Vol. 45 No. 2
SCIENCE IN CHINA (Series B)
April 2002
Binding equilibrium study of phosphotungstic acid and
HSA or BSA with UV spectrum, fluorescence spectrum
and equilibrium dialysis
HUANG Jin, YUAN Yuzhou& LIANG Hong
Institute of bioinorganic Chemistry, Department of Chemistry, Guangxi Normal University, Guilin 541004, China
Correspondence should be addressed to Liang Hong (email: [email protected])
Received September 6, 2001
Abstract The binding equilibrium between phosphotungstic acid (H7[P(W2O7)6]CXH2O;PTA) and
human serum albumin (HSA) or bovine serum albumin (BSA) has been studied by UV-Vis, fluorescence spectroscopies and equilibrium dialysis. It has been observed that UV absorption enhanced and the fluorescence quenched as the PTA binding to HSA or BSA at physiological pH
7.43(±0.02). The Scatchard analysis indicated that there exists a strong binding site of PTA in both
HSA and BSA, and the successive stability constants of these two systems are obtained by
nonlinear least-squares methods fitting Bjerrum formula.
Keywords: serum albumin, phosphotungstic acid, UV spectrum, fluorescence spectrum, equilibrium dialysis.
As one of the necessary nonmetal elements in human body, phosphor has very important
physiological functions. Phosphowolframate has long-range activity and selectivity to anti
DNA-virus and RNA-virus[1]. In recent years, the important applied value and good developing
prospect of polyoxometalate have made the pharmaceutical chemistry of polyoxometallate become an important branch of polyacid chemistry[2]. Serum albumin has very important physiological functions, such as transportation, buffering and nutrition. The interactions of metal ions,
ü
medicines, little molecules and simple anion with HSA or BSA have been reported[3 11], but no
reports about the behavior of complex heteropolyanion binding to serum albumin. Studying on the
binding equilibrium of PTA to HSA or BSA has an important sense to find out the absorption,
transportation and physiological functions of PTA and its salt as medicine in human body. In this
paper, the binding equilibrium of PTA and HSA or BSA has been studied by using UV spectrum,
fluorescence spectrum and equilibrium dialysis, and the difference of the weak binding of I − to
HSA or BSA[11] is found. The binding of PTA and HSA or BSA is much stronger at physiological
pH(7.43). PTA has peculiar binding sites in HSA or BSA, and their binding can enhance the absorbance of UV absorption peaks and quench the characteristic fluorescence peaks.
1
Experimental
1.1 Materials and reagents
HSA and BSA(> 98%), fatty acids less than 0.005%, both were electrophoretic purity grade
reagents. Tris was biochemical reagent (> 99.5%), and the major impurity was moisture. They
No. 2
BINDING EQUILIBRIUM OF PHOSPHOTUNGSTIC ACID & HSA OR BSA
201
were all originally packed by Sigma Chemical Company, purchased from Sino-American Biotechnology Company Shanghai Branch, mailed freshly, and used without further purification.
Other reagents were of analytical grade. All the solutions were prepared with double deionized
water. All of the solutions contained 0.10 mol/L NaCl and 0.01 mol/L tris-HCl to maintain the
same ionic strength and pH 7.43(±0.02). pHS-10A precision acidometer was used for the pH
measurements. The concentrations of HSA and BSA were determined by Varian CARY 100
Conc-type UV-visible spectrophotometer[12]. The data of fluorescence were obtained with a Shimadzu RF-540 spectrofluorophotometer (Kyoto Japan). The concentrations of free phosphotungstic acid before and after dialysis were all determined by spectrophotometric method[13] with Varian CARY 100 Conc-type UV-visible spectrophotometer. The pH of PTA solution was buffered
with 0.1 mol/L tris-NaOH (pH = 7.43±0.02), and the addition of PTA has no obvious effect on the
pH of serum albumin solutions.
1.2 Methods
1.2.1 UV spectrum. The PTA solution with different concentrations was added into 1.0 × 10−4
mol/L serum albumin solution. The samples were scanned in the range of 200 400 nm after being mixed homogeneously. The UV-spectra were recorded at room temperature and the corresponding protein solutions without PTA of the same concentrations as reference.
1.2.2
Fluorescence spectrum.
Add different volumes of PTA solution into 4 mL of 1.0 × 10−4
mol/L serum albumin solution with trace injector and carry out fluorosphotometric titrate. With λex
= 296 nm, and scanning the emission wavelength region of 250 1000 nm, we obtained the fluorescence emission spectra after intensive mixing 20 min.
1.2.3 Equilibrium dialysis. The concentration of PTA solution was determined by spectrophotometric method[13]. Then the PTA solution was diluted to different concentration dialysis solutions with the buffer solutions. Both the concentrations of HSA and BSA solutions were about 1.0
× 10−4 mol/L, prepared freshly, and dialyzed for 12 h at 20 in the darkness, and others were the
same as described in ref.[3].
2 Results and discussion
2.1 UV spectra of PTA binding to serum albumin
According to sec. 1.2.1, the UV absorption spectra of PTA binding to HSA or BSA are obtained (fig. 1). As shown in fig. 1, the absorbance at the peak around 250 nm increases rapidly
with the concentration increase under the conditions of such lower concentration rates ([PTA]
[serum albumin] < 11). When [PTA][serum albumin] > 11, the absorbance at the peak
around 250 nm increases slightly, while the absorbance at the peak around 295 nm increases rapidly. The absorbance at the peak around 250 and 295 nm has no obvious increase when [PTA]
[serum albumin] ≈ 71. What should be pointed out is the PTA solution which do not bind with
202
SCIENCE IN CHINA (Series B)
Vol. 45
serum albumin has no absorption peak.
Fig. 1. UV spectra of HSA(a) and BSA(b) at different PTA concentrations. [PTA]/[serum albumin]: 1, 0.4/1; 2, 0.6/1; 3, 0.8/1;
4, 1/1; 5, 2/1; 6, 3/1; 7, 4/1; 8, 5/1.
The absorption peak around 290 nm of serum albumin is caused by aromatic heterocyclic
π→ π* transition of one or two Trp-residues and nineteen Tyr-residues in the peptide chain, and
the absorbance around 250 nm is caused by π→ π* transition of peptide bond’s carboxide. The
absorbance around 250 nm relates to the content of α-helix in serum albumin[14]. When the concentration rate is lower ([PTA][serum albumin] < 11), the absorbance around 250 nm increases rapidly while the concentration of PTA increases. This indicates that when PTA at a lower
concentration, the binding of the PTA and serum albumin is benefit to coacervation in and among
the serum albumin molecule, thus to change the conformation of serum albumin, reduce the content α-helix and make the absorption peak around 250 nm have a red shift[15]. As the concentration
of PTA increases further, the PTA molecule induces the peptide chain of serum albumin molecule
extense such as the phenomenon caused by the lower pH[14]. At this time, the tertiary structure of
serum albumin changes rapidly, but the secondary structure of serum albumin changes little. The
alteration of serum albumin’s tertiary structure makes the aromatic heterocyclic hydrophobic
group of Trp-residues and Tyr-residues which are in the serum albumin formerly exposed, and
thus increases the absorbance around 290 nm[15]. At the same time, the hydrophobic interaction
among the hydrophobic groups was enhanced, the transition energy of π→ π* increases, and the
absorption peak has a red shift. When [PTA] [serum albumin] ≈ 71, the reinforcing of absorption peak tends to gentle. Maybe the binding of PTA and serum albumin reaches to saturation,
and there are about 7 binding sites of PTA in serum albumin.
2.2
Fluorescence quenching of PTA binding to serum albumin
With fluorescence excitation λex = 296 nm, there are characterized fluorescence emission
ü
peaks of HSA and BSA for the Trp-residue as the intrinsic fluorescence sites in serum albumin[8 10].
According to sec. 1.2.2, the fluorescence emission spectra of PTA binding to HSA or BSA were
obtained (fig. 2). From fig.2, there are characteristic emission peaks at about 296, 350, 592, 700
and 888 nm. The peaks at 350 and 700 nm are the first and second multiple-frequency fluores-
No. 2
BINDING EQUILIBRIUM OF PHOSPHOTUNGSTIC ACID & HSA OR BSA
203
cence emission peak respectively, because of the Trp214 in HSA, and Trp213 and Trp134 in BSA[9]. The
peaks at 296, 592 and 888 nm are first, second and third resonance Rayleigh scattering (RRS) peaks
respectively[11]. As shown in fig. 2, the adding of PTA has not induced obvious alternation to the
situation of serum albumin excitation peak, maximum fluorescence peak and scattering peaks, but
made the intensity of peaks quenched in different degrees. We can neglect dilution effect because the
volume of serum albumin (4 mL) is far more larger than that of PTA(200 µL).
Fig. 2. Fluorescence quenching plot of PTA vs. HSA(a) and BSA(b). The concentration of PTA increases from up to
down, [serum albumin] =1.0D10−4molCL−1.
Using the fluorescence spectra in physical conditions (fig. 3), plotting PTA versus HSA or
BSA can obtain the Stern-Volmer quenching curvilinear. In fig. 3, the fluorescence quenching of
serum albumin which induced by PTA has an obvious break point at about [PTA]/[serum albumin]
≈ 1, indicating that there exist two types of binding sites in PTA-serum albumin systems, and the
fluorescence quenching of the first binding sites (strong binding sites) is weaker than that of the
secondary binding sites. We also inferred from fig. 3 that PTA has more than 5 binding sites in
HSA, and has at least 6 binding sites in BSA.
Fig. 3. Stern-Volmer diagram PTA vs. serum albumin fluorescence quenching. F0 is the fluorescence intensity without quenchers and F is the fluorescence intensity after adding quenchers. (a) PTA-HSA, (b) PTA-BSA, [serum albumin] = 1.0D10−4
molCL−1.
204
SCIENCE IN CHINA (Series B)
Vol. 45
When pH and temperature are in strict control, the fluorescence quenching of serum albumin
can be attributed to these two causes: dynamic quenching and effect of non-radiation energy
transfer. According to fig. 3 and Stern-Volmer equation:
F0/F = 1 + Kqτ 0[Q],
(1)
where [Q] is the concentration of quencher, and τ 0 is fluorescence lifetime of biomolecular without quenchers and about 10 ns[16]. From this, we can calculate the apparent quenching constant
Kqmol/L)−1s−1) of serum albumin: HSA is 4.89×1011(mol/L)−1s−1, and BSA is 4.3×1011
(mol/L)−1s−1. The Kq of these two systems are both much larger than the quenching constant (2.0
1010(mol/L)−1s−1)[17] controlled by diffusion, and indicates that the serum albumin fluorescence
quenching is not induced by dynamic collision.
Determining the absorption spectra of PTA, and comparing it with the emission spectra of
HSA or BSA, we found that the two spectra overlap in some degree, as shown in fig. 4. So, the
fluorescence quenching of serum albumin can be attributed to non-radiation energy transfer.
Fig. 4. Spectra overlap between the absorption spectra of PTA and the emission spectra of HSA (a) or BSA (b). üü, Fluorescence spectra; - - - - , absorption spectra.
According to Fster non-radiation energy transfer theory, the degree of luminophore fluorescence quenching is determined by the distance between the luminophore (the energy donor) and
quencher (the energy acceptor). Assuming that the binding of PTA and serum albumin is sequential binding, we can acquire the distance between the binding site and fluorescence group which in
the protein from Fster non-radiation energy transfer theory[17,18].
E=
R06
,
(2)
E = 1−F / F0 ,
(3)
R06 = 8.8 × 10−25 J K 2ϕ n −4 ,
(4)
∞
∫
J= 0
R06 + r 6
F (λ )ε λ 4 dλ
∞
∫0
F ( λ ) dλ
,
(5)
No. 2
BINDING EQUILIBRIUM OF PHOSPHOTUNGSTIC ACID & HSA OR BSA
205
where R0 is the distance corresponding to E of 50%, orientation factor K2 is 2/3, the average of
that of donor (Trp) and acceptor (PTA), the reference material of ϕ is Trp(ϕ = 0.14), the refractive
index n = 1.36, the average of that of H2O and organics, and J is the overlap integral of the spectra.
Applying formula (5) to integral the overlap in fig. 4, we can obtain the overlap integral J of
the spectra: JHSA = 1.276 × 10−15(cm)6(mol−1)−1, JBSA = 0.998×10−15(cm)6(molL−1)−1.
When [PTA][serum albumin] ≈ 1, if the serum albumin fluorescence quenching induced by PTA
is attributed to non-radiation energy transfer, according to fig. 3 and formula (3), we can obtain
that the efficiency of non-radiation energy transfer between HSA Trp214-residue and the first binding site of PTA EHSA is 0.10826, and that between BSA Trp212-residue and the first binding site of
PTA EBSA is 0.08759. From formula (4), we can infer R0 = 1.769 nm (HSA), and R0 = 1.698 nm
(BSA). According to the value of E, R0 and formula (2), we can calculate that the distance r between HSA Trp214-residue and the first binding site of PTA is 2.514 nm, and that between BSA
Trp212-residue and the first binding site of PTA is 2.509 nm.
We can also see from fig. 3 that the fluorescence quenching of the secondary binding site in
serum albumin is stronger than that of the first binding site, that is, the efficiency of non-radiation
energy transfer E of the secondary binding is larger than that of the first binding site. So the secondary binding site is more close to the Trp-residue.
2.3 Analysis of equilibrium dialysis result on PTA binding to HSA or BSA
According to sec. 2.2.3, we can obtain the
saturation curvilinear of PTA binding with HSA
or BSA, as shown in fig. 5. The abscissa of fig.
5 represents the concentration of free PTA after
dialysis, and the vertical coordinate is formation
function nc which is always called average
ligand number and represents the number of
Fig. 5. nc vs. [PTA] (the concentrations of HSA and BSA
PTA that every serum albumin binds.
[19]
are both 1.0×10− 4molL−1).
From the Scatchard plot , we obtain the
type and number of binding sites of PTA in HSA and BSA (fig. 6). As shown in fig. 6, there exist
two types of binding sites in both PTA-HSA and PTA-BSA systems. There are one strong binding
site and five weak binding sites in PTA-HSA, and one strong binding site and seven weak binding
sites in PTA-BSA. This is in accordance with the result obtained from fluorescence analysis and
UV analysis. The successive stability constants of PTA-serum albumin are obtained by nonlinear
least square methods fitting Bjerrum formula and summarized in table 1. The degree of fitting experimental data is expressed by Hamilton R-factor (table 1)[3].
There exist the specific binding sites of PTA in HSA and BSA, but the situation and conformation of binding sites need further study.
206
SCIENCE IN CHINA (Series B)
Vol. 45
Table 1 Successive stability constants of PTA-HSA and PTA-BSA systems
Systems
HSA(1.062
×10−4molL−1)
BSA(1.091×
10−4mol−1)
Successive stability constants
K4
K5
K1
K2
K3
1.905×104
3.632×103
1.208×103
8.507×102 7.413×102 7.079×102
1.787×104
3.806×103
1.953×103
1.382×103 9.331×102 8.911×102 8.509×102 8.423×102
K6
K7
K8


R-factor
0.042
0.030
Fig. 6. Scatchard plot. Concentrations of HSA and BSA are both 1.0×10− 4molL−1. (a) PTA-HSA, (b) PTA-BSA.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No.
29961001), the Cross-Century Talents Foundation of Guangxi Zhuang Autonomous Region and Science Foundation of Guangxi
University.
References
1.
Toshihiro Yamase, Biological activities of polyoxometalates, Chemical Industry (in Japanese), 1990, 41(10): 4046.
2.
3.
Wang Enbo, Hu Changwen, Xu Lin, The Introduction of Heteropolyacid Chemistry (in Chinese), Beijing: Press of Chemical Industry, 1998, 146.
Liang Hong, Xing Bengang, Wang Xioujian et al., Equilibrium dialysis study on the interaction between Cu(II) and HSA
4.
Sadler, P. J., Viles, J. H., 1H and 13C NMR investigation of Cd2+ and Zn2+ binding sites on serum albumin: competition
or BSA, Chinese Science Bulletin, 1998, 43(5): 404409.
with Ca2+, Ni2+, Cu2+, and Zn2+, Inorg. Chem., 1996, 35: 44904496.
5.
Hu Xueying, Song Zhongrong, Shu Xiandong et al., The binding equilibrium of metal-serum albuminII. The conformational transition effect of Ni(II)-serum albumin system, Science in China, Series B, 1997, 40(2): 122127.
6.
Tu Chuqiao, Zhang Hongzhi, Liang Hong et al., Study on the binding equilibrium between Cd(II) and HSA or BSA, Chinese Journal of Chemistry (in Chinese), 2000, 58(2): 229234.
7.
Liang Hong, Tu Chuqiao, Zhang Hongzhi et al., Binding equilibrium study between Mn(II) and HSA or BSA, Chinese
8.
Mateen, A. K., Salman, M., Javed, M., Interactions of photosensitized tetracycline with serum albumin, Biochemistry and
9.
Liang Hong, Xing Bengang, Wu Qinxuan et al., Study on the interaction of human serum albumin with Cu(II) and Fe(III)
10.
Yang Binsheng, Yang Pin, Fluorescence quenching studies on the action of metal ions with human serum albumin, Pro-
Journal of Chemistry (in Chinese), 2000, 18(1): 3541.
Molecular Biology International, 1998, 46(5): 943950.
by fluorescence method, Acta Chimica Sinica (in Chinese), 1999, 57(2): 161165.
gress in Biochemistry and Biophysics (in Chinese), 1992, 19(2): 110113.
11.
Liang Hong, Shen Xingcan, Jiang Zhiliang et al., Binding equilibrium study of I− to serum albumin with resonance
12.
Edward, F. B., Rombauer, R. B., Campbell, B. J., Thiol-disulfide interchange reactions between serum albumin and disul-
Rayleigh scattering, Science in China, Series B, 2000, 43(6): 600608.
No. 2
BINDING EQUILIBRIUM OF PHOSPHOTUNGSTIC ACID & HSA OR BSA
207
fides, Biochim. Biophys. Acta, 1969, 194(1): 234245.
13.
Qi Xiaolin, Shao Guangdi, Chen Huaxu, Colour associated compound of phosphoomlybdotungstate with crystal violet and
14.
Shahid, F., Gomez, J. E., Birnbaum, E. R., The lanthanide-induced NF transition and acid expansion of serum albumin,
15.
Li Xiaojing, Wang Zhiqiang, Chen Ji et al., UV spectral study on the interaction of RE ions with BSA, Chinese Journal of
16.
Lakowicz, J. R., Weber, G., Quenching of fluorescence by oxygen, Probe for structural fluctuations in macromolecules,
17.
Berde, C. B., Hudson, B. S., Simoni, R. D. et al., Human serum albumin, spectroscopic studies of binding and proximity
18.
Sklar, L. A., Hudson, B. S., Simoni, R. D., Conjugated polyene fatty acids as fluorescent probes: synthetic phospholipid
19.
Scatchard, G., Scheinberg, I. H., Armstrong, S. H., Physical chemistry of protein solutions, IV. The combination of humin
its analytical application, Analytical Chemistry (in Chinese), 1990, 18(2): 164166.
J. Biol. Chem., 1982, 257(10): 56185622.
Applied Chemistry (in Chinese), 1998, 15(1): 58.
Biochemistry, 1973,12(21): 41614170.
relationships for fatty acids and bilirubin, J. Biol. Chem., 1979, 254(2): 391400.
membrane studies, Biochemistry, 1977, 16(5): 819828.
serum albumin with chloride ion, J. Am. Chem. Soc., 1950, 72: 535540.