Characterization of the Interaction between Acetylisovaleryltylosin Tartrat and Bovine Serum Albumin without or with Zn2+ and Cu2+ by Spectroscopic Analysis

DENG Feng-yu, HU Tao-ying, ZHOU Shan-shan,2, LIU Ying,2*

1. College of Life and Environmental Sciences, Minzu University of China, Beijing 100081, China

2. Beijing Engineering Research Center of Food Environment and Public Health, Minzu University of China, Beijing 100081

Characterization of the Interaction between Acetylisovaleryltylosin Tartrat and Bovine Serum Albumin without or with Zn2+and Cu2+by Spectroscopic Analysis

DENG Feng-yu1, HU Tao-ying1, ZHOU Shan-shan1,2, LIU Ying1,2*

1. College of Life and Environmental Sciences, Minzu University of China, Beijing 100081, China

2. Beijing Engineering Research Center of Food Environment and Public Health, Minzu University of China, Beijing 100081

Acetylisovaleryltylosin tartrate (ATLL) is a new macrolide veterinary antibiotic, it is necessary to study the binding of ATLL to protein, which will directly correlate with the efficiency in vivo. Bovine serum albumin (BSA) is structure homologous with human serum albumin (HSA), and is commonly chosen as a model to investigate drug-protein interaction.There are many metal ions in plasma, as yet, the studies on mainly focus on single metal ion. In this study, the multiple systems formed by ATLL and BSA without or with Zn2+and Cu2+have been studied by mult-spectroscopy. The results showed that, the fluorescence of BSA was quenched by ATLL through a static quenching mechanism. The effective quenching constant (Ka) of ATLL to BSA decreased with Zn2+and increased with Cu2+. Thermodynamic parameters revealed that hydrogen bonds and hydrophobic forces played significant roles in the binding of ATLL to BSA. The polarity of tryptophan and tyrosine residues changed when adding ATLL with or without Zn2+and Cu2+. FT-IR spectra showed that ATLL changedα-helix andβ-sheet of BSA intoβ-turn and random structure. The UV-Vis spectra indicated that the effects of Zn2+on ATLL binding to BSA may cause by a competition binding, and Cu2+possibly formed Cu2+-ATLL complex via metal ion bridge. All the knowledge obtained in this work will be helpful to understand the transport mechanism of ATLL with BSA and the effect of metal ions on the interaction of drug-protein in vivo.

Acetylisovaleryltylosin tartrat; Bovine serum albumin; Metal ion; Spectroscopy

Introduction

Acetylisovaleryltylosin tartrate (ATLL) is a new macrolide veterinary antibiotic, which has good effect on staphylococcus aureus, streptococcus pyogenes, streptococcus pneumoniae, etc., and overcomes the shortcoming of poor effect on pleural actinobacillus[1]. Its activity is higher than tylosin in anti-bacterial and anti-mycoplasma, and has special effect on mycoplasma which is resistant to tylosin and many other antibiotics[2]. ATLL is also used as food additive to promote animal growth. However, as a new drug, to our knowledge, there are few reports on the interaction of ATLL with protein. Therefore, it is necessary to study the binding of ATLL to protein, which will directly correlate with the efficiency in vivo.

Serum albumin is the most common soluble protein in the body and the most prominent protein in the plasma. It acts as the depot and transport protein for numerous endogenous and exogenous substances such as fatty acids, hormones, metals and drugs. The affinity of a drug toward plasma protein is an important determinant of its pharmacokinetics profile affecting its absorption, distribution, metabolism, excretion and consequently levels of the drug’s activity and toxicity[3]. Bovine serum albumin (BSA) is structure homologous with human serum albumin (HSA), and thus is commonly chosen as a model to investigate drug-protein interaction[4].

In plasma, the essential trace metal ions participate many biochemical processes, affect the reactions between drugs and serum albumin, influence the distribution, pharmacological property, metabolism of drugs and so on[4]. It is important to study the effect of metal ions on the binding of the drug with serum albumin. As yet, the studies on this aspect mainly focus on single metal ion, while there are many metal ions in the body and they may have more complex effects on drugs binding to serum albumin. It is important to investigate drug-serum albumin interaction with multiple metal ions. Cu2+is the third most abundant trace mineral, and Zn2+is one of the most essential life trace elements in body. They or their complexes can react with serum albumins, which then can affect the interaction of drugs and serum albumin[5-6]. Hence, it is necessary to investigate the interaction of ATLL with BSA in the presence of Zn2+and Cu2+. In this study, the multiple systems formed by ATLL and BSA without or with Zn2+and Cu2+have been offered to research with mult-spectroscopy.

1 Experimental

1.1 Apparatus

F-4500 spectrofluorimeter (Hitachi, Japan) equipped with a 1.0 cm quartz cell and a Xenon lamp, the VERTEX70 FT-IR spectrometer (Brooke, Germany) equipped with a germanium attenuated total reflection (ATR) accessory, a DTGS KBr detector and KBr beam splitter and UV-2800 spectrophotometer (Hitachi, Japan) were used.

1.2 Reagents

BSA (Fraction V, Genview) and ATLL (ECO Animal Health Ltd., UK) stock solutions were prepared with the concentrations of 1.0×10-4and 1.0×10-3mol·L-1, respectively and kept in dark at 0～4 ℃. 0.1 mol·L-1NaCl, CuSO4and ZnCl2solution (1.0×10-4mol·L-1) were used, pH 7.40, 0.05 mol·L-1Tris-HCl buffer solution.

1.3 Procedure

Tris-HCl buffer solution, NaCl, appropriate amount of BSA solution, Cu2+, Zn2+and ALTT solutions were added into 10 mL tubes in turn. The mixture was diluted to 10 mL with ultrapure water, vortex-mixed and incubated for 1 h at 298 K or 310 K, respectively, and then, the characterization of interaction between ALTT and BSA without or with Zn2+and Cu2+were studied by spectroscopic analysis.

2 Results and discussions

2.1 Fluorescence quenching of BSA by ATLL in absence and presence of Zn2+, Cu2+

The fluorescence spectra of BSA with different concentrations of ATLL in the absence and presence of Zn2+and Cu2+were measured (Fig.1). It could be seen that BSA had a strong fluorescence emission at around 350 nm and the fluorescence intensity of BSA decreased regularly with a red shift on addition of ATLL regardless without or with Zn2+and Cu2+, which suggested that ATLL could interact with BSA. The emission peak position and shape with Zn2+or Cu2+at different concentrations of ATLL were similar to that without Zn2+and Cu2+, while the fluorescence quenching extent was larger than that without metal ions. When the same final concentration of ATLL (1.2×10-5mol·L-1) was added to BSA, BSA-Zn2+, BSA-Cu2+and BSA-Zn2+-Cu2+systems, respectively, the fluorescence intensity was decreased about 40.7%, 42.9%, 56.8%, 51.5%, successively. The results indicated that the presence of Zn2+or Cu2+had effects on the interaction of ATLL with BSA in varying degrees, and both Zn2+and Cu2+enhanced the quenching effect of BSA fluorescence induced by ATLL.

Fig.1 Fluorescence spectra of BSA-ATLL systems without or with Zn2+ and Cu2+

cBSA=1.0×10-6mol·L-1;cZn2+=cCu2+=1.0×10-5mol·L-1;cATLL(1～7): 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 (×10-5mol·L-1);T=310 K

2.2 Quenching mechanism analysis and effecting of Zn2+ and Cu2+ on the binding constants for ATLL binding to BSA

In order to elucidate the quenching mechanism, the fluorescence quenching data were analyzed with the Stern-Volmer equation, The physical meaning of the formula refers to literature[7]

The Stern-Volmer plots were shown in Fig.2 and the error bars were calculated using the standard error of the means of three independent experiment. The calculated quenching constantsKSVat corresponding temperatures were listed in Table 1. The results showed that in all systemsKSVis inversely correlated with temperature, that is, the fluorescence quenching of BSA was static quenching.

Fig.2 Stern Volmer plots of BSA-ATLL systems without or with Zn2+ and Cu2+cBSA=1.0×10-6 mol·L-1. Data are mean values±standard errors of three independent experiments

Table 1 Stern-Volmer quenching constants of BSA-ATLL systems without or with Zn2+and Cu2+

SystemsT/KKSV/(L·mol-1)RaATLL?BSA2983105 03×1044 31×1040 99570 9977ATLL?BSA?Zn2+2983104 83×1043 96×1040 99950 9965ATLL?BSA?Cu2+2983105 27×1044 58×1040 99880 9969ATLL?BSA?Zn2+?Cu2+2983105 12×1044 51×1040 99760 9992

a:Ris the correlation coefficient

For static quenching, the modified Stern-Volmer equation[7]was applied to determine the quenching way

The physical meaning of the formula refers to literature[7]. As shown in Table 2, when the temperature was increased, decreasing trend ofKawas in accordance withKSV’s dependence on temperature no matter without or with Zn2+or Cu2+, which coincided with the static quenching mechanism. From Table 2 we can also see that the presence of Zn2+decreased theKaof ATLL to BSA. This would shorten the storage time of ATLL and hence more amount of free ATLL would be available in plasma, and enhance ATLL’s maximum effects, but might lead to the need for more doses of ATLL to achieve the desired therapeutic effect. TheKaincreased when Cu2+was added, which would decrease the concentration of ATLL

in plasma, indicating longer storage time of ATLL in blood plasma. The binding constants of ATLL to BSA in the presence of both Zn2+and Cu2+were higher than that of without Zn2+or Cu2+and higher than that of with Zn2+but lower than that of with Cu2+. Furthermore, the variation ofKawith both Zn2+and Cu2+was not the sum of changes with Zn2+and with Cu2+.

Table 2 Effective quenching constants and thermodynamic parameters

a:Ris the correlation coefficient

2.3 Thermodynamic parameters and the binding force

The thermodynamic parameters can then be calculated from the Van’s Hoff equation[8]

ΔG=ΔH-TΔS=-RTlnKa

As shown in Table 2, the negative values for free energy (ΔG) in all systems suggested that the binding process was spontaneous, the hydrogen bonds and hydrophobic forces played significant roles in the binding of ATLL to BSA[9].

2.4 Synchronous fluorescence investigation in the absence and presence of Zn2+ and Cu2+

2.5 FT-IR investigation on BSA secondary structure

In infrared spectra of proteins, the amide I is the most widely used in studies of protein secondary structures as it is more sensitive to the changes than amide Ⅱ[10]. The secondary structure changes of BSA as a consequence of ATLL binding without or with Zn2+and Cu2+were studied through evaluation of amide Ⅰ band of the BSA FT-IR spectra. By means of secondary derivative and fourier self-deconvolution, curve-fitting process, the secondary structure components of BSA were estimated (Fig.3, Table 3). After adding ATLL at the study concentration in the absence of Zn2+and Cu2+, the amount ofα-helix decreased markedly (from 48.3% to 45.6%) accompanied withβ-sheet little decreasing (from 21.3% to 20.9%),β-turn increasing (from 19.0% to 20.3%) and random structure increasing (from 11.4% to 13.2%). The effects of Zn2+were mainly on theβ-helix and random structure, and Cu2+almost had the same effects trend in the BSA second structure change. Zn2+together with Cu2+further promotedα-helix,β-sheet structure changing intoβ-turn and random structure.

Table 3 Percentages of BSA secondary structure

2.6 UV-vis spectra investigation and effect of Zn2+ and Cu2+ on the binding model

Fig.3 Curve-fitted infrared spectra of BSA amide Ⅰ band before and after adding of ATLL, Zn2+ and Cu2+

The results ofKa(Table 2) showed the decreased binding constant of ATLL-BSA complex in the presence of Zn2+, which were consistent with two distinct interpretations: there is the existence of the competition of Zn2+and ATLL binding to BSA, which may decrease the binding capability of ATLL to BSA; or Zn2+induced the conformational changes of BSA, which is more difficult for ATLL binding to BSA. The increased binding constant of ATLL-BSA complex with Cu2+possibly results from two aspects as follows: Cu2+-ATLL complex was formed via metal ion bridge, which may enhance the binding capability of ATLL to BSA; or Cu2+induced the conformational changes of BSA, which is easier for ATLL binding to BSA.

To explore the influence of Zn2+and Cu2+on the interaction between ATLL and BSA, the UV-vis spectra of ATLL with different concentrations of Zn2+and Cu2+were measured (Fig.5). The absorption intensity and peak position of ATLL almost had no change with increasing concentration of Zn2+. This implied that ATLL didn’t interact with Zn2+. Comparing the UV-Vis spectra of BSA and BSA-Zn2+, they were overlapped (shown in the insert of Fig. 4 BSA-ATLL-Zn2+), which indicated that Zn2+at the research concentration did not change the conformation of BSA. Therefore, it is reasonable to assume that the effect of Zn2+on BSA-ATLL binary system was through a competition binding with ATLL rather than conformational change of BSA induced by Zn2+, and this resulted in the lower binding constant of BSA and ATLL. While the ATLL absorption decreased markedly with obvious red shift following the addition of Cu2+, indicating the formation of compound between Cu2+and ATLL. From the insert of Fig.4 BSA-ATLL-Cu2+, we found that the spectra of BSA had apparent change when Cu2+was added into BSA solution. This implied that Cu2+could bind to BSA forming Cu2+-BSA complex. Thus in the BSA-ATLL-Cu2+ternary system, it is possible that Cu2+-ATLL complex formed via metal ion bridge, and enhanced the binding capability of ATLL to BSA.

Fig.4 UV-Vis spectra of BSA-ATLL systems without or with Zn2+ and Cu2+

Fig.5 UV-Vis spectra of ATLL with Zn2+ or Cu2+

3 Conclusions

In this paper, the interaction between ATLL and BSA was studied by multi-spectroscopic techniques in the absence and presence of Zn2+and Cu2+. All results indicated that the interaction of BSA and ATLL was a static quenching. The presence of Zn2+decreased the binding constant, while Cu2+increased the binding constant of ATLL to BSA. ATLL changed the polarity of tryptophan and tyrosine residues of BSA no matter without or with Zn2+and Cu2+. FT-IR spectra indicated that BSA secondary structure changed when adding ATLL withα-helix andβ-sheet structure turning intoβ-turn and random structure. The UV-Vis spectra revealed that the effect of Zn2+on ATLL binding to BSA may through competition binding, and Cu2+may form Cu2+-ATLL complex via metal ion bridge.

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*通訊聯(lián)系人

O657.3

A

光譜法研究酒石酸乙酰異戊酰泰樂菌素與牛血清白蛋白的相互作用及Zn2+，Cu2+的影響

鄧鳳玉1, 胡濤英1, 周珊珊1,2, 劉 穎1,2*

1. 中央民族大學(xué)生命與環(huán)境科學(xué)學(xué)院，北京 100081

2. 中央民族大學(xué)北京市食品環(huán)境與健康工程技術(shù)研究中心，北京 100081

酒石酸乙酰異戊酰泰樂菌素(ATLL)是一種新的大環(huán)內(nèi)酯類獸用抗菌藥，研究ATLL與蛋白質(zhì)的相互作用非常重要，這直接與ATLL在體內(nèi)的藥效相關(guān)。牛血清白蛋白(BSA)結(jié)構(gòu)上與人血清白蛋白(HSA)同源，因此，常用它作研究藥物與蛋白質(zhì)相互作用的蛋白模型。血液中有許多金屬離子，已有關(guān)于體內(nèi)單一離子對藥物與蛋白質(zhì)相互作用影響的研究，采用多光譜方法對ATLL與BSA相互作用及Zn2+和Cu2+的影響進(jìn)行研究。結(jié)果表明，BSA的熒光猝滅屬于靜態(tài)猝滅，Zn2+和Cu2+分別使有效猝滅常數(shù)降低和增大。主要的相互作用力為氫鍵和疏水作用力。ATLL改變蛋白質(zhì)色氨酸和酪氨酸的微環(huán)境極性。紫外光譜分析發(fā)現(xiàn)，Cu2+可能是通過Cu2+-ATLL復(fù)合物以金屬離子架橋作用來影響ATLL與BSA的作用，Zn2+可能通過與ATLL的競爭作用結(jié)合BSA。紅外光譜分析表明，ATLL使BSA的β-折疊和α-螺旋結(jié)構(gòu)向β-轉(zhuǎn)角和無規(guī)卷曲轉(zhuǎn)變。這些基礎(chǔ)數(shù)據(jù)有助于闡明在生理?xiàng)l件下，有無Zn2+，Cu2+時(shí)ATLL與BSA的相互作用機(jī)制及金屬離子在藥物與蛋白質(zhì)作用過程中對蛋白質(zhì)功能的影響。

酒石酸乙酰異戊酰泰樂菌素； 牛血清白蛋白； 金屬離子； 光譜法

2015-06-18，

2015-10-13)

Foundation item： The National Natural Science Foundation of China (21177163), 111 Project B08044, First-class University First Class Academic Program of Minzu University of China (YLDX01013), Coordinate Development of First-Class and First-Class University Discipline Construction Funds(10301-0150200604), The Academic Team Construction Project of Minzu University of China (2015MDTD25C&13C), 2015MDTD08C

10.3964/j.issn.1000-0593(2016)07-2351-07

Received： 2015-06-18; accepted： 2015-10-13

Biography： DENG Feng-yu, (1983—), Master of College of Life and Environmental Sciences, Minzu University of China e-mail: dengfengyu0627@163.com *Corresponding author e-mail: liuying4300@163.com