Published: April 27, 2011 r2011 American Chemical Society 6312 dx.doi.org/10.1021/jp2008978 | J. Phys. Chem. B 2011, 115, 6312–6320 ARTICLE pubs.acs.org/JPCB Exploring the Mechanism of Fluorescence Quenching in Proteins Induced by Tetracycline Uttam Anand, Chandrima Jash, Ravi Kiran Boddepalli, Aseem Shrivastava, and Saptarshi Mukherjee* Department of Chemical Sciences, Indian Institute of Science Education and Research, Bhopal ITI Campus (Gas Rahat) Building, Govindpura, Bhopal 462 023, Madhya Pradesh, India ABSTRACT: The binding of the antibiotic tetracycline hydrochloride (TC) to three proteins was investigated by steady-state, time-resolved, and circular dichroism spectroscopy. The tryptophan (Trp) amino acid residues were used as an intrinsic fluorophore to decipher the structurefunction relationship. As monitored by CD spectroscopy, the addition of TC causes the protein to alter some of its helical content although such changes are only marginal. The gradual decrease in fluorescence intensity of Trp can be ascribed to static quenching which takes place by the interaction of the drug with the protein. Besides Trp quenching, there is evidence of fluorescence resonance energy transfer (FRET) in all three proteins with different values of efficiency of energy transfer. Various quenching/binding and thermodynamic parameters associated with such drugprotein interactions have been estimated. The results thus obtained can provide guidelines to synthetic chemists to design and synthesize target-oriented drugs. ’ INTRODUCTION The binding of drugs to proteins has been recognized as an important factor in drug availability, drug efficacy, and drug transport for many years. 1 Although the topic of drugprotein interactions has drawn considerable research interest of late and has been extensively studied, 26 the mechanism of such interac- tions is still not clear. Among such drugprotein interactions, the use of tetracyclines (TC) occupies a seminal position. 7,8 TC molecules consist of four linearly fused tetracyclic nuclei (rings designated as A, B, C, and D, Scheme 1) to which a variety of functional groups are attached. TCs are a group of broad spectrum antibiotics which act as protein synthesis inhibitors, inhibiting the binding of aminoacyl-tRNA to the mRNAribosome com- plex, mainly by binding to the 30S ribosomal subunit in the mRNA complex. 9 Photosensitive and phototoxic properties 10 of TC causing skin lesions, 11 papular eruptions, 12 and formation of multinucleated giant cells 13 have been documented. These drugs after ingestion are solubilized inside the gut and are subsequently transported to the target sites through blood, bound to serum proteins. Human serum albumin (HSA) is the most abundant protein in the circulatory system and is responsible for the binding and transport of a wide variety of fatty acids, drug molecules, and metabolites to their molecular targets. 14 It is a single polypeptide chain having 585 amino acid residues, characterized by low tryptophan and high cysteine content. 15 The secondary structure of the protein consists of 67% R-helix having 6 turns and 17 disulfide bridges. 16 Under physiological conditions, HSA adopts a heart-shaped three-dimensional structure having three homo- logous domains IIII. 15 Each of these three domains are further subdivided into two subdomains A and B that consist of 4 and 6 R-helices, respectively. 14,15 Using X-ray crystallography, He and Carter showed that the two halves of the albumin molecule form a 10 Å wide and 12 Å deep crevice that house a single tryptophan (W214) residue in the binding site IIA of the protein. 15 In spite of the complexity in shape and size, HSA has only a single tryptophan (Trp) residue, which makes it very convenient to study the structure and dynamics of the protein using intrinsic fluorescence. Received: January 27, 2011 Revised: April 7, 2011