Colloids and Surfaces B: Biointerfaces 112 (2013) 374–379 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb Binding study of novel anti-diabetic pyrimidine fused heterocycles to -lactoglobulin as a carrier protein Mohammad Hossein Mehraban a , Reza Yousefi a,∗ , Asghar Taheri-Kafrani b , Farhad Panahi c , Ali Khalafi-Nezhad c a Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran b Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan 81746-73441, Iran c Department of Chemistry, College of Sciences, Shiraz University, Shiraz, Iran article info Article history: Received 12 June 2013 Received in revised form 2 August 2013 Accepted 9 August 2013 Available online 28 August 2013 Keywords: -Lactoglobulin Pyrimidine-fused heterocycles Fluorescence intensity Circular dichroism Thermodynamic parameters abstract Bovine milk -lactoglobulin (-LG) demonstrates significant resistance against both gastric- and simu- lated duodenal digestions. Therefore, it seems a realistic protein candidate for safe delivery and protection of particularly pH sensitive drugs in stomach. Recently, pyrimidine fused heterocycles (PFHs) revealed inhibitory properties against -glucosidase (-Gls) which is an important target enzyme for those drugs playing significant role in treatment of type-II diabetes and HIV/AIDS infection. The delivery of these compounds to small intestine where the enzyme plays its biological function is of great importance. Therefore, in this work the interaction of PFH compounds with -LG, as a carrier protein has been inves- tigated. Fluorescence, circular dichroism (CD) and UV–vis spectroscopic studies were used to examine the binding parameters and binding modes of the interaction. Moreover, the effects of PFH complexation on the secondary structures of -LG were studied. All of these compounds significantly quenched the fluorescence intensity of -LG due to a ground state complex formation. The binding and thermodynamic parameters were calculated. While hydrophobic interactions were proved to play significant role in the interaction of L 1 , L 2 and L 3 , hydrogen bonding was shown to be important in the complexation of L 4 . The secondary structures of -LG were preserved upon interaction of these synthetic compounds. Based on the achieved results, these potentially therapeutic agents can significantly bind to -LG. Consequently, this protein might be useful for delivery of PFH compounds to small intestine where representing their potential ability to inhibit -Gls and to reduce the postprandial hyperglycemia in diabetic patients. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Bovine whey protein -lactoglobulin (-LG) is a small globular protein (MW = 18 kDa) with known primary, secondary and three- dimensional structure but with still unknown biological functions [1]. At the physiological conditions, this protein normally exists in dimeric form, whereas at acidic pH it dissociates into monomers due to electrostatic repulsion between the subunits [2]. -LG is a well-known member of the hydrophobic transporter superfamily, termed lipocalin. According to the crystal structure of this trans- porter, its three dimensional structure consists of eight anti-parallel -sheets, forming a -barrel calyx which is responsible for the major binding properties of this protein [3,4]. -LG demonstrates significant affinity towards specific binding to a wide variety of ∗ Corresponding author. Tel.: +98 7116137617/7116137665; fax: +98 7112280916. E-mail address: ryousefi@shirazu.ac.ir (R. Yousefi). hydrophobic ligands such as retinoids, steroids, fatty acids, vita- mins and cholesterol [5–9]. This protein is an exceptional transporter due to its stability in acidic milieu of the stomach. At pH 2.0, it reversibly dissociates to monomers, while its native structure remains intact [10]. Fur- thermore, the native structure of -LG is considerably resistant to pepsin degradation at low pH of stomach [11,12]. Because of these exceptional properties, it is possible to detect intact -LG in small intestine, where most of its digestion occurs [13]. Therefore this protein may serve as a carrier for safe delivery of especially non- soluble ligands which their target molecules are existed in the small intestine. Various biological activities such as anticancer, anti- inflammatory, antioxidant, antiviral and antimicrobial activities have been ascribed to pyrimidine fused heterocycles (PFHs) [14–16]. These compounds consist of a pyrimidine ring which in turn is a crucial chemical moiety, as it is existed in a wide number of alkaloids, drugs, antibiotics and antimicrobial agents [17]. Also, the simple bioactive PFHs such as purine and pteridine can be found in the structure of natural compounds [18,19]. Furthermore, 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.08.013