acid (pK a = 4.0) with a protein-binding ability of ~ 99% and is utilized in the form of a sodium or potassium salt (11). The interaction of BSA with Dic.Na is very important for under- standing the pharmacokinetics and pharmacodynamics of both components. The binding of Dic.Na with BSA plays a large part in its bioavailability, because the drug fraction bound to BSA act as a depot or reservoir, whereas the free drug fraction shows pharmaco- logical effects. In summary, the absorption, distribution, metabo- lism and excretion of a drug can be significantly affected by its binding to the BSA (12). Previous studies have mainly focused on the traditional interaction of Dic.Na with proteins (11,13), and the interaction of Dic.Na with urea-induced proteins has been studied only rarely (14). Urea is a by-product of amino acid catabolism acid and is transported in the plasma from the liver for excretion in urine, sweat and tears (15). Therefore, in this study, we investigated the effect of a low urea concentration (i.e. 0.05 and 0.1 μM) on the binding between Dic.Na and BSA using various spectroscopic techniques, i.e. UV–vis fluorescence, time-resolved fluorescence and circular dichroism (CD) spectroscopy. Experimental Materials BSA (96% purity, batch no. A2153), Dic.Na salt (batch no. D6899) and urea (batch no. V800441) were purchased from Sigma Aldrich (St. Louis, USA), and were all used without further purification. All * Correspondence to: R. Patel, Biophysical Chemistry Laboratory, Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (A Central Uni- versity), New Delhi-110025, India. Tel.: +91-8860634100; Fax: +91-1126983409. E-mail: rpatel@jmi.ac.in Biophysical Chemistry Laboratory, Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India Luminescence 2016; 31: 945–951 Copyright © 2015 John Wiley & Sons, Ltd. Research article Received: 8 July 2015, Revised: 16 September 2015, Accepted: 6 October 2015 Published online in Wiley Online Library: 13 November 2015 (wileyonlinelibrary.com) DOI 10.1002/bio.3055 945