Interaction Studies of Coumaroyltyramine with Human Serum Albumin and Its Biological Importance Satyabala Neelam, † Mahesh Gokara, † Babu Sudhamalla, † Damu G. Amooru, ‡ and Rajagopal Subramanyam* ,† Department of Biochemistry, School of Life Sciences, UniVersity of Hyderabad, Hyderabad, Andhrapradesh 500046, India, and Department of Chemistry, Yogi Vemana UniVersity, Kadapa, Andhrapradesh 516003, India ReceiVed: October 24, 2009; ReVised Manuscript ReceiVed: January 21, 2010 N-trans-p-Coumaroyltyramine (CT) isolated from Physalis minima is a phenolic substance exhibiting many pharmacological activities like potent inhibition of acetyl cholinesterase, cell proliferation, platelet aggregation, and also antioxidant activity. Here, we have studied the binding of CT with HSA at physiological pH 7.2 by using fluorescence, circular dichroism spectroscopy, mass spectrometry, and molecular docking methods. From the fluorescence emission studies, the number of binding sites and binding constant were calculated to be 2 and (4.5 ( 0.01) × 10 5 M -1 , respectively. The free energy change was calculated as -7.6 kcal M -1 at 25 °C, which indicates the hydrophobic interactions of CT with HSA and is in well agreement with the computational calculations and molecular docking studies. The changes in the secondary structure of HSA after its complexation with the ligand were studied with CD spectroscopy, which indicated that the protein became partially unfolded. Also, temperature did not affect the HSA-CT complexes. The binding of CT with HSA was detected as 2 molecules bound to HSA was determined using micro TOF-Q mass spectrometry. Further, molecular docking studies revealed that CT was binding at subdomain IIA with hydrophobic interactions and also by hydrogen-bond interactions between the hydroxyl (OH) group of carbon-16 and carbon-2 of CT and Arg222, Ala291, Val293, and Met298 of HSA, with hydrogen-bond distances of 2.488, 2.811, 2.678, and 2.586 Å, respectively. I. Introduction Human serum albumin (HSA) is the principal extracellular protein of blood plasma, synthesized and secreted from liver cells. 1,2 It plays an important role in maintaining normal osmolarity in plasma as well as in interstitial fluids. It is a globular protein consisting of a single peptide chain of 585 amino acids, largely helical (∼67%) and having 67 kDa mass. It is composed of three structurally homologous domains (I, II, and III), each containing subdomains A and B stabilized by 17 disulfide bridges. Each domain contains 10 helices; helices 1-6 form the respective subdomains A, and helices 7-10 comprise subdomains B. Aromatic and heterocyclic ligands were found to bind within two hydrophobic pockets in subdomains IIA and IIIA, described as site I and site II. 1-7 Seven binding sites are localized for fatty acids in subdomains IB, IIIA, IIIB, and on the subdomain interfaces. 7 HSA has the high affinity metal binding site at the N-terminus. 1 It binds a variety of substrates, including hormones, metal ions, fatty acids, amino acids, and diverse drugs. 1,8,9 The abundance of HSA in the circulatory system and its extraordinary acceptor capabilities makes it an important tool in the prognosis of pharmacokinetic behavior of several drugs. 8,9 It has been shown that the distribution, free concentration, and the metabolism of various drugs can be significantly altered as a result of their binding to HSA. 1 HSA is a widely studied protein because its primary structure is well- known and its tertiary structure has been determined by X-ray crystallography. 1 There are many reports containing studies on HSA structure and its interactions with different ligands. 10-18 Often, more than 90% of the drugs are bound to the protein, which significantly influences the drug efficacy, the rate of drug delivery, and its elimination. Thus, interactions with plasma proteins, especially HSA, are important factors to be considered in drug development. Very recent reports from our group 19,20 showed that the natural products of pentacyclic triterpenoids, betulinic acid and feruloyl maslinic acid isolated from Tephrosia calophylla and Tetracera asiatica, respectively, were binding strongly to HSA, leading to ligand-HSA complexation. 19,20 The importance of phytochemicals is rapidly being recog- nized, and extensive research is consequently focused on finding their beneficial effects and on evaluating their potential impact on human health. Phenolic substances are widely distributed in the plant kingdom, the products of higher plants secondary metabolism in response to external stimuli. Among these, hydroxycinnamate-tyramine conjugates derived from the phe- nol propanoid pathway are considered to be defense chemicals that protect plants against pathogen and herbivore attack. 21 Phenolic amides have cytotoxic, anti-inflammatory, antineoplas- tic, cardiovascular, mild analgesic activity, and antifungal properties. 21 N-trans-p-Coumaroyltyramine (CT) is a phenolic amide, mainly produced in plants during wounding and defense mechanisms by the formation of reactive oxygen species during suberization, pathogen attack. 22 The in vitro studies suggest that CT has the growth suppress- ing activity of tumor cell lines and arrests the cells at the S-phase of the cell cycle by inhibition of protein tyrosine kinases. 23 CT can inhibit Mycobacterium tuberculosis growth rate, and also this compound showed significant inhibitory effects on platelet * Corresponding author. Tel.: +91-40-23134572. Fax: +91-40-23010120. E-mail: srgsl@uohyd.ernet.in. † University of Hyderabad. ‡ Yogi Vemana University. J. Phys. Chem. B 2010, 114, 3005–3012 3005 10.1021/jp910156k  2010 American Chemical Society Published on Web 02/05/2010