Journal of Pharmaceutical and Biomedical Analysis
Study of curcumin and genistein interactions with human serum albumin
Introduction
Curcumin 1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiens-3,5-dione (Scheme 1) the main yellow pigment of the powedered rhiome (turmeric) of the herb Curcuma longa has been used for centuries as a spice and food coloring agent [1]. It has also been used to treat diseases such as inflammation, skin wounds and tumors as traditional medicine [2]). Curcumin exhibits antioxidant activity both in vivo and in vitro [1]. Apart from its anti-inflammatory, antimicrobial and antiviral properties curcumin is considered as cancer chemopreventive agent [3], [4]. Mechanisms by which curcumin prevent cancer were attributed to several effects including anti-angiogenic action, up-regulation of enzymes detoxifying carcinogens and inhibition of certain signal transduction pathways for tumor cell growth and neutralization of carcinogenetic free radicals [1], [2]. Curcumin–protein interactions have been studied and the effect of curcumin on inhibition and activation of protein kinase C is reported [5].
Genistein 4′,5,7-trihydroxy isoflavone (Scheme 1) presents in soybean and chick peas has a wide spectrum of physiological and pharmacological functions. It is known to antagonize human melanoma cell growth at G2/M transition [6] and found to inhibit H2O2/Cu(II) mediated DNA strand breaks acting as a direct scavenger of reactive oxygen species with the OH group at C-4 position responsible for its antioxidant activity [7]. Genisten intercalation into DNA and RNA duplexes is known [8], [9], [10] and the effects of genistein on activation of protein phosphatases is well investigated [11].
Human serum albumin (Scheme 2) is the most abundant serum protein, which carries several endogenous compounds including fatty acids. HSA has long been the center of attention of pharmaceutical industry due to its ability to bind various drug molecules and alters their pharmacokinetic properties [12]. HSA is a globular protein composed of three structurally similar domains (I, II and III), each containing two subdomains (A and B) and stabilized by 17 disulphide bridges [13], [14], [15], [16], [17], [18], [19], [20] Aromatic and heterocyclic ligands were found to bind within two hydrophobic pockets in subdomains IIA and IIIA, namely site I and site II [13], [14], [15], [16], [17], [18], [19], [20]. Seven binding sites for fatty acids are localized in subdomains IB, IIIA, IIIB and on the subdomain interfaces [13]. HSA has also a high affinity metal binding site at the N-terminus [14]. These multiple binding sites underline the exceptional ability of HSA to interact with many organic and inorganic molecules and make this protein an important regulator of intercellular fluxes, as well as the pharmacokinetic behaviour of many drugs [13], [14], [15], [16], [17], [18], [19], [20], [21]. Therefore, it was of interest to study the binding of curcumin and genistein on protein secondary structure, conformation and stability.
In this report, the spectroscopic analysis of HSA complexation with curcumin and genistein was carried out in aqueous solution at physiological conditions, using constant protein concentration and various pigment contents. Structural information regarding pigment binding mode and the effects of complexation on the protein stability and secondary structure is reported here.
Section snippets
Materials
HSA fraction V and curcumin and genistein were purchased from Sigma Chemical Company (St-Louis, MO) and used as supplied. Other chemicals were of reagent grade and used without further purification.
Preparation of stock solutions
Human serum albumin was dissolved in aqueous solution (40 mg/ml or 0.5 mM) containing 10 mM Tris–HCl buffer (pH 7.4). The protein concentration was determined spectrophotometrically using the extinction coefficient of 36,500 M−1 cm−1 at 280 nm [22]. In this study, HSA did not have its fatty acids removed,
FTIR spectra of curcumin– and genistein–HSA complexes
The pigment–HSA interaction was characterized by infrared spectroscopy and its derivative methods. Since there was no major spectral shifting for the protein amide I band at 1656 cm−1 (mainly CO stretch) and amide II band at 1544 cm−1 (C–N stretching coupled with N–H bending modes) [24], [25], [26] upon pigment interaction, the difference spectra [(protein solution + pigment solution) − (protein solution)] were obtained, in order to monitor the intensity variations of these vibrations and the results
Summary
Based on our spectroscopic results curcumin and genistein bind human serum albumin via both hydrophilic and hydrophobic interactions with stronger affinity for curcumin than genistein. However, larger binding of genistein induces more perturbations of HSA secondary structure with a partial protein unfolding. The pigment binding site is mainly in the subdomain II A (site I), where tryptophan 214 located.
Acknowledgment
This work is supported by a grant from Natural Sciences and Engineering Research Council of Canada (NSERC).
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