Interaction of weakly bound antibiotics neomycin and lincomycin with bovine and human serum albumin: biophysical approach Received March 5, 2010; accepted March 22, 2010; published online March 30, 2010 Neelam Keswani, Sinjan Choudhary and Nand Kishore* Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India *Nand Kishore, Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India. Tel: þ91-222-5767-157, Fax: þ91-222-5767-152, E-mail: nandk@chem.iitb.ac.in The thermodynamics of interaction of neomycin and lincomycin with bovine serum albumin (BSA) and human serum albumin (HSA) has been studied using isothermal titration calorimetry (ITC), in combination with UVvisible, steady state and time resolved fluor- escence spectroscopic measurements. Neomycin is observed to bind weakly to BSA and HSA whereas lincomycin did not show any evidence for binding with the native state of these proteins, rather it interacts in the presence of surfactants. The ITC results suggest 1 : 1 binding stoichiometry for neomycin in the studied temperature range. The values of the van’t Hoff en- thalpy do not agree with the calorimetric enthalpy in the case of neomycin, suggesting conformational changes in the protein upon ligand binding, as well as with the rise in the temperature. Experiments at differ- ent ionic strengths, and in the presence of tetrabutyl ammonium bromide and surfactants suggest the pre- dominant involvement of electrostatic interactions in the complexation process of neomycin with BSA and HSA, and non-specific interaction behaviour of linco- mycin with these proteins. Keywords: binding of antibiotics to serum albumin/ bovine serum albumin/isothermal titration calorim- etry/lincomycin/Neomycin. Abbreviations: BSA, bovine serum albumin; HSA, human serum albumin; HTAB, hexadecyltrimethyl ammonium bromide; SDS, sodium dodecyl sulphate; TBAB, tetrabutylammonium bromide; TX-100, triton X-100. Knowledge of the mechanisms of interaction between drugs and plasma proteins is of crucial importance in understanding the pharmacodynamics and pharma- cokinetics of a drug (1). Therefore studies on drug protein interactions have increasingly attracted the research attention of chemists, pharmacists and biologists (2). Microcalorimetry has been widely used in understanding the thermodynamic aspects of drugprotein interactions (39). Serum albumin is the major extracellular protein of plasma, accounting for 60% of the total plasma protein content, having a concentration of 3450 g dm 3 [(500750)  10 6 mol dm 3 ]. Serum albumin possesses a unique capability to bind, covalently or reversibly, a great number of various endogenous and exogenous com- pounds (10). An understanding of the features of drug interactions with albumin can provide insights into drug therapy and design. Bovine serum albumin (BSA) has been one of the most extensively studied proteins, particularly because of its 76% structural homology with human serum albumin (HSA) (11). It has been shown that the distribution, free concen- tration and the metabolism of various drugs may be strongly affected by drugprotein interactions in the blood stream (12). Neomycin (Fig. 1A) has been a widely used antibiot- ic which belongs to the water soluble aminoglycoside family, produced by Streptomyces fradiae. It is known to prohibit the growth of Gram-positive bacteria and Gram-negative bacteria (13, 14), though it may cause ototoxicity and nephrotoxicity. Neomycin can disturb protein synthesis in bacteria by binding 30S subunit of ribosomal RNA. Therefore, it can cause misreading of the genetic code and inhibit translation (15). It is also used as a preventive measure for hepatic encephalop- athy and hypercholesterolemia. Neomycin is used to treat gastrointestinal infections of cattle, sheep, pigs, goats and poultry by the oral route and to treat mas- titis by intramammary administration (16). Therefore the interaction behaviour of neomycin with the plasma proteins is crucial to the health of humans and animals. Lincomycin (Fig. 1B) is a lincosamide antibiotic isolated from the bacteria of the genus Streptomyces. Although similar in structure, antibacterial spectrum, and in mechanism of action to macrolides they are also effective against other species as well i.e. actino- mycetes, mycoplasma (17), and some species of Plasmodium (18). A lincosamide antibiotic produced by S. lincolnensis is used as the hydrochloride salt (18). Lincomycin hydrochloride is a well-established antibiotic drug used in human and veterinary medi- cine. It is effective primarily against Gram-positive pathogens and has been used in a considerable variety of illnesses, including infections of the mouth and upper respiratory tract, as well as skin infections (19). To the best of our knowledge the binding thermo- dynamics of neomycin and lincomycin with serum albumin has not been reported in literature, though some qualitative studies are available (20, 21). J. Biochem. 2010;148(1):71–84 doi:10.1093/jb/mvq035 ß The Authors 2010. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved 71 at Indian Institute of Technology - Bombay on January 2, 2011 jb.oxfordjournals.org Downloaded from