Research article Received: 16 June 2014,
Revised: 9 September 2014,
Accepted: 24 September 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2805
Comparative studies on the interaction of cefixime with bovine serum albumin by fluorescence quenching spectroscopy and synchronous fluorescence spectroscopy Lihui Zhang, Baosheng Liu,* Zhiyun Li and Ying Guo ABSTRACT: Under simulated physiological conditions, the reaction mechanism between cefixime and bovine serum albumin at different temperatures (293, 303 and 310 K) was investigated using a fluorescence quenching method and synchronous fluorescence method, respectively. The results indicated that the fluorescence intensity and synchronous fluorescence intensity of bovine serum albumin decreased regularly on the addition of cefixime. In addition, the quenching mechanism, binding constants, number of binding sites, type of interaction force and energy-transfer parameters of cefixime with bovine serum albumin obtained from two methods using the same equation were consistent. The results indicated that the synchronous fluorescence spectrometry could be used to study the binding mechanism between drug and protein, and was a useful supplement to the conventional method. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: fluorescence quenching spectroscopy; synchronous fluorescence; cefixime; bovine serum albumin; interaction
Introduction Serum albumin, the most abundant protein in blood plasma, can combine with many endogenous and exogenous compounds and plays a fundamental role in the disposition and transportation of various molecules. Investigations into the binding mechanism between endogenous or exogenous compounds and serum albumin are of great value in the life sciences, chemistry, pharmacy and clinical medicine. Conventional fluorescence spectroscopy is used to study the reaction mechanism for small-molecule drugs and proteins, mainly by measuring changes in the fluorescence intensity of the protein at the maximum emission wavelength before and after the addition of drugs. The method is used to derive the binding constants, binding sites and donor-to-acceptor distance, and other information, between proteins and drugs (1,2). Synchronous fluorescence spectrometry was first proposed by Lloyd (3), and the biggest difference between this and the fluorescence measurement method is that the excitation and emission monochromators are scanned simultaneously. Compared with conventional fluorescence spectroscopy, synchronous fluorescence spectrometry shows good selectivity, high sensitivity and less interference (4,5), and it can be used for the simultaneous determination of components in a multicomponent mixture (6). However, use of this method to study the binding constant and binding sites between the protein and the drug has not been reported. Cefixime (CFX) (Fig. 1) is an oral third-generation cephalosporin, and has some advantages such as a broad antimicrobial generation, strong antimicrobial effects and a strong stability to β-lactamases (7). Here, we study the binding mechanism of CFX and proteins by utilizing conventional fluorescence quenching and the synchronous fluorescence method. The data
Luminescence 2014
were obtained and analyzed. The results show that the new method is a useful supplement to the application of fluorescence spectroscopy in the field.
Experimental Apparatus All fluorescence spectra were recorded with a Shimadzu RF-5301PC spectrofluorophotometer. Absorption was measured with an UV/vis recording spectrophotometer (UV-265, Shimadzu, Japan). All pH measurements were carried out with a pHS-3C precision acidity meter (Leici, Shanghai, China). All temperatures were controlled by a CS501 superheated water bath (Nantong Science Instrument Factory).
Materials CFX was purchased from Shanghai Boyle Chemical Co., Ltd. Bovine serum albumin (BSA) was purchased from Sigma Co. and was of the purity grade inferior 99%. Stock solutions of BSA (1.0 × 10–5 M) and CFX (2.0 × 10–3 M) were prepared. All the stock solutions were further diluted for use as working solutions. Tris/HCl buffer (0.05 M Tris, 0.15 M NaCl) was used to maintain
* Correspondence to: B. Liu, Key Laboratory of Medical Chemistry and Molecular Diagnosis, Ministry of Education, College of Chemistry & Environmental Science, Hebei University, Baoding 071002, Hebei Province, People’s Republic of China. E-mail: [email protected] Key Laboratory of Medical Chemistry and Molecular Diagnosis, Hebei University, Baoding People’s Republic of China
Copyright © 2014 John Wiley & Sons, Ltd.
L. Zhang et al.
Figure 1. Chemical structure of cefixime.
the pH of the solution at 7.40, and NaCl solution was used to maintain the ionic strength of the solution. All other reagents were of analytical grade and all aqueous solutions were prepared with newly double-distilled water and stored at 277 K. The fluorescence intensities were corrected for the absorption of excitation light and reabsorption of emitted light to decrease the inner filter effect using the following relationship (8): F cor ¼ F obs eðAex þAem Þ=2
(1)
Where, Fcor and Fobs are the corrected and observed fluorescence intensities, respectively. Aex and Aem are the absorbance values for CFX at excitation and emission wavelengths, respectively. The fluorescence intensity used in this paper was corrected. Procedures Fluorescence measurements. In a typical fluorescence measurement, 1.0 mL of Tris/HCl, pH 7.40, 0.4 mL of 1.0 × 10–5 M BSA solution and different concentrations of CFX were successively added to a 10-mL colorimetric tube. The samples were diluted to scaled volume with water, mixed thoroughly by shaking, and kept static for 30 min at different temperatures (293, 303 and 310 K). The excitation wavelength for BSA was 280 nm and 295 nm, respectively, with a 10 mm path length cell. The excitation and emission slits were set at 5 nm. The solution was subsequently scanned on the fluorophotometer and we recorded the fluorescent intensity at 340 nm. Synchronous fluorescence measurements. Solution preparation was as detailed above; we recorded the fluorescence spectra of the BSA–CFX system when the difference between the excitation and emission wavelengths, Δλ, was 15 and 60 nm.
–5
Figure 2. Absorption spectra of the CFX–BSA system (T =310 K). CCFX =4.0 × 10 M, –6 –7 1–8 CBSA = (0, 0.4, 0.8, 1.2, 2.0, 2.5, 3.0, 3.5) × 10 M, 9 CBSA =4 × 10 M.
the intensity of the peak at 201 nm decreases with a significant red shift of 12 nm. The results indicate that the interaction between CFX and BSA resulted in the formation of a complex between the drug and protein, and the microenvironment around BSA was changed (10).
Fluorescence quenching spectra of BSA-CFX system Proteins are considered to have intrinsic fluorescence due to the presence of amino acids, mainly tryptophan (Trp) and tyrosine (Tyr). When the excitation wavelength was 280 nm (or 295 nm), BSA shows a strong fluorescence emission peak at 340 nm. The fluorescence spectra of the BSA–CFX system are shown in Fig. 3. As shown in Fig. 3, the fluorescence intensity of BSA decreased regularly on addition of CFX when the excitation wavelength is 280 nm (similar to 295 nm). This result showed that CFX could quench the intrinsic fluorescence of BSA strongly and there
UV/vis measurements. Tris/HCl (1.0 mL, pH 7.40), 2.0 mL of 2.0 × 10–4 M CFX solution and different concentrations of BSA were successively added to a 10-mL colorimetric tube. The reference was the corresponding concentration of BSA solution. The samples were diluted to the scaled volume with water, mixed thoroughly by shaking, and kept static for 30 min at 310 K. The UV/vis absorption spectra of CFX in the presence and absence of BSA were scanned with 1 cm quartz cells over the range 190–450 nm.
Results and discussion UV/vis absorption spectra studies UV/vis absorption measurement is a very simple and applicable method to explore structural and microenvironmental changes in proteins (9). The UV/vis absorption spectra of CFX in the absence and presence of BSA are shown in Fig. 2. As shown in Fig. 2, BSA has two absorption peaks: the strong absorption peak at 220 nm and a weak absorption peak at 280 nm. CFX has two absorption peaks: a strong absorption peak at 201 nm and another at 280 nm. On gradual addition of BSA to CFX solution,
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Figure 3. Fluorescence spectra of the CFX–BSA system (λex =280 nm, T =310 K). -7 -7 CBSA =4.0 × 10 M; 1–10 CCFX = (0, 2.0, 3.0, 4.0, 8.0, 20, 40, 60, 80, 100) × 10 M.
Copyright © 2014 John Wiley & Sons, Ltd.
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Studies on the interaction of CFX with BSA by fluorescence was an interaction between CFX and BSA; it also revealed that a new complex is formed (11). To confirm the quenching mechanism, the fluorescence quenching data were analyzed using the Stern–Volmer equation (12): F 0 =F ¼ 1 þ K q τ 0 ½L ¼ 1 þ K sv ½L
(2)
Where, F0 and F represent the fluorescence intensities in the absence and presence of quencher, respectively. τ 0 is the average lifetime of the fluorescence without quencher, and is 10–8 s. Ksv is the Stern–Volmer quenching constant. Kq is the bimolecular quenching constant and [L] is the concentration of the quencher. Fig. 4 shows the Stern–Volmer plots for the quenching of BSA fluorescence by CFX. The curves show a linear relationship with increasing concentrations of CFX. Based on the linear fit plot of F0/F vs. [L], values of Kq could be obtained. The calculated results are shown in Table 1. The different quenching mechanisms are usually classified as dynamic quenching and static quenching. Dynamic and static quenching can be distinguished by their different dependence on temperature. Quenching rate constants decrease with increasing temperature for the static quenching, but the reverse
is observed for dynamic quenching (13). As shown in Table 1, the values of Ksv decreased with increasing temperature in all systems, which indicated that the probable quenching mechanism of the interaction between BSA and CFX was initiated by complex formation rather than by dynamic collision (14). In addition, all values of Kq were much greater than the maximum scatter collision quenching constant of various quenchers (2 × 1010 M–1 · s-1), this also suggested that the quenching was a static process (15). For static quenching, the relationship between the fluorescence intensity and the concentration of quencher could be usually described by equation (3) (16) to obtain the binding constant (Ka) and the number of binging sites (n) in most of the previously published papers: lg½ðF 0 F Þ=F ¼ nlg½L þ lg K a
(3)
Where, Ka is the binding constant of CFX with BSA, Ka, which can be determined from the plot of lg [(F0 – F)/F] vs. lg [L]. Thus we could obtain binding constant Ka and number of binding sites n for CFX with BSA from equation (3), and the calculated results are shown in Table 1. The values of n were all ~1 which suggested that just one binding site for CFX existed in BSA. Meanwhile, the binding constants Ka decreased with increasing temperature, further suggesting that the quenching was a static process (17). The primary binding site studies
Figure 4. Stern–Volmer plots for the quenching of BSA by CFX at different tem–7 -7 -6 peratures. CBSA =4.0 × 10 M, CCFX =4.0 × 10 M and 6.0 × 10 M.
At 280 nm wavelength, the Trp and Tyr residues in BSA are excited, whereas a wavelength of 295 nm excites only Trp residues (18). In BSA, sub-hydrophobic domain IIA (containing both Trp and Tyr) and IIIA (containing only Tyr) are the major binding sites for small molecule ligands (19). Based on the Stern–Volmer equation, comparing the fluorescence quenching of BSA excited at 280 nm and 295 nm allows us to estimate the participation of Trp and Tyr groups in the system (20). As seen in Fig. 5, in the presence of CFX, the quenching curves of BSA excited at 280 nm and 295 nm overlap. This showed that only Trp residues played an important role in the interaction of CFX with BSA. Therefore, it may be implied that the primary binding site for CFX was sub-hydrophobic domain IIA (21). From Table 1, at the same temperature, Ka values at excitation wavelengths of 280 nm and 295 nm were similar, which also suggests that only Trp residues played an important role in the interaction between CFX and BSA.
Table 1. Quenching reactive parameters of BSA and CFX at different temperatures λex (nm)
T (K)
Kq (M–1 · s–1)
Ksv (M–1)
r1
Ka (M-1)
n
r2
280
293 303 310 293 303 310
3.91 × 1012 3.73 × 1012 3.67 × 1012 3.74 × 1012 3.47 × 1012 3.44 × 1012
3.91 × 104 3.73 × 104 3.67 × 104 3.74 × 104 3.47 × 104 3.44 × 104
0.9979 0.9918 0.9981 0.9993 0.9910 0.9984
4.72 × 104 4.20 × 104 3.66 × 104 4.54 × 104 4.31 × 104 3.81 × 104
0.97 0.93 0.96 0.88 0.95 0.94
0.9931 0.9927 0.9908 0.9931 0.9959 0.9950
295
Kq is the quenching rate constant; Ka is the binding constant; n is the number of binding site. r1 is the linear relative coefficient of F0/F – [L]; r2 is the linear relative coefficient of lg(F0 – F)/F – lg[L].
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Copyright © 2014 John Wiley & Sons, Ltd.
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L. Zhang et al.
-7
Figure 5. The curve of F/F0 with CFX/BSA of CFX–BSA (T =310 K). CBSA =4.0 × 10 M, –7 -5 CCFX =2.0 × 10 –1.0 × 10 M.
Synchronous fluorescence spectra studies When the value of Δλ between the excitation and emission wavelengths is stabilized at either 15 or 60 nm, synchronous fluorescence gives characteristic information for tyrosine or tryptophan residues, respectively. It can be seen from Fig. 6 that when Δλ was 15 nm, the fluorescence intensities of BSA–CFX showed no obvious decrease, with no shifts with increasing CFX concentration; this indicated that Tyr residues do not have an important role in the interaction between CFX and BSA. When Δλ was 60 nm, the synchronous fluorescence intensities of CFX–BSA decreased regularly with some shifts, indicating that the interaction of BSA with CFX changed the microenvironment of the tryptophan residues (22), which was coincident with the primary binding site studies. The polarity of the hydrophobic environment was enhanced and hydrophobicity was reduced in the BSA cavity due to changes in the microenvironment of the tryptophan residues on insertion of CFX (23). This led to a conformation change in BSA. Protein molecules were extended by high concentrations of drugs, which reduced energy transfer among the amino acid residues and their fluorescence intensity. The corresponding results for Δλ =60 nm, according to equations (2) and (3), are shown in Table 2. From Table 2, it can be seen the values of Ksv decreased with increasing temperature for all systems, which indicated that the probable quenching mechanism of the interaction between BSA and CFX was static. The value of Kq was in the order of 1012 M–1 · s–1. Obviously, the Kq value for the protein-quenching procedure initiated by CFX was >2 × 1010 M–1 · s–1, which indicated that the quenching was not initiated by dynamic collision but from the formation of a complex. The n value approaches unity, suggesting that one molecule of CFX combines with one molecule of BSA; the trend for decreasing Ka values with increasing temperature was in accordance with dependence of the binding constants on the temperature, as mentioned above, which indicated that CFX–BSA would partly decompose with increasing temperature; this also indicated the process was static quenching. The quenching mechanism obtained by synchronous fluorescence method was coincident with that obtained by fluorescence
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Figure 6. Fluorescence spectrum of CFX–BSA system (T =310 K) (A) Δλ =15 nm; –7 (B) Δλ =60 nm. CBSA =4.0 × 10 M; 1–10 CCFX = (0, 2.0, 3.0, 4.0, 8.0, 20, 40, 60, 80, -7 100) × 10 M.
method. Comparing Tables 1 and 2 shows that the quenching parameters obtained using the two methods are of the same order of magnitude. Type of interaction force in BSA–CFX systems In general, interactions between a small drug molecule and a biological macromolecule include hydrogen bonds, Van der Waal’s forces, electrostatic interactions and hydrophobic force. Ross and Subramanian (24) have characterized the sign and magnitude of the thermodynamic parameters, enthalpy change (ΔH), free energy (ΔG) and entropy change (ΔS), associated with various types of interaction. When temperature varies over a small range, ΔH can be considered constant (25). Many researchers think that a negative value for ΔH (ΔH is very small, almost zero) and a positive value for ΔS indicate that electrostatic interactions have a major role in the binding reaction. Positive ΔH and ΔS values are generally considered to be evidence of typical hydrophobic interactions. In addition, Van der Waal’s forces and hydrogen bond formation in low dielectric media are characterized by negative values of ΔH and ΔS (26). However, from the point of view of water, a
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014
Studies on the interaction of CFX with BSA by fluorescence Table 2. Quenching reactive parameters of CFX and BSA at different temperatures (Δλ =60 nm) T (K)
Kq (M-1 · s-1) 12
293 303 310
4.34 × 10 3.60 × 1012 3.59 × 1012
Ksv (M-1)
r3
4
4.34 × 10 3.60 × 104 3.59 × 104
0.9990 0.9920 0.9997
Ka (M-1) 4
4.88 × 10 4.65 × 104 3.31 × 104
n
r4
0.93 0.95 0.95
0.9967 0.9911 0.9905
Kq is the quenching rate constant; Ka is the binding constant; n is the number of binding site. r3 is the linear relative coefficient of F0/F – [L]; r4 is the linear relative coefficient of lg(F0 – F)/F – lg[L]. positive ΔS value is frequently taken as evidence of hydrophobic interactions. The thermodynamic parameters can be calculated on the basis of equations (4) and (5) (27): Rln K ¼ ΔS ΔH=T
(4)
ΔG ¼ ΔH TΔS ¼ RT lnK
(5)
The type of interaction force for small molecule drugs and biological macromolecules can be obtained from the relevant thermodynamic parameters. The values of ΔH were obtained from the linear van’t Hoff plot (Fig. 7) and are listed in Table 3 along with ΔS and ΔG. From Table 3, it can be seen that the reaction of CFX with BSA was a spontaneous molecular interaction in which entropy increased and Gibb’s free energy decreased (28). From the point of view of the polarity and ionization of CFX, the main interaction of CFX and BSA was hydrophobic
Figure 7. Van’t Hoff plot of the interaction of CFX and BSA in Tris/HCl; pH =7.4.
contact. The conclusions drawn from the synchronous fluorescence method were consistent with the fluorescence method.
Energy transfer from BSA to CFX According to the Förster nonradioactive resonance energy transfer theory (29), the effective energy transfer from donor to acceptor will occur when two molecules meet the following preconditions: (1) the donor is a fluorophore; (2) the overlap is sufficient between the fluorescence emission spectrum of the donor and UV/vis absorption spectrum of the acceptor; and (3) the distance between the donor and the acceptor is within 2–7 nm. The energy transfer effect is not only related to the distance between the donor (tryptophan residue) and acceptor, but also influenced by the critical energy transfer distance R0 and is described by equations (6) and (7) (30,31):
Figure 8. Overlap of the fluorescence spectrum of BSA (λex =280 nm) (1) and (Δλ =60 nm) (2) with the absorption spectrum of CFX (3). (T =310 K) CCFX = CBSA –7 =4.0 × 10 M.
Table 3. The thermodynamic parameters of CFX–BSA at different temperatures T (K) Δλ =60 nm λex =280 nm
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293 303 310 293 303 310
Ka (M-1) 4
4.88 × 10 4.65 × 104 3.31 × 104 4.72 × 104 4.20 × 104 3.66 × 104
ΔH (kJ/mol)
ΔS (J/mol/K)
ΔG (kJ/mol)
–16.14
34.66 36.08 34.46 51.65 51.93 51.61
–26.30 –27.07 –26.82 –26.22 –26.82 –27.08
–11.08
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L. Zhang et al. Table 4. Parameters of E, J, r, R0 between CFX and BSA at different temperatures Method
T (K)
Fluorescence quenching
293 303 310 293 303 310
Synchronous fluorescence
E (%) 4.22 2.86 2.84 2.90 2.87 2.79
J (cm3 · M–1) -15
8.95 × 10 8.94 × 10-15 8.71 × 10-15 8.38 × 10-15 8.40 × 10-15 8.44 × 10-15
R0 (nm)
r (nm)
2.41 2.40 2.39 2.37 2.38 2.38
4.04 4.32 4.31 4.27 4.28 4.30
R0 is the critical distance when E is 50%; r is the distance between the acceptor and donor; J is the overlap integral between the fluorescence emission spectrum of donor and the absorption spectrum of the acceptor. E ¼ 1 F=F 0 ¼ R0 6 = R0 6 þ r 6 R0 ¼ 8:7810 6
25 2
4
K ΦN J
(6)
Acknowledgements
(7)
The authors gratefully acknowledge the financial support of National Science Foundation of China (Grant no. 20675024) and Hebei Provincial Key Basic Research Program (Grant no. 10967126D).
Where R0 is the critical distance when the transfer efficiency is 50%, r is the distance between the acceptor and the donor, and E is the energy-transfer efficiency. K is the spatial orientation factor of the dipole, N is refractive index of the medium, Φ is the fluorescence quantum yield of the donor and the overlap integral (J) between the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor can be calculated using equation (8) (32): J¼
X
X F ðλÞεðλÞλ4 Δλ= F ðλÞΔλ
(8)
Where F(λ) is the fluorescence intensity of the fluorescent donor at a known wavelength and ε(λ) is the molar absorbance of the acceptor at wavelength λ. Figure 8 shows the overlap integral of the fluorescence emission spectrum of BSA and the absorption spectrum of CFX. In this case, K2 = 2/3, N =1.336 and Φ =0.118 (33), according to equations (6)–(8); the corresponding results are shown in Table 4. From Table 4, it can be seen that the donor-to-acceptor distance r
Revised: 9 September 2014,
Accepted: 24 September 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2805
Comparative studies on the interaction of cefixime with bovine serum albumin by fluorescence quenching spectroscopy and synchronous fluorescence spectroscopy Lihui Zhang, Baosheng Liu,* Zhiyun Li and Ying Guo ABSTRACT: Under simulated physiological conditions, the reaction mechanism between cefixime and bovine serum albumin at different temperatures (293, 303 and 310 K) was investigated using a fluorescence quenching method and synchronous fluorescence method, respectively. The results indicated that the fluorescence intensity and synchronous fluorescence intensity of bovine serum albumin decreased regularly on the addition of cefixime. In addition, the quenching mechanism, binding constants, number of binding sites, type of interaction force and energy-transfer parameters of cefixime with bovine serum albumin obtained from two methods using the same equation were consistent. The results indicated that the synchronous fluorescence spectrometry could be used to study the binding mechanism between drug and protein, and was a useful supplement to the conventional method. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: fluorescence quenching spectroscopy; synchronous fluorescence; cefixime; bovine serum albumin; interaction
Introduction Serum albumin, the most abundant protein in blood plasma, can combine with many endogenous and exogenous compounds and plays a fundamental role in the disposition and transportation of various molecules. Investigations into the binding mechanism between endogenous or exogenous compounds and serum albumin are of great value in the life sciences, chemistry, pharmacy and clinical medicine. Conventional fluorescence spectroscopy is used to study the reaction mechanism for small-molecule drugs and proteins, mainly by measuring changes in the fluorescence intensity of the protein at the maximum emission wavelength before and after the addition of drugs. The method is used to derive the binding constants, binding sites and donor-to-acceptor distance, and other information, between proteins and drugs (1,2). Synchronous fluorescence spectrometry was first proposed by Lloyd (3), and the biggest difference between this and the fluorescence measurement method is that the excitation and emission monochromators are scanned simultaneously. Compared with conventional fluorescence spectroscopy, synchronous fluorescence spectrometry shows good selectivity, high sensitivity and less interference (4,5), and it can be used for the simultaneous determination of components in a multicomponent mixture (6). However, use of this method to study the binding constant and binding sites between the protein and the drug has not been reported. Cefixime (CFX) (Fig. 1) is an oral third-generation cephalosporin, and has some advantages such as a broad antimicrobial generation, strong antimicrobial effects and a strong stability to β-lactamases (7). Here, we study the binding mechanism of CFX and proteins by utilizing conventional fluorescence quenching and the synchronous fluorescence method. The data
Luminescence 2014
were obtained and analyzed. The results show that the new method is a useful supplement to the application of fluorescence spectroscopy in the field.
Experimental Apparatus All fluorescence spectra were recorded with a Shimadzu RF-5301PC spectrofluorophotometer. Absorption was measured with an UV/vis recording spectrophotometer (UV-265, Shimadzu, Japan). All pH measurements were carried out with a pHS-3C precision acidity meter (Leici, Shanghai, China). All temperatures were controlled by a CS501 superheated water bath (Nantong Science Instrument Factory).
Materials CFX was purchased from Shanghai Boyle Chemical Co., Ltd. Bovine serum albumin (BSA) was purchased from Sigma Co. and was of the purity grade inferior 99%. Stock solutions of BSA (1.0 × 10–5 M) and CFX (2.0 × 10–3 M) were prepared. All the stock solutions were further diluted for use as working solutions. Tris/HCl buffer (0.05 M Tris, 0.15 M NaCl) was used to maintain
* Correspondence to: B. Liu, Key Laboratory of Medical Chemistry and Molecular Diagnosis, Ministry of Education, College of Chemistry & Environmental Science, Hebei University, Baoding 071002, Hebei Province, People’s Republic of China. E-mail: [email protected] Key Laboratory of Medical Chemistry and Molecular Diagnosis, Hebei University, Baoding People’s Republic of China
Copyright © 2014 John Wiley & Sons, Ltd.
L. Zhang et al.
Figure 1. Chemical structure of cefixime.
the pH of the solution at 7.40, and NaCl solution was used to maintain the ionic strength of the solution. All other reagents were of analytical grade and all aqueous solutions were prepared with newly double-distilled water and stored at 277 K. The fluorescence intensities were corrected for the absorption of excitation light and reabsorption of emitted light to decrease the inner filter effect using the following relationship (8): F cor ¼ F obs eðAex þAem Þ=2
(1)
Where, Fcor and Fobs are the corrected and observed fluorescence intensities, respectively. Aex and Aem are the absorbance values for CFX at excitation and emission wavelengths, respectively. The fluorescence intensity used in this paper was corrected. Procedures Fluorescence measurements. In a typical fluorescence measurement, 1.0 mL of Tris/HCl, pH 7.40, 0.4 mL of 1.0 × 10–5 M BSA solution and different concentrations of CFX were successively added to a 10-mL colorimetric tube. The samples were diluted to scaled volume with water, mixed thoroughly by shaking, and kept static for 30 min at different temperatures (293, 303 and 310 K). The excitation wavelength for BSA was 280 nm and 295 nm, respectively, with a 10 mm path length cell. The excitation and emission slits were set at 5 nm. The solution was subsequently scanned on the fluorophotometer and we recorded the fluorescent intensity at 340 nm. Synchronous fluorescence measurements. Solution preparation was as detailed above; we recorded the fluorescence spectra of the BSA–CFX system when the difference between the excitation and emission wavelengths, Δλ, was 15 and 60 nm.
–5
Figure 2. Absorption spectra of the CFX–BSA system (T =310 K). CCFX =4.0 × 10 M, –6 –7 1–8 CBSA = (0, 0.4, 0.8, 1.2, 2.0, 2.5, 3.0, 3.5) × 10 M, 9 CBSA =4 × 10 M.
the intensity of the peak at 201 nm decreases with a significant red shift of 12 nm. The results indicate that the interaction between CFX and BSA resulted in the formation of a complex between the drug and protein, and the microenvironment around BSA was changed (10).
Fluorescence quenching spectra of BSA-CFX system Proteins are considered to have intrinsic fluorescence due to the presence of amino acids, mainly tryptophan (Trp) and tyrosine (Tyr). When the excitation wavelength was 280 nm (or 295 nm), BSA shows a strong fluorescence emission peak at 340 nm. The fluorescence spectra of the BSA–CFX system are shown in Fig. 3. As shown in Fig. 3, the fluorescence intensity of BSA decreased regularly on addition of CFX when the excitation wavelength is 280 nm (similar to 295 nm). This result showed that CFX could quench the intrinsic fluorescence of BSA strongly and there
UV/vis measurements. Tris/HCl (1.0 mL, pH 7.40), 2.0 mL of 2.0 × 10–4 M CFX solution and different concentrations of BSA were successively added to a 10-mL colorimetric tube. The reference was the corresponding concentration of BSA solution. The samples were diluted to the scaled volume with water, mixed thoroughly by shaking, and kept static for 30 min at 310 K. The UV/vis absorption spectra of CFX in the presence and absence of BSA were scanned with 1 cm quartz cells over the range 190–450 nm.
Results and discussion UV/vis absorption spectra studies UV/vis absorption measurement is a very simple and applicable method to explore structural and microenvironmental changes in proteins (9). The UV/vis absorption spectra of CFX in the absence and presence of BSA are shown in Fig. 2. As shown in Fig. 2, BSA has two absorption peaks: the strong absorption peak at 220 nm and a weak absorption peak at 280 nm. CFX has two absorption peaks: a strong absorption peak at 201 nm and another at 280 nm. On gradual addition of BSA to CFX solution,
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Figure 3. Fluorescence spectra of the CFX–BSA system (λex =280 nm, T =310 K). -7 -7 CBSA =4.0 × 10 M; 1–10 CCFX = (0, 2.0, 3.0, 4.0, 8.0, 20, 40, 60, 80, 100) × 10 M.
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014
Studies on the interaction of CFX with BSA by fluorescence was an interaction between CFX and BSA; it also revealed that a new complex is formed (11). To confirm the quenching mechanism, the fluorescence quenching data were analyzed using the Stern–Volmer equation (12): F 0 =F ¼ 1 þ K q τ 0 ½L ¼ 1 þ K sv ½L
(2)
Where, F0 and F represent the fluorescence intensities in the absence and presence of quencher, respectively. τ 0 is the average lifetime of the fluorescence without quencher, and is 10–8 s. Ksv is the Stern–Volmer quenching constant. Kq is the bimolecular quenching constant and [L] is the concentration of the quencher. Fig. 4 shows the Stern–Volmer plots for the quenching of BSA fluorescence by CFX. The curves show a linear relationship with increasing concentrations of CFX. Based on the linear fit plot of F0/F vs. [L], values of Kq could be obtained. The calculated results are shown in Table 1. The different quenching mechanisms are usually classified as dynamic quenching and static quenching. Dynamic and static quenching can be distinguished by their different dependence on temperature. Quenching rate constants decrease with increasing temperature for the static quenching, but the reverse
is observed for dynamic quenching (13). As shown in Table 1, the values of Ksv decreased with increasing temperature in all systems, which indicated that the probable quenching mechanism of the interaction between BSA and CFX was initiated by complex formation rather than by dynamic collision (14). In addition, all values of Kq were much greater than the maximum scatter collision quenching constant of various quenchers (2 × 1010 M–1 · s-1), this also suggested that the quenching was a static process (15). For static quenching, the relationship between the fluorescence intensity and the concentration of quencher could be usually described by equation (3) (16) to obtain the binding constant (Ka) and the number of binging sites (n) in most of the previously published papers: lg½ðF 0 F Þ=F ¼ nlg½L þ lg K a
(3)
Where, Ka is the binding constant of CFX with BSA, Ka, which can be determined from the plot of lg [(F0 – F)/F] vs. lg [L]. Thus we could obtain binding constant Ka and number of binding sites n for CFX with BSA from equation (3), and the calculated results are shown in Table 1. The values of n were all ~1 which suggested that just one binding site for CFX existed in BSA. Meanwhile, the binding constants Ka decreased with increasing temperature, further suggesting that the quenching was a static process (17). The primary binding site studies
Figure 4. Stern–Volmer plots for the quenching of BSA by CFX at different tem–7 -7 -6 peratures. CBSA =4.0 × 10 M, CCFX =4.0 × 10 M and 6.0 × 10 M.
At 280 nm wavelength, the Trp and Tyr residues in BSA are excited, whereas a wavelength of 295 nm excites only Trp residues (18). In BSA, sub-hydrophobic domain IIA (containing both Trp and Tyr) and IIIA (containing only Tyr) are the major binding sites for small molecule ligands (19). Based on the Stern–Volmer equation, comparing the fluorescence quenching of BSA excited at 280 nm and 295 nm allows us to estimate the participation of Trp and Tyr groups in the system (20). As seen in Fig. 5, in the presence of CFX, the quenching curves of BSA excited at 280 nm and 295 nm overlap. This showed that only Trp residues played an important role in the interaction of CFX with BSA. Therefore, it may be implied that the primary binding site for CFX was sub-hydrophobic domain IIA (21). From Table 1, at the same temperature, Ka values at excitation wavelengths of 280 nm and 295 nm were similar, which also suggests that only Trp residues played an important role in the interaction between CFX and BSA.
Table 1. Quenching reactive parameters of BSA and CFX at different temperatures λex (nm)
T (K)
Kq (M–1 · s–1)
Ksv (M–1)
r1
Ka (M-1)
n
r2
280
293 303 310 293 303 310
3.91 × 1012 3.73 × 1012 3.67 × 1012 3.74 × 1012 3.47 × 1012 3.44 × 1012
3.91 × 104 3.73 × 104 3.67 × 104 3.74 × 104 3.47 × 104 3.44 × 104
0.9979 0.9918 0.9981 0.9993 0.9910 0.9984
4.72 × 104 4.20 × 104 3.66 × 104 4.54 × 104 4.31 × 104 3.81 × 104
0.97 0.93 0.96 0.88 0.95 0.94
0.9931 0.9927 0.9908 0.9931 0.9959 0.9950
295
Kq is the quenching rate constant; Ka is the binding constant; n is the number of binding site. r1 is the linear relative coefficient of F0/F – [L]; r2 is the linear relative coefficient of lg(F0 – F)/F – lg[L].
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L. Zhang et al.
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Figure 5. The curve of F/F0 with CFX/BSA of CFX–BSA (T =310 K). CBSA =4.0 × 10 M, –7 -5 CCFX =2.0 × 10 –1.0 × 10 M.
Synchronous fluorescence spectra studies When the value of Δλ between the excitation and emission wavelengths is stabilized at either 15 or 60 nm, synchronous fluorescence gives characteristic information for tyrosine or tryptophan residues, respectively. It can be seen from Fig. 6 that when Δλ was 15 nm, the fluorescence intensities of BSA–CFX showed no obvious decrease, with no shifts with increasing CFX concentration; this indicated that Tyr residues do not have an important role in the interaction between CFX and BSA. When Δλ was 60 nm, the synchronous fluorescence intensities of CFX–BSA decreased regularly with some shifts, indicating that the interaction of BSA with CFX changed the microenvironment of the tryptophan residues (22), which was coincident with the primary binding site studies. The polarity of the hydrophobic environment was enhanced and hydrophobicity was reduced in the BSA cavity due to changes in the microenvironment of the tryptophan residues on insertion of CFX (23). This led to a conformation change in BSA. Protein molecules were extended by high concentrations of drugs, which reduced energy transfer among the amino acid residues and their fluorescence intensity. The corresponding results for Δλ =60 nm, according to equations (2) and (3), are shown in Table 2. From Table 2, it can be seen the values of Ksv decreased with increasing temperature for all systems, which indicated that the probable quenching mechanism of the interaction between BSA and CFX was static. The value of Kq was in the order of 1012 M–1 · s–1. Obviously, the Kq value for the protein-quenching procedure initiated by CFX was >2 × 1010 M–1 · s–1, which indicated that the quenching was not initiated by dynamic collision but from the formation of a complex. The n value approaches unity, suggesting that one molecule of CFX combines with one molecule of BSA; the trend for decreasing Ka values with increasing temperature was in accordance with dependence of the binding constants on the temperature, as mentioned above, which indicated that CFX–BSA would partly decompose with increasing temperature; this also indicated the process was static quenching. The quenching mechanism obtained by synchronous fluorescence method was coincident with that obtained by fluorescence
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Figure 6. Fluorescence spectrum of CFX–BSA system (T =310 K) (A) Δλ =15 nm; –7 (B) Δλ =60 nm. CBSA =4.0 × 10 M; 1–10 CCFX = (0, 2.0, 3.0, 4.0, 8.0, 20, 40, 60, 80, -7 100) × 10 M.
method. Comparing Tables 1 and 2 shows that the quenching parameters obtained using the two methods are of the same order of magnitude. Type of interaction force in BSA–CFX systems In general, interactions between a small drug molecule and a biological macromolecule include hydrogen bonds, Van der Waal’s forces, electrostatic interactions and hydrophobic force. Ross and Subramanian (24) have characterized the sign and magnitude of the thermodynamic parameters, enthalpy change (ΔH), free energy (ΔG) and entropy change (ΔS), associated with various types of interaction. When temperature varies over a small range, ΔH can be considered constant (25). Many researchers think that a negative value for ΔH (ΔH is very small, almost zero) and a positive value for ΔS indicate that electrostatic interactions have a major role in the binding reaction. Positive ΔH and ΔS values are generally considered to be evidence of typical hydrophobic interactions. In addition, Van der Waal’s forces and hydrogen bond formation in low dielectric media are characterized by negative values of ΔH and ΔS (26). However, from the point of view of water, a
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Studies on the interaction of CFX with BSA by fluorescence Table 2. Quenching reactive parameters of CFX and BSA at different temperatures (Δλ =60 nm) T (K)
Kq (M-1 · s-1) 12
293 303 310
4.34 × 10 3.60 × 1012 3.59 × 1012
Ksv (M-1)
r3
4
4.34 × 10 3.60 × 104 3.59 × 104
0.9990 0.9920 0.9997
Ka (M-1) 4
4.88 × 10 4.65 × 104 3.31 × 104
n
r4
0.93 0.95 0.95
0.9967 0.9911 0.9905
Kq is the quenching rate constant; Ka is the binding constant; n is the number of binding site. r3 is the linear relative coefficient of F0/F – [L]; r4 is the linear relative coefficient of lg(F0 – F)/F – lg[L]. positive ΔS value is frequently taken as evidence of hydrophobic interactions. The thermodynamic parameters can be calculated on the basis of equations (4) and (5) (27): Rln K ¼ ΔS ΔH=T
(4)
ΔG ¼ ΔH TΔS ¼ RT lnK
(5)
The type of interaction force for small molecule drugs and biological macromolecules can be obtained from the relevant thermodynamic parameters. The values of ΔH were obtained from the linear van’t Hoff plot (Fig. 7) and are listed in Table 3 along with ΔS and ΔG. From Table 3, it can be seen that the reaction of CFX with BSA was a spontaneous molecular interaction in which entropy increased and Gibb’s free energy decreased (28). From the point of view of the polarity and ionization of CFX, the main interaction of CFX and BSA was hydrophobic
Figure 7. Van’t Hoff plot of the interaction of CFX and BSA in Tris/HCl; pH =7.4.
contact. The conclusions drawn from the synchronous fluorescence method were consistent with the fluorescence method.
Energy transfer from BSA to CFX According to the Förster nonradioactive resonance energy transfer theory (29), the effective energy transfer from donor to acceptor will occur when two molecules meet the following preconditions: (1) the donor is a fluorophore; (2) the overlap is sufficient between the fluorescence emission spectrum of the donor and UV/vis absorption spectrum of the acceptor; and (3) the distance between the donor and the acceptor is within 2–7 nm. The energy transfer effect is not only related to the distance between the donor (tryptophan residue) and acceptor, but also influenced by the critical energy transfer distance R0 and is described by equations (6) and (7) (30,31):
Figure 8. Overlap of the fluorescence spectrum of BSA (λex =280 nm) (1) and (Δλ =60 nm) (2) with the absorption spectrum of CFX (3). (T =310 K) CCFX = CBSA –7 =4.0 × 10 M.
Table 3. The thermodynamic parameters of CFX–BSA at different temperatures T (K) Δλ =60 nm λex =280 nm
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293 303 310 293 303 310
Ka (M-1) 4
4.88 × 10 4.65 × 104 3.31 × 104 4.72 × 104 4.20 × 104 3.66 × 104
ΔH (kJ/mol)
ΔS (J/mol/K)
ΔG (kJ/mol)
–16.14
34.66 36.08 34.46 51.65 51.93 51.61
–26.30 –27.07 –26.82 –26.22 –26.82 –27.08
–11.08
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L. Zhang et al. Table 4. Parameters of E, J, r, R0 between CFX and BSA at different temperatures Method
T (K)
Fluorescence quenching
293 303 310 293 303 310
Synchronous fluorescence
E (%) 4.22 2.86 2.84 2.90 2.87 2.79
J (cm3 · M–1) -15
8.95 × 10 8.94 × 10-15 8.71 × 10-15 8.38 × 10-15 8.40 × 10-15 8.44 × 10-15
R0 (nm)
r (nm)
2.41 2.40 2.39 2.37 2.38 2.38
4.04 4.32 4.31 4.27 4.28 4.30
R0 is the critical distance when E is 50%; r is the distance between the acceptor and donor; J is the overlap integral between the fluorescence emission spectrum of donor and the absorption spectrum of the acceptor. E ¼ 1 F=F 0 ¼ R0 6 = R0 6 þ r 6 R0 ¼ 8:7810 6
25 2
4
K ΦN J
(6)
Acknowledgements
(7)
The authors gratefully acknowledge the financial support of National Science Foundation of China (Grant no. 20675024) and Hebei Provincial Key Basic Research Program (Grant no. 10967126D).
Where R0 is the critical distance when the transfer efficiency is 50%, r is the distance between the acceptor and the donor, and E is the energy-transfer efficiency. K is the spatial orientation factor of the dipole, N is refractive index of the medium, Φ is the fluorescence quantum yield of the donor and the overlap integral (J) between the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor can be calculated using equation (8) (32): J¼
X
X F ðλÞεðλÞλ4 Δλ= F ðλÞΔλ
(8)
Where F(λ) is the fluorescence intensity of the fluorescent donor at a known wavelength and ε(λ) is the molar absorbance of the acceptor at wavelength λ. Figure 8 shows the overlap integral of the fluorescence emission spectrum of BSA and the absorption spectrum of CFX. In this case, K2 = 2/3, N =1.336 and Φ =0.118 (33), according to equations (6)–(8); the corresponding results are shown in Table 4. From Table 4, it can be seen that the donor-to-acceptor distance r
