The interactions between biological macromolecules and small molecules have attracted great interest in recent years.1–3 Among bio-macromolecules, serum albumin is the major soluble protein http://www.cusabio.com/catalog-13-1.html constituent of the circulatory system and has many physiological functions. It acts as a transport protein for many endogenous and exogenous compounds and plays a pharmacological role in the colloid blood pressure and maintenance of blood pH value.4–6 Therefore, studies on the binding of drugs with albumin can provide useful information of the metabolism and transporting process of drugs, and hence become an important research field in chemistry, life sciences and clinical medicine.7,8 In addition, drug–albumin complex may be considered as a model for gaining general fundamental insights into drug–protein binding, because of the availability, stability, and extraordinary binding capacity of albumin.9 In this regard, bovine serum albumin (BSA) has been extensively studied because of its structural homology with human serum albumin (HSA).10–12
Polyphenols, the biggest group of natural anti-oxidants, are widely distributed in the plant kingdom and are present in considerable amounts in fruits, vegetables, and beverages in the human diet.13 These compounds have attracted much attention due to their physiological functions, namely, in the prevention of coronary heart disease, cancer, and inflammation.14–16 Hydroxy-cinnamic acid derivatives (HCAs), such as chlorogenic, caffeic, m, p-coumaric, ferulic and sinapic acid, also known as phenolic compounds, comprise one of the largest and most ubiquitous groups of plant metabolites,17 and have been confirmed to possess multiple biological and pharmacological properties including antioxidant,18,19 antiviral,20 antimicrobial,21 antityrosi-nase,22 hepatoprotective actions23 and modulation of signal transduction pathway.24 Interestingly, the difference of their structures leads to different effects in function. For example, Chiang et al.25 showed that caffeic acid and chlorogenic acid which contain two hydroxyl groups in the phenyl ring exhibit more potent activity against herpesviruses and adenoviruses infections than those bearing one hydroxyl group such as ferulic acid and p-coumaric acid. Moreover, Wen et al.26 determined the antimicrobial activity of several phenolic acids against five strains of L. monocytogenes, and found that cinnamic acid exhibited the strongest activity, followed by p-coumaric, ferulic and caffeic acids, while chlorogenic acid was ineffective even at the maximum concentration tested (1.0% (w/v)). Their results suggested that increased hydroxylation of cinnamic acid molecule clearly reduced activity. These studies show that HCAs biological properties may depend both on their chemical structure, such as the number and position of the hydroxyl groups in the aromatic ring,27,28 and on their affinity with the molecules of enzymes and other biological macromolecules.29,30 Therefore, investigations on HCAs–BSA recognition processes may throw light on studies about drug metabolism and drug–protein interaction. In fact, there have been several studies on the interaction between various phenolic acids and BSA by means of fluorescence method.31,32 However, so far none of these investigations determined the binding epitope and evaluated binding modes and structure affinity in detail, which may limit our proper and comprehensive understanding of the interaction between HCAs and BSA.
NMR spectroscopy offers a variety of approaches for the characterization of drug–protein Interactions,33–36 such as the saturation transfer difference (STD) NMR experiments for the analysis of binding epitopes at atomic resolution and the relaxation rate experiments for the investigation of binding affinity.37–40 In addition, fluorescence spectroscopy is also an appropriate method to study the interaction between drugs and proteins http://www.cusabio.com/.
In the present work, we have employed a combination of NMR, fluorescence and computational techniques, in an attempt to determine where and how HCAs bind to BSA in solution. Several structural analogues of HCAs were investigated to identify the binding epitopes and their binding sites on BSA by STD NMR. 1H NMR relaxation and fluorescence experiments ranked the analogues according to their relative binding affinity yielding detailed structure–affinity relations. Molecular docking was utilized to validate experimental data and to more accurately characterize the models of BSA–HCAs complexs. Our work thus is intended to provide a framework for elucidating the mechanisms of BSA–HCAs binding.
