Differential binding of tetracyclines with serum albumin and induced structural alterations in drug-bound protein
Introduction
Tetracycline (TC) and its derivatives including 5α-hydroxytetracycline (OTC), 6α-deoxy-5α-hydroxy-tetracycline (DOTC), 6-demethyl-7-chlorotetracycline (DMTC), and 7-chloro-tetracycline (CTC) are known for their photo-reactive properties [1], [2], [3], [4], [5]. The photosensitivity and phototoxicity [6], [7] of these pharmacological drugs are manifested as skin lesions [1], onycholysis [3], papular eruptions [6] and formation of multinucleated giant cells [8]. These drugs after ingestion are solubilized in guts and transported to the target sites through blood, bound to serum proteins. The serum albumin as one of the most abundant carrier proteins plays an important role in the transport and deposition of variety of endogenous and exogenous ligands in blood [9], [10], [11], [12], [13], [14], [15], [16]. Indeed, there are two major specific drug binding sites (site I and II) on serum albumin [15], [17]. Albeit, the high-resolution crystal structures revealed the major ligand binding sites on serum albumin [14], [18] the exact location of many ligands is still obscure. Since the overall distribution [19], [20], metabolism and efficacy [21], [22] of many drugs in the body are correlated with their affinities towards serum albumin, the investigation of pharmaceuticals with respect to albumin–drug binding is imperative and of fundamental importance.
Several pharmaceutical drugs including certain antibiotics viz. penicillin, cefoxitin, cefazolin, cephalosporins have been reported to bind reversibly with serum albumin to different extents, depending on their side chain functional groups [23], [24], [25], [26]. Also, the percent binding of TC in human plasma has been extensively studied using ultrafiltration, equilibrium dialysis and spectrophotometric techniques [27], [28], [29]. However, the earlier reported data are inconsistent primarily due to differences in the sensitivity and specificity of methods used to determine the extent of binding [25], [26], [27]. In addition, there are contradictory reports in the literature regarding the role of some divalent metal ions in the binding of TCs with biological macromolecules [28], [30], [31].
Our earlier studies have clearly demonstrated the high binding affinity (Ka=4.6×106 l/mole) of serum albumin with TC molecule [32]. However, the quantitative binding of other clinically important TC derivatives with serum albumin and the resultant structural alterations in the carrier protein are rarely reported. This prompted us to investigate the (i) extent and nature of interactions of TC derivatives with serum albumin, (ii) binding constant (Ka) and capacity (n) of serum albumin for these derivatives, (iii) influence of binding specificities of metal ions on drug–albumin interactions, and (iv) drug-induced conformational changes in protein, using the sensitive techniques such as fluorescence spectroscopy and circular dichroism (CD). Indeed, a better understanding of important binding parameters influencing the transport, metabolism, elimination, bioavailability and toxicity of these drugs in the body is needed. In this regard, the knowledge of the binding characteristics of serum albumin for TCs in presence and/or absence of metal ions will provide useful evidence for establishing a predictable correlation between the drug distribution, stability, pharmacokinetic and phototoxic behavior.
Section snippets
Materials and methods
Bovine serum albumin (BSA, grade V) and warfarin were purchased from Sigma Chemical Co., St. Louis, MO, USA. The TC derivatives viz. OTC, DOTC, DMTC, CTC were obtained from Hi-Media, India. Bilirubin and diazepam were obtained from Sisco Research Laboratory and Ranbaxy, India. All other reagents used were of analytical grade. Protein concentration was determined spectrophotometrically using E1%1cm of 6.61 at 280 nm [33] on a Cecil (model CE 594) double beam spectrophotometer.
Results and discussion
The interactions of TC derivatives with albumin were analyzed by measuring the changes in the intrinsic fluorescence of BSA at different drug/protein molar ratios. Fig. 1 shows the representative fluorescence emission spectra of BSA–OTC complex at 300–400 nm in the range of 0–3 protein/drug molar ratios. Similarly, spectra exhibiting proportionate reductions in the intrinsic fluorescence of BSA were obtained with other derivatives viz. DOTC, DMTC and CTC at different molar ratios under
Conclusions
Serum albumin as target protein possesses multiple binding sites for TC derivatives and exhibited differential binding affinity for the ligands. Competitive binding of derivatives in presence of marker ligands, warfarin, bilirubin and diazepam suggests that the binding sites for TCs on BSA are located in vicinity of site I within sub-domain IIA of BSA. Enhanced binding among the BSA and TC derivatives in presence of transition metal ions signifies the formation of metal-ion bridge formation,
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Present address: Department of Biology, Johns Hopkins University, Baltimore, MD, USA.