Spectrochimica Acta Part A 74 (2009) 1135–1137 Contents lists available at ScienceDirect Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa Spectrophotometric analysis of flavonoid–DNA binding interactions at physiological conditions Naveed Kausar Janjua a,∗ , Asima Siddiqa b , Azra Yaqub a , Sana Sabahat a , Rumana Qureshi a , Sayed ul Haque c a Department of Chemistry, Quaid-i-Azam University, Islamabad 45230, Pakistan b National Centre for Physics, Quaid-i-Azam University, Islamabad, Pakistan c Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan article info Article history: Received 13 January 2009 Received in revised form 11 August 2009 Accepted 12 September 2009 Keywords: Flavonoids ds.DNA Binding constants Physiological conditions Body temperature abstract Mode of interactions of three flavonoids [morin (M), quercetin (Q), and rutin (R)] with chicken blood ds.DNA (ck.DNA) has been investigated spectrophotometrically at different temperatures including body temperature (310 K) and at two physiological pH values, i.e. 7.4 (human blood pH) and 4.7 (stomach pH). The binding constants, K f , evaluated using Benesi–Hildebrand equation showed that the flavonoids bind effectively through intercalation at both pH values and body temperature. Quercetin, somehow, showed greater binding capabilities with DNA. The free energies of flavonoid–DNA complexes indicated the spontaneity of their binding. The order of binding constants of three flavonoids at both pH values were found to be K f(Q) > K f(R) > K f(M) and at 310 K. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Reactivity of deoxyribonucleic acid (DNA) offers the analyti- cal chemist a powerful tool in the recognition and monitoring of many biologically important compounds [1]. DNA contains all of the genetic information related to cellular function, including DNA replication and gene expression. However, DNA molecule can be easily damaged, e.g., by reactive oxygen species (ROS) and reactive nitrogen species (RNS) [2,3]. Therefore, in a health pre- venting perspective, there is a deep interest in identifying free radical scavengers or antioxidants that inhibit oxidative DNA dam- age. Flavonoids are non-nutritive compounds of plants which pos- sess broad pharmacological activities like antioxidant, antiallergic, anti-inflammatory, antimicrobial, anticancer, antiviral, antitumor, antimutagenic and antiviral [4,5]. The nature and dynamics of binding of small molecules (drugs and flavonoids) to biomacro- molecules like DNA represent an active area of investigation which can lead to rational drug design [6]. In the present work, interactions of three flavonoids namely; morin, quercetin and rutin with chicken blood ds-DNA have been investigated via spectrophotometric method at two physiological pH values, i.e. 4.7 (stomach pH) and 7.4 (blood pH) and differ- ∗ Corresponding author. Tel.: +92 51 90642146; fax: +92 51 90642241. E-mail address: nkausarjanjua@yahoo.com (N.K. Janjua). ent temperatures including body temperature (310 K). Formation constant of flavonoid–DNA complex and free energy were calcu- lated which showed the strength of bonding between flavonoid and DNA. It was inferred from UV spectral changes that DNA deformation occurred upon interaction with flavonoids proba- bly via intercalation although quercetin formed stronger complex [7]. 2. Experimental The electronic absorption spectra were recorded on Shimadzu 1601 spectrophotometer equipped with a Julabo F-34 thermostat (±0.1 ◦ C) using 1.0 cm matched quartz cells. Chicken blood DNA extracted in lab was dissolved in auto- claved distilled water and its concentration was determined spectrophotometrically at 260 nm using molar extinction coeffi- cient, ε 260 = 6600 cm -1 M -1 . A ratio of absorbance at 260 nm to that at 280 nm, A 260 /A 280 > 1.8 indicated that DNA was sufficiently pure and free from protein [8]. Three flavonoids, quercetin hydrate (Acros Organics), morin (BDH Biochemicals), and rutin trihydrate (Sigma) were used without purification. Experiments were con- ducted in (a) 0.1 M acetic acid–sodium acetate buffer solution, containing 50 mM KCl with pH 4.7 and (b) 0.1 M sodium phos- phate buffer solution with pH 7.4. The concentration of flavonoid was kept constant and the amount of DNA was varied at pH 4.7 and 7.4 and at different temperatures; 293, 298, 303, 310, 313 and 318 K. 1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2009.09.022