Interaction of 4-Nitroaniline with Serum Albumin

Transcription

1 Applied Mechanics and Materials Online: ISSN: , Vols , pp doi:1.428/ 214 Trans Tech Publications, Switzerland Interaction of 4-Nitroaniline with Serum Albumin Yanqiu LIANG a, Ying ZHANG b Shenyang University of Chemical Technology, Shenyang 11142, China a Dalin417@163.com, b zhangyingamour@126.com Keywords: 4-nitroaniline, HSA, BSA, fluorescence spectroscopy, thermodynamic parameters. Abstract. Bovine serum albumin (BSA) and human serum albumin (HSA) interaction with 4-nitroaniline were investigated by fluorescence spectroscopy respectively. 4-Nitroaniline can strongly quench intrinsic fluorescence of BSA and HSA. 4-Nitroaniline exhibits a high affinity to bovine and human serum albumins. The binding constants K and the number binding site n were obtained by double-log regression equation. Negative enthalpy (ΔH) and positive entropy (ΔS) values indicated that both hydrogen bond and hydrophobic forces played a major role in the binding of 4-nitroaniline and SA. The results of synchronous fluorescence showed the polarity around tryptophan residues was decreased and the hydrophobicity was increased. Introduction 4-Nitroaniline is the most important medium of many chemical products widely used in many industrial processes such as the manufacture of pharmaceuticals, dyes and synthetic colors [1]. It is strongly toxic and has damage to growth of aquatic lives. As a result, it has been included in the list of priority pollutants in China [2]. Toxicity about 4-nitroaniline was generally assessed according to acute toxicity to living things, such as white rats commonly used. Serum albumin is the main constituent of plasma protein responsible for the binding and transport of many endogenous and exogenous substances such as hormones, fatty acids and the foreign molecule of drugs. Therefore, the interaction between album and low-molecular weight organic compounds such as drugs, pesticides has been investigated successfully [3,4]. However, the binding of poisonous compounds to proteins has seldom been investigated [5]. In our studies, we have a detailed investigation of interaction of 4- nitroaniline of SA by fluorescence spectra. Experimental 4-Nitroaniline were obtained from Shanghai Reagent Co. Ltd., China. Bovine serum album and human serum album were purchased from Sigama. The stock solution of mol/l were prepared by dissolving the solid BSA and HSA in tris-hcl respctively, and diluted to mol/l using ph 7.4 Tris-HCl buffer solution (.5 mol/l Tris,.1 mol/l NaCl). The fluorescence spectra and synchronous fluorescence spectroscopy was performed with Cary Eclipse spectrofluorometer (Varian) equipped with temperature controller and a 15 W Xenon lamp source and 1. cm quartz cell. A 3. ml solution, containing appropriate concentration of BSA (HSA), was titrated by successive additions of a mol/l 4-nitroaniline stock solution of (to give a final concentration of mol/l). Titrations were done manually by using trace syringes. The addition volume of quencher is far less than total volume. Therefore, the change of volume can be ignorable. The fluorescence spectra were then measured (excitation at 28 nm and emission wavelengths of (38-44 nm) at three temperatures (298, 31 and 318 K). Results and discussion Fluorescence quenching. The fluorescence of HSA and BSA with the addition of 4-Nitroaniline as quencher was obtained respectively. (shown in Fig. 1) and the results showed that a gradual decrease in the fluorescence intensity of SA was caused by quenching. In addition, we observed that there was All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications, (ID: , Pennsylvania State University, University Park, USA-5/3/16,23:8:49)

2 F F 338 Environmental Protection and Sustainable Development significant emission wavelength blue shift with the addition of 4-nitroaniline, which indicated that the microenvironment around tryptophan in SA had changed after interacting with 4-nitroaniline. To interpret the data from fluorescence quenching studies, it is important to understand what kind of interaction takes place between the fluorophore (HSA and BSA respectively) and the quencher (4-nitroaniline). If it is assumed that the fluorescence quenching of SA induced by 4-nitroaniline are dynamic quenching process, fluorescence quenching is described by the Stern Volmer equation [6,7]: F /F=1+K SV [Q]=1+K q τ [Q] (1) where F and F are the fluorescence intensities before and after the addition of the quencher, respectively. K q, K SV, τ, and [Q] are the quenching rate constant of the biomolecule, the Stern-Volmer dynamic quenching constant, the average lifetime of the biomolecule without quencher (τ = 1-8 s) and the concentration of the quencher, respectively BSA 25 HSA Fig. 1 Effect of 4- nitroaniline on fluorescence spectra of serum albumin (T=298K) c BSA =c HSA =1.μmol/L in all cases ; peak from up to down, c Q (1.μmol/L ):,.8,1.6,2.4,3.2,4,4.8,5.6,6.4,7.2,8,8.8,9.6,1.4,11.2,12,12.8,13.6 The fluorescence quenching data are usually analysed by the Stern Volmer equation. The results were shown in fig. 2. In many cases, the fluorophore can be quenched both by collision and by complex formation with the same quencher. When this is the case, the Stern Volmer plot exhibits an upward curvature, concave toward the y-axis at high [Q] [8-1]. In our research, For BSA interaction with 4-nitroaniline, both dynamic and static quenching were involved, which was demonstrated by the fact that the Stern Volmer plot slightly deviated from linearity toward the y-axis at high 4-nitroaniline concentrations (Fig. 2). Quenching can also be caused by the formation of a complex between the two compounds after returning from the excited state and that is due to a specific interaction. In contrast, for HSA, interaction with 4-nitroaniline, only static quenching occurs. BSA molecule is formed by 582 amino acid residues, and contains a first tryptophan reside in position 134, in subdomain IB of the albumin molecule, and a second tryptophan residue in position 212, in sub domain IIA. HSA contains 585 amino acid residues with only one tryptophan located at position 214 along the chain, in sub domain IIA [11]. These differences between these two protein molecules help understanding our results in fluorescence quenching studies. Binding constants of BSA and HSA with 4-nitroaniline.we can assume that there are similar and independent binding sites in the serum albumin in the static quenching interaction, the binding constant (K) and the number sites (n) can be obtained from the double logarithm regression curve of log(f F)/F versus log[q] based on the following, which was deduced in previous report [12]:

3 F/a.u. F/a.u. Applied Mechanics and Materials Vols lg(( F F) / F)=nlgK-nlg(1/([Q t ]- ( F F) [P t ]/ F )) (2) K can be determined by the slope of double logarithm regression curve of log(f F)/F versus log[q] based on the Eq. (2). The double logarithm regression curve of SA 4-nitroaniline was linear in the whole small molecule concentration range measured and the binding constant K and binding sites n of 4-nitroaniline are shown in Table 1. The value of n approximately equal to 1 indicates the existence of just a single binding site in BSA for 4-nitroaniline. The system of 4-nitroaniline and HSA was found by setting n = 1.. HSA also has one site for binding 4-nitroaniline. Table 1 The relevant constants and R at different temperature for albumin-4-nitroaniline system albumin BSA HSA Double-lg Thermodynamic parameters T(K) K ΔH θ ΔG θ ΔS θ R n (L mol -1 ) (KJ mol -1 ) (KJ mol -1 ) (J mol -1 K -1 ) Thermodynamic parameters and nature of the binding forces. The interaction forces between drugs and biomolecules may include hydrogen bonds, van der Waals forces, electrostatic, and hydrophobic bonds. They could play a role in ligand binding to proteins[13]. To obtain such information, the thermodynamic parameters were calculated according to the vant Hoff equations[14].table 1 shows the values of ΔH and ΔS obtained for the binding site from the slopes and ordinates at the origin of the fitted lines. Negative enthalpy (ΔH) and positive entropy (ΔS) values indicated that both hydrogen bond and hydrophobic forces played a major role in the binding of 4-nitroaniline and SA[12]. Effect of 4-nitroaniline on the protein conformation.the effect of 4-nitroaniline on SA synchronous fluorescence spectroscopy is shown in Fig.3. It is apparent from Fig. 3 that the maximum emission wavelength does not apparently shift when Δλ= 15 nm, whereas the maximum emission wavelength blueshifts (from 283 to 277nm for BSA; from 281nm to 273 nm for HSA) at the investigated concentration range when Δλ= 6 nm, which is consistent with the fact that the conformation of SA was changed. It is also indicated that the polarity around the tryptophan residues was increased and the hydrophobicity was decreased HSA (a) 8 BSA (b) Fig.3 Effect of 4-nitroaniline on synchronous fluorescence spectra of SA (a) Δλ=15 nm; (b) Δλ=6 nm

4 34 Environmental Protection and Sustainable Development References [1] G. H. Lu, X. Yuan and Y. H. Zhao: Environ. Chem. Vol. 19 (2), p. 225 [2] D. L. Xi, Y. S. Sun: Environmental Monitoring ( Higher Education press, China 21) [3]A. Papadopoulou, R.J. Green and R.A. Frazier: J. Agric. Food Chem Vol. 53 (212), p. 158 [4] C. Dufour, O. Dangles: Biochim. Biophys. Acta Vol (29) p. 164 [5]G. Colmenarejo: Med. Res. Rev. Vol. 23 (23) p [6] J.H. Tang, F. Luan and X.G. Chen: Bioorg. Med. Chem Vol. 14 (26) p. 321 [7] Y.J. Hu, Y. Liu and R.M. Zhao: J. Biol. Macromol Vol. 37 (25) p. 122 [8] E.L. Gelamo, C.H.T.P. Silva, and H. Imasato: Biochim. Biophys. Acta Vol (22) p.84 [9] J.R. Lakowicz: Principles of Fluorescence Spectroscopy (Plenum Press,America 1999) [1]B. Sengupta, P.K. Sengupta: Biochem. Biophys Res. Commun Vol. 299 (22) p.4 [11] X.M. He, D.C. Carter: Nature Vol. 358 (1992) p.29 [12] Y.J. Hu, Y. Liu and J. Wang: J. Pharm. Biomed. Anal Vol. 36 (24) p.915 [13] A. Mallick, S.C. Bera and S. Maiti: Biophys. Chem. Vol. 112 (24) p. 9 [14] S. Bi, D. Song, Y. Tian and X. Zhou: Spectrochim. Acta. Part A Vol. 61 (25) p.629

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