Studies on the Binding Mechanism of Several Antibiotics and Human Serum Albumin

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1 Vol. 63, , ACTA CHIMICA SINICA No. 23, a,b a a a *,a ( a ) ( b ), 26 K A , , , L mol 1, n 1.16, 0.86, 1.19, ; ; ; ; ; ; Studies on the Binding Mechanism of Several Antibiotics and Human Serum Albumin BI, Shu-Yun a,b DING, Lan a SONG, Da-Qian a TIAN, Yuan a ZHANG, Han-Qi*,a ( a College of Chemistry, Jilin University, Changchun ) ( b Chemistry Department, College of Science and Engineering, Yanbian University, Yanji ) Abstract The binding characteristics of antibiotics, cefotaxime sodium, oxacillin, amoxicillin, norfloxacin and enoxacin with human serum albumin (HSA) were studied by fluorescence spectrometry. These drugs quenched the HSA fluorescence via a nonradiative energy transfer mechanism. The binding constants (K A ) and binding sites (n) for drug-hsa were obtained by an improved analysis method to process the experimental data. The values of K A obtained were , , , and L mol 1 for cefotaxime sodium-hsa, oxacillin-hsa, amoxicillin-hsa, norfloxacin-hsa and enoxacin-hsa, respectively, at 26. The values of n were 1.16, 0.86, 1.19, 0.91 and 0.93, respectively, for these drugs in the above order. The electrostatic interaction was found to be main acting force in the studied systems. Keywords human serum albumin; cefotaxime sodium; oxacillin; amoxicillin; norfloxacin; enoxacin; fluorescence quenching (HSA),, [1].,., [2 5], [6]. (cefotaxime sodium) β-, (oxacillin) (amoxicillin) β-, (norfloxacin) (enoxacin)., 1. ph HSA, * analchem@mail.jlu.edu.cn Received ebruary 18, 2005; revised May 8, 2005; accepted August 23, (No ) (No ).

2 2170 Vol. 63, (1) (2) (3) (4) (5) igure 1 Molecular structures of cefotaxime sodium (1), oxacillin (2), amoxicillin (3), norfloxacin (4) and enoxacin (5). [7 10], [11], R-5301PC ( ); GBC Cintra 10e UV-vis ( ); phs-3c ( ); ( ). HSA (, 95%) mol L 1, ( ) mol L 1 ; 0.05 mol L 1 ph 7.4 Tris-HCl 0.1 mol L 1 NaCl;,. 1.2 HSA 10 ml, ph 7.4 Tris-HCl,, 5 min. 2.5 ml, 280 nm, nm, 5 nm, HSA, 280 nm, 340 nm. HSA mol L 1, 2. 2,, HSA 340 nm,,., 280 nm, nm,,. HSA, HSA. 280 nm, (Try-214) (Tyr) nm., 340 nm, Try-214., 340 nm, HSA Try-214. HSA. Stern-Volmer [12] : 0 / 1 K q τ 0 [Q] 1 K sv [Q],, 0 [Q], τ 0,, τ s [13], K q, K sv Stern-Volmer. HSA, Stern-Volmer, 0 / [Q], K q , , , , L s 1 mol L s 1 mol 1 [14], HSA K q. HSA,

3 No (1) (2) (3) (4) (5) HSA igure 2 Effects of cefotaxime sodium (1), oxacillin (2), amoxicillin (3), norfloxacin (4) and enoxacin (5) on the fluorescence spectra of human serum albumin (HSA) c cefotaxime sodium from top to bottom: 0, 2.00, 4.00, 6.00, 8.00, 10.00, and ( 10 5 mol L 1 ); c oxacillin from top to bottom: 0, 0.15, 0.74, 1.47, 2.94, 5.89, and ( 10 4 mol L 1 ); c amoxicillin from top to bottom: 0, 0.50, 1.00, 2.00, 4.00, 6.00, 8.00 and ( 10 4 mol L 1 ); c norfloxacin from top to bottom: 0.00, 2.04, 4.08, 6.12, 8.16, 10.20, and ( 10 6 mol L 1 ); c enoxacin from top to bottom: 0.00, 2.00, 4.00, 6.00, 8.00, 10.00, and ( 10 6 mol L 1 ); c HSA mol L 1 HSA., 26, 37 HSA, 26 HSA K A ( 1) 37 K A : , , , L mol 1., K A. HSA.

4 2172 Vol. 63, Table 1 Binding constants and binding sites of the drugs and HSA (26 ) Drug Method 1 Method 2 Method 3 K A /(L mol 1 ) n K A /(L mol 1 ) n K A /(L mol 1 ) Cefotaxime sodium Oxacillin Amoxicillin Norfloxacin Enoxacin HSA K A n, Scatchard [7],,. [8 10],., [11] : log 0 nlog 0 KA nlog([d t ] n[p t ] )( 1), 0 [D t ] [P t ].,, log 0 log([dt ] m 0 ), m 0 n[p t ], K A n. log 0 logka nlog[d f ] [18] ( 2) [9] ( 3) K A n, K [D ] 0 0 A 0 f [D f ], [D f ], [D t ],,,., , 1 K A 2 K A, 3 K A HSA HSA, [4,5] ln K A1 /K A2 (1/T 1 1/T 2 ) G RTln K A H T S H, G, S , 26 37, H 1, H 2, H 3, G 1, G 2, G 3 S 1, S 2, S 3, 2. 3 log[( 0 )/] log[[d t ] m( 0 )/ 0 ] igure 3 Plots of log[( 0 )/] vs. log[[d t ] m( 0 )/ 0 ] for (1) cefotaxime sodium, (2) oxacillin, (3) amoxicillin, (4) norfloxacin and (5) enoxacin at 26 4 log[( 0 )/] log[d f ] igure 4 Plots of log[( 0 )/] vs. log[d f ] for (1) cefotaxime sodium, (2) oxacillin, (3) amoxicillin, (4) norfloxacin and (5) enoxacin at 26, H S, [15], HSA.

5 No Drug H 1 / Table 2 H 2 / 2 -HSA Thermodynamic parameters of drug-hsa binding reaction H 3 / G 1 / G 2 / G 3 / S 1 / S 2 / S 3 / Cefotaxime sodium Oxacillin Amoxicillin Norfloxacin Enoxacin /( 0 ) 1/( 0 [D f ]) igure 5 Plots of 1/( 0 ) vs. 1/( 0 [D f ]) for (1) cefotaxime sodium, (2) oxacillin, (3) amoxicillin, (4) norfloxacin and (5) enoxacin at 26 References 1 Kragh, H. U. Pharmacol. Rev. 1981, 33, Dilson, S.; C lia, M. C.; Sônia, R. W. L. Spectrochim. Acta, Part A 2004, 60, Lilianna, T.-L.; Aldona, K.; Bernhard, K. K.; Henryk, K. J. Inorg. Biochem. 2000, 78, Bi, S.-Y.; Ding, L.; Tian, Y.; Song, D.-Q.; Zhou, X.; Liu, X.; Zhang, H.-Q. J. Mol. Struct. 2004, 703, Bi, S.-Y.; Song, D.-Q.; Tian, Y.; Zhou, X.; Liu, Z.-Y.; Zhang, H.-Q. Spectrochim. Acta, Part A 2005, 61, Iwatsubo, T.; Hirota, N.; Ooie, T.; Uzuki, H.; Sugiyama, Y. Biopharm. Drug Dispos. 1996, 17(4), Scatchard, G.; Ann, N. Y. Acad. Sci. 1949, 51, Alain, M.; Michel, B.; Michel, A. J. Chem. Educ. 1986, 63(4), Jiang, C.-Q.; Gao, M.-X.; Meng, X.-Z. Spectrochim. Acta, Part A 2003, 59, Yi, P.-G.; Liu, J.-.; Shang, Z.-C.; Yu, Q.-S. J. Spectrosc. Spec. Anal. 2001, 21, 826 (in Chinese). (,,,,, 2001, 21, 826.) 11 Bi, S.-Y.; Song, D.-Q.; Kan, Y.-H.; Xu, D.; Tian, Y.; Zhou, X.; Zhang, H.-Q. Spectrochim. Acta, Part A 2005, in press. 12 Biophysical and Biochemical Aspects of luorescence Spectroscopy, Ed.: Deway, T. G., Plenum, New York, 1991, pp Lakowica, J. R.; Weber, G. Biochemistry 1973, 12, Ware, W. R. J. Phys. Chem. 1962, 66, Ross, D. P.; Sabramanian, S. Biochemistry 1981, 20, (A CHENG, B.; AN, Y. Y.)