Binding Interaction Between Prazosin and Immobilized Receptor by Frontal Analysis
Xinfeng Zhao, Haiyan Lu, Jing Huang, Jianbin Zheng, Xiaohui Zheng & Youyi Zhang Chromatographia An International Journal for Rapid Communication in Chromatography, Electrophoresis and Associated Techniques ISSN 0009-5893 Volume 75 Combined 7-8 Chromatographia (2012) 75:411-415 DOI 10.1007/s10337-012-2198-4
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Author's personal copy Chromatographia (2012) 75:411–415 DOI 10.1007/s10337-012-2198-4
SHORT COMMUNICATION
Binding Interaction Between Prazosin and Immobilized Receptor by Frontal Analysis Xinfeng Zhao • Haiyan Lu • Jing Huang • Jianbin Zheng • Xiaohui Zheng • Youyi Zhang
Received: 8 November 2011 / Revised: 25 January 2012 / Accepted: 26 January 2012 / Published online: 25 February 2012 Ó Springer-Verlag 2012
Abstract Binding interaction between ligand and G-protein coupled receptor plays an important role in the several steps of exertion of therapeutic effect by a drug and has become increasingly interesting to pharmacists, chemists, biologists and companies. a1-Adrenoceptor (a1-AR) was purified from cell line which stably expressed a1-AR and evenly immobilized on the surface of macroporous silica gel to prepare a novel stationary phase for investigating the interaction between drug and receptor. Control drugs of a1-AR including phentolamine, terazosin and urapidil were used to characterize the retention properties of the stationary phase containing the receptor. Further work was performed to calculate the binding sites and association constant of prazosin binding to the immobilized receptor. The results presented a value of 4.55 9 10-7 M-1 for binding sites and of 2.2 9 104 M for the association constant during the interaction between prazosin and a1-AR. The proposed affinity method was stable at least in seven consecutive days, as well as had the primary bioactivities of recognizing and binding the ligand, providing an alternative for representing the interaction between drug and functional protein.
X. Zhao H. Lu J. Huang X. Zheng (&) College of Life Sciences, Northwest University, 195#, No. 229, Taibai North Road, Xi’an 710069, Shaanxi, China e-mail:
[email protected] J. Zheng Institute of Analytical Science, Northwest University, Xi’an 710069, China Y. Zhang (&) Institute of Vascular Medicine, Peking University, Third Hospital and Key Laboratory of Molecular Cardiovascular Sciences Ministry of Education, Beijing 100083, China e-mail:
[email protected]
Keywords Affinity chromatography Immobilized receptor Association constant Prazosin a1-Adrenoceptor
Introduction Traditional Chinese medicine increasingly interests chemists, pharmacists, biologists and companies as an important resource of drug discovery [1–3]. Classic strategies using animal model [4], cell or enzyme-based assays [5, 6] and fractionation approach [7] have been successfully applied in screening bioactive compounds from traditional Chinese medicine and have made great contribution to drug discovery. Technologies are continuously in demand for rapidly identifying new leads and reducing the number of potential candidates, to counter the disadvantages of timeconsuming, labor-intensive and environment unfriendly traditional assays [8–11]. As is widely known, the key sequential steps for drugs to exert their pharmaceutical activities involve specifically being recognized and combining with the receptors on the membrane to activate signal transmission. A fast and stable affinity chromatographic method needs to be established for investigating the interaction between receptor and ligand, and screening the bioactive compounds specifically binding to the receptor in a complex system by immobilizing the receptor on the surface of the stationary phase. The proposed assay will enable the use of non-volatile buffers in the receptor–ligand binding reaction, generating the properties of being environment friendly, as well as having the advantages of high specificity and separating capacity. In previous reports, Wainer and his colleagues [12–14] constructed a novel immobilized receptor-based stationary phase using physical methods and applied it in screening
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receptor ligands and studying interaction between ligand and receptors. Schriemer and co-workers [15, 16] developed a frontal affinity chromatography–mass spectrometry platform for lead discovery. In the above studies, a sample containing a set of ligands and a non-binding ‘marker’ compound are passed through a column onto which a receptor has been attached through physical adsorption. The non-binding marker compounds elute in the void volume, while each ligand elutes at a later time depending on its affinity to the immobilized receptor. The superfamily of G-protein coupled receptors (GPCRs) is the largest one of all receptor types [17]. In general, GPCRs represent the major proportion of targets for drug discovery and most of the medicines in the current market target GPCRs. Full understanding of the interaction between drugs and GPCRs reveals the biologic basis and activity of the ligands and enables designing new bioactive compounds [18]. a1-Adrenoceptor (a1-AR) is a member of GPCRs, mainly distributed in blood vessels and heart, which is closely related to signal mediation of catecholamines, adrenaline and noradrenaline [19]. These neurotransmitters play important roles in fighting diseases of the cardiovascular system [20, 21]. In our previous work, beta2-adrenoceptor was immobilized on the surface of macroporous silica gel to construct a new kind of stationary phase [22]. The assay proves to be an alternative for investigating the interaction between drug and receptor and screening bioactive compounds from complex system such as traditional Chinese medicine [23, 24]. This study was designed to develop another stationary phase attaching a1-AR for exploring the interaction between prazosin hydrochloride and the receptor.
Experimental Instrument and Reagents ZZXT-A packing machine was supplied by Dalian Yilite Analytic Instruments Company Limited (Dalian, China). The chromatographic system consisted of an Agilent Technologies Series 1100 binary pump, a Series 1100 column oven, a Series 1100 diode array detector (Waldbronn, Germany) and a Chemistation 5.2 software installation used for data acquisition and processing. Digitoxin, epichlorohydrin and 4-(1-(4-amino-6, 7-dimethoxyquinazoline-2-yl)piperazine-4-carbonyl)benzaldehyde were from Shanghai Chemical Reagents Supplier Company Limited (Shanghai, China), 6B agarose from Bio-sep Technique Company Limited (Xi’an, China) and ˚ , parmacroporous silica gel (SPS 300-7, pore size 300 A ticle size 7.0 lm) from Fuji Silysia Chemical Company Limited (Tokyo, Japan).
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Preparation of Immobilized a1-AR To purify a1-AR, a new affinity resin was synthesized by the following method. As much as 100 mL of agarose 6B resin was rinsed six times with double distilled water to remove the ethanol. The wet resin was then activated by 200 mL of solution containing 0.3 M NaOH, 0.3 g NaBH3 and 10 mL epichlorohydrin, and stirred for 2.0 h before the addition of 10.0 mL 0.3 M NaOH and 9.0 mL epichlorohydrin. The suspension was filtered and washed to a pH of 7.0 after 12 h. An aliquot of 40.0 mL of 2.0 M NaCO3–NaHCO3 buffer (pH 10.5) and 4.0 mL ethylenediamine was added to 40.0 mL of activated resin and stirred for 24 h in a 558C water bath. The resulting resin was modified by 1.0 g 4-(1(4-amino-6,7-dimethoxyquinazoline-2-yl)piperazine-4-carbonyl)benzaldehyde with stirring for 24 h at 608C. Finally, the resin was suspended in ethanol with 0.25 g NaBH4 and stirred overnight before being packed in a glass column (20 9 2.0 cm). A solution containing 50 mM NaCl, 10 mM Tris–HCl, 2 mM EDTA and 0.1% digitoxin was used as mobile phase to equilibrate the column. Human embryonic kidney 293 (HEK293) cells stably expressing a1-AR were prepared and cultured according to a previous report [25]. A lysate of a1-AR cells was loaded into the column and washed with Tris–HCl buffer (50 mM, pH 7.2) containing 0.5 M NaCl, 2 mM EDTA and 0.05% (w/v) digitalin, at a flow rate of 1.0 mL min-1. When the baseline achieved stability, the column was eluted with 200 mL Tris–HCl buffer (50 mM, pH 7.2) in the presence of 500 mM NaCl, 2.0 mM EDTA and 0.1% (w/v) digitoxin. The column was rinsed with 200 mL of the same buffer solution with the addition of 80 lM terazosin in a 0–100% linear gradient, because more than one protein may have bound to the resin in the lysate. Then the solution eluted by terazosin was collected and processed by a size exclusion column (Sephadex G50) to remove the free terazosin. Total protein in the solution was determined by bicinchoninic acid. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the purity and molecular weight of a1-AR. According to our previous reports [22], the a1-AR was immobilized on the surface of macroporous silica gel to construct a new stationary phase consisting of the receptor. Retention characters of columns were studied by the chromatographic behaviors of control drugs. Chromatographic Profile of Control Drugs on a1-AR Column Retention behaviors of phentolamine, terazosin and urapidil on the a1-AR column were triply studied to characterize the chromatographic properties of the immobilized receptor. The mobile phase was Tris–HCl buffer (10 mM, pH 7.2)
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Association Constants Determination of Prazosin with a1-AR As a well-known theory for studying the interaction between immobilized functional protein and drugs, frontal analysis was used to investigate the affinity interaction between prazosin and the receptor. The binding constant between the ligand and the receptor could be calculated using Scatchard equation: 1 1 ½A ¼ þ Vr V0 KA mL mL
ð1Þ
MW / kDa
consisting of 1.0 mM EDTA and 2.0 mM NaCl. The flow rate was 0.6 mL min with a detecting wavelength of 278 nm for phentolamine, 246 nm for terazosin and 268 nm for urapidil. 94.0→ 66.2→ 45.0→ 35.0 → 26.0→ 20.0→ 14.4→
where Vr represents the total volume of the mobile phase at inflexion on the breakthrough curve; V0 denotes the dead volume of the system; mL is the number of the active sites within the column; [A] is the mole concentration of the analyzed ligand in the mobile phase; KA is the association constant between the receptor and the ligand [22]. Experiments of frontal analysis were all performed at 37 °C. The mobile phase consists of two kinds of solutions. One contains 10.0 mM Tris–HCl, 1.0 mM EDTA and 2.0 mM NaCl. The other is the same solution, but with 40 lM prazosin. The column was equilibrated for 30 min using the former mobile phase before being eluted by the mixture of the two solutions until a stable platform was achieved. The void time of the chromatographic system was determined by NaNO2 using the same method.
The purified a1-AR was immobilized on the surface of macroporous silica gel by the method in a previous publication [22]. An aliquot of 1.4 g of stationary phase containing a1-AR was obtained and further packed in a stainless steel column tubule (50 9 4.6 mm; particle size, 7.0 lm) using 5.0 mM Tris–HCl (pH 7.2) as a slurry and propulsive agent.
Results and Discussion
Chromatographic Profile of Control Drugs on a1-AR Column
Preparation of Immobilized a1-AR The calibration curve was plotted to measure total protein in the solution using bovine serum albumin as a standard protein. The regressing equation was y = 0.0301x ? 0.041 with a correlation coefficient of 0.9994. The average concentration of protein in a1-AR elution was determined as 800.8 ± 23.4 ng mL. SDS-PAGE was utilized to measure the molecular weight and the purity of a1-AR. The SDSPAGE of a1-AR solution gave an obvious band corresponding to the standard protein with molecular weight of 66.0 kDa (Fig. 1). This value highly agreed with the characters of a1-AR reported in the literature [26], which confirmed that the proposed protein in the elution was a1-AR. The purity of the protein was further determined as 90.2%.
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Fig. 1 SDS-PAGE of a1-AR solution. Lane 1, protein molecular weight marker (the molecular weights from top to bottom are 94.0, 66.2, 45.0, 35.0, 26.0, 20.0 and 14.4 kD, respectively). Lane 2, 10.0 lL of a1-AR solution. Lane 3, 10.0 lL of cell lysate. The molecular weight (MW) of a1-AR was determined as 66.0 kDa
In the affinity chromatography, the force between sample and stationary phase proved to be the hydrogen bond, hydrophobic interaction and Van der Waals force. The interaction between ligands and immobilized receptor substantially belongs to the affinity process. The mobile phase was accordingly modified through the addition of Tris, EDTA and NaCl to ensure weak interactions between drug and immobilized receptors on the stationary phase, because these reagents were expected to clearly decrease the electrostatic forces between drugs and non-reacting aminos and silanols on the surface of the stationary phase containing the receptor. The retention times of phentolamine, terazosin and urapidil on the a1-AR column were 58.1, 5.7 and 6.8 min, respectively. The corresponding capacity factors were calculated as 35.1, 2.5 and 2.6, which indicate that the
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immobilized receptor retains its bioactivity and has the properties of specifically recognizing and validly separating the ligands in the mixture. Additionally, yohimbine, metoprolol and salbutamol, specific ligands of a2-AR, b1-AR and b2-AR, are not retained on the a1-AR column, which confirmed the specificity of the stationary phase again. The stability of a1-AR was studied by determining the retention times of phentolamine, terazosin and urapidil in 7 consecutive days. The relative standard deviations (RSD) of the retention times on different days were calculated as 0.4%, 4.3% and 1.2%, indicating that retention times of the three drugs remained stable over the 7 days. This was further confirmed by the absence of substantial changes of peak profiles in those days. As a conclusion, the receptor stationary phase had good stability for at least 7 days. Association Constant of Prazosin on a1-AR Column In practice, prazosin is well known for relaxing vascular smooth muscle, expanding peripheral vascular muscle and decreasing peripheral vascular resistance and blood pressure. As a derivative of quinoline compounds, prazosin proved to be a selective blocker of a1-AR by previous report [26], demonstrating the existence of the interaction between prazosin and the receptor. Figure 2 presents the chromatogram of prazosin on the stationary phase containing the receptor. The affinity interaction between prazosin and immobilized a1-AR was again confirmed by the value of 7.3 min for prazosin on the column. This result demonstrates that the immobilized receptor has the capacity to recognize and react with its ligand. Under the proposed chromatographic condition, all the capacity factors of prazosin were more than 1.0 with the
values of 2.3–2.6, which presented a proof of affinity interaction between prazosin and the receptor. In addition, the relative standard deviations of the retention times and capacity factors were 3.4% and 4.3%, respectively, which indicated the good stability and high precision of the method as well as the feasibility of applying the assay in the investigation of the interaction between ligand and receptor. As shown in Fig. 3, different concentrations of 1.0, 2.0, 4.0, 6.0, 8.0, 12.0, 16.0 and 20.0 lM prazosin were prepared and analyzed at 37 °C through frontal analysis. The breakthrough times of prazosin decreased when the concentration increased. The regression of the reciprocal of the breakthrough volume to the concentrations gave a linear curve with the equation of y = 2.2 9 106x ? 99.2 and the coefficient correlation of 0.9863. The binding sites of prazosin on the immobilized receptor were calculated as 4.55 9 10-7 M-1 through the slope of the linear curve. The association constant was further accounted as 2.2 9 104 M using the intercept and the calculated binding sites.
Conclusion This study presented a novel stationary phase by immobilizing a1-AR on the surface of macroporous silica gel. The immobilized receptor had the properties of being stable for at least 7 days, specifically recognizing and binding to its ligand as well as showing high separating capacity from chromatography. The proposed assay will probably provide an alternative for investigating the interaction between ligand and receptor and screening bioactive compounds of the receptor from complex systems such as medicinal plants and traditional Chinese medicine.
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Fig. 2 Chromatogram of prazosin on the a1-AR column. The injection volume was 10.0 lL. The concentration of prazosin was 8.0 lM
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Fig. 3 Frontal analysis of different concentrations of 1.0, 2.0, 4.0, 6.0, 8.0, 12.0, 16.0 and 20.0 lM prazosin. The chromatographic experiments were performed at 37 °C
Author's personal copy Binding Interaction Between Prazosin and Immobilized Receptor Acknowledgments Financial support was kindly provided from the National Natural Sciences Foundation of China (Nos. 21005060), the Chinese Ministry of Education (No. 20106101110001), Technology Support Plan Project (No. 2008BAI51B01) the Department of Education of the Shaanxi Province (No. 11JK0661), and the Key Innovative Project (2010ZDKG-46) of Science and Technology from Shaanxi Province.
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