Studies on the interaction between neutral red and bovine hemoglobin by fluorescence spectroscopy and molecular modeling

Journal of Molecular Liquids 211 (2015) 584–590

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Studies on the interaction between neutral red and bovine hemoglobin by fluorescence spectroscopy and molecular modeling Ruiqiang Wang a,⁎, Zhigang Li b, Lingling Yang b, Ting Ren b, Lijiao Zhang b, Ruiyong Wang b,⁎ a b

First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China Department of Chemistry, Zhengzhou University, Zhengzhou 450001, China

a r t i c l e

i n f o

Article history: Received 22 April 2015 Received in revised form 19 July 2015 Accepted 25 July 2015 Available online xxxx Keywords: Fluorescence spectroscopy Bovine hemoglobin Neutral red Interactions

a b s t r a c t The binding interaction of neutral red (NR) with bovine hemoglobin (BHb) was studied by fluorescence spectroscopy in combination with molecular modeling. NR quenched the intrinsic fluorescence of BHb via a static mechanism. According to relevant data, the binding constants were calculated at two different temperatures. The thermodynamic parameters obtained from the fluorescence data showed that the hydrophobic and electrostatic interactions played a major role in stabilizing the complex. Synchronous and three-dimensional fluorescence spectra of BHb were investigated in the presence of NR. The results showed that the environment of tryptophan and tyrosine residues was altered by the dye. The fluorescence experimental results were in agreement with the results obtained by molecular modeling study. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Hemoglobin (Hb, Mr = 64,500 Da), an important functional protein for reversible oxygen carrying and storage in the vascular system of animals, consists of two identical α-chains of 141 amino acids each, and two identical β-chains of 146 amino acids each [1]. Hb emits intrinsic fluorescence mainly due to Trp and Tyr residues [2]. The fluorescence intensity and the location of fluorescence peak reflect the microenvironment of the chromophore group of protein [3]. Except for albumin, as a kind of intracellular protein, Hb can also function as binders of drugs [4]. What's more, the concentration of Hb is 330 mg/mL [5], bigger than the serum albumin 40 mg/mL [6]. Hb can reversibly bind with many kinds of small bioactive molecules, such as alkaloids [7], analogs of biphenyldicarboxylate [8], Troxerutin [9], tannic acid [10], FNC [11], artemisinin [12], dihydromyricetin [13], herbicide [14] and surfactant [15], and so on. It is known that organic dyes are widely used as a working medium in dye lasers [16], in analytical chemistry to determine the various trace elements [17]. Moreover, dyes are applied to textile, cosmetic, printing and food processing industries extensively [18]. If the wastewater enters the environment, the dye-containing wastewaters will pose a great threat to the environment, that affects the health of living beings and fertility of the soil. Therefore, investigation of the dye–protein complexes is of vital importance. There have been a number of previous studies on the interaction between dyes and proteins, such as

phenosafranin [19], tricarbocyanine dyes [20]. The interactions of hemoglobin with some dyes have been reported, such as C.I. acid red 27 [21], the food additive amaranth [22], Toluidine blue [23], tartrazine [24], Sudan dyes [25] and gentian violet [26]. Neutral red (NR, Fig. 1) is a kind of mixed anthracycline-based cationic dye, which not only used for cytosol dyeing and cell identification, but also can be used as pH indicator, adsorption indicator, redox indicator and biological fluorescent probe. The interaction of neutral red with bovine serum albumin has been reported [27,28]. However, the interaction mechanism between neutral red and Hb has not been reported. Furthermore, NR is an interacting mode spectroscopic probe [29]. NR gives an emission maximum at 650 nm (λex = 540 nm), while NR had no intrinsic fluorescence from 310 nm to 500 nm at the excitation wavelength of 290 nm. The present paper deals with the mechanism of binding of NR with bovine hemoglobin (BHb) at simulated physiological conditions by fluorescence, UV/vis absorption and molecular modeling. The energy transfer between NR and BHb and the characteristics of resonance light-scattering spectra (RLS) are also reported. Moreover, the conformational changes of BHb occurring in the presence of NR have been analyzed by using synchronous and three-dimensional fluorescence techniques. This paper helps understand the hemoglobin's binding mechanisms to dyes and provides clues to the biological effects and functions of dyes in body. 2. Materials and methods 2.1. Materials

⁎ Corresponding authors. E-mail addresses: [email protected] (R. Wang), [email protected] (R. Wang).

http://dx.doi.org/10.1016/j.molliq.2015.07.066 0167-7322/© 2015 Elsevier B.V. All rights reserved.

BHb (Sigma) was dissolved in ultra pure water to form 1.0 × 10−4 mol · L−1 solution, then preserved at 4 °C and diluted as required.

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Fig. 1. The molecule structure of NR.

NR (Three Elsevier, Shanghai, China) was dissolved in ultra pure water to form 1.0 × 10−3 mol · L−1 solution, then diluted as required. The buffer tris (Shanpu, Shanghai, China), NaCl and HCl were all of analytical purity. 2.2. Equipment and spectral measurements The UV/vis spectra were recorded on an UV-1800 spectrophotometer (Mapada, Shanghai, China) equipped with 1.0 cm quartz cells. Fluorescence quenching and synchronous fluorescence spectra were recorded on a 970-CRT spectrofluorimeter (San Ke, Shanghai, China) equipped with 1.0 cm quartz cells. The widths of excitation and emission slits were set to 5.0 nm/10.0 nm respectively. RLS and threedimensional fluorescence measurements were performed on an F-4500 fluorescence spectrophotometer (Hitachi, Japan) equipped with 1.0 cm quartz cell, using 10.0 nm/20.0 nm slit widths. 2.3. Procedures The fluorescence measurements were carried out as follows: to each of a series of 10 mL test-tube, 2.0 mL Tris–HCl buffer (pH 7.4), 2.0 mL of 0.5 mol · L−1 NaCl and 1.0 mL of 2.0 × 10−5 mol · L−1 BHb were added, followed by 1.0 mL of different concentrations NR. The fluorescence spectra were then measured (excitation at 290 nm and emission wavelengths range 310–500 nm) at two temperatures (307 K, 322 K). The synchronous fluorescence spectra were obtained through simultaneous scanning of the excitation and emission monochromators while maintaining a constant wavelength interval between them (Δλ, 15 nm and 60 nm). The three-dimensional fluorescence spectrum was performed under the following conditions: the emission wavelengths at 280–470 nm, the excitation at 200 nm with an increment of 10 nm, excitation and emission slit widths were 10 nm/20 nm respectively, and the scan speed was 1200 nm · min−1, PMT (Photo Multiplier Tube) voltage was 700 V. The UV/vis absorbance spectra of BHb with different concentrations NR were recorded at room temperature. RLS were obtained by synchronous scanning with the wavelength range of 200–800 nm on the spectrofluorophotometer at room temperature.

Fig. 2. Absorption spectra of NR bound to BHb at pH 7.4. (a) Absorption spectra of NR only, c(NR) = 16.0 μM; (b) absorption spectra of BHb only, c(BHb) = 2.0 μM; (c) absorption spectra of BHb–NR; (d) absorption spectra of [BHb–NR]–NR.

3. Results and discussion 3.1. UV/vis absorption spectra studies UV/vis absorption measurement is a simple but efficacious method to explore the structural changes of protein and investigate protein–ligand complex formation [31]. Hence, absorption spectra of NR and NR-BHb system were recorded. Fig. 2 showed the absorption spectral changes of BHb in the presence of NR in the wavelength 200–600 nm. It can be seen that BHb has three absorption peaks. The strong absorption peak at 210 nm not only reflects the framework conformation of protein but also corresponds to the peptide bond [32]. The weak absorption peak at 278 nm appears due to the aromatic amino acids (Trp, Tyr and Phe) [33]. The peak at 405 nm corresponds to the porphyrin Soret band of BHb [34]. Spectra b and d should be identical in Fig. 2 if no interaction occurred between BHb and NR. The UV/vis absorption spectrum of BHb shows a strong band in the near-UV region with a maximum at 210 nm, which appears due to peptide bond absorption of tryptophan. By comparing the spectra b with d, it can be found that the absorbance at 210 nm of BHb decreases. This result indicates that the binding interactions occur between BHb and NR, which may cause the slight change of the conformation of protein [35].

2.4. Molecular modeling study The AutoDock4.2 [30] program was used to calculate the interaction modes between NR and BHb. Lamarckian genetic algorithm (LGA) implemented in AutoDock was applied to calculate the possible conformation of NR that binds to the protein. The structure of NR was downloaded on website (http://zinc.docking.org/). The crystal structure of BHb was taken from the Brookhaven Protein Data Bank (http://www. rcsb.org/pdb) (PDB ID: 1G09). Water molecules were removed, and hydrogen atoms were added. A grid map of 126 × 106 × 126 grid points in size with a grid-points pacing of 0.553 Å was created for the protein. The scoring functions of the empirical free energies for the docked configurations have been tested for all docking models. According to the binding energy and the geometry matching after 250 runs, the most favorable docking model was selected for further analysis.

Fig. 3. Effect of NR on fluorescence spectra of BHb (T = 307 K, pH = 7.40 and λex = 290 nm). Curves (1–7): c(BHb) = 2.0 μM, c(NR): 0, 4.0, 8.0, 12.0, 16.0, 20.0, 24.0 μM, respectively.

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Fig. 4. Stern–Volmer plots for the quenching of BHb caused by NR at different temperatures. c(BHb) = 2.0 μM, pH = 7.40, λex = 290 nm, λem = 332 nm.

Fig. 5. Modified Stern–Volmer plots for the quenching of BHb by NR at two different temperatures. c(BHb) = 2.0 μM, pH = 7.40, λex = 290 nm, λem = 332 nm.

3.2. Fluorescence quenching of BHb by NR

quenching, but the reverse effect would be observed for dynamic quenching [41]. The Stern–Volmer linear curves for the binding of NR to BHb are displayed in Fig. 4. The plots of F0/F versus the concentration of NR show well linear relation at both experimental temperatures. The values of KSV at different temperatures are shown in Table 1. The quenching constant KSV values decreased with an increase in temperature and kq was of the order of 1012 L · mol−1 · S−1. Obviously, this indicated that the quenching was not initiated from dynamic collision but from the formation of a compound.

Induced by a variety of molecular interactions with quencher molecule, such as excited-state reaction, molecules rearrangement, energy transfer, ground state complex formation and collision quenching, fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore. At the same time, it is an important method to study the interaction of substances with protein because it is sensitive and relatively easy to use. Fluorescence quenching types can be divided into dynamic and static. The collision quenching, or dynamic quenching, results from collision between fluorophores and a quencher. Dynamic quenching is related to temperature. Higher temperature result in larger diffusion coefficient and the bimolecular quenching constants are expected to increase with increasing temperature [36]. The static quenching is due to the formation of ground state complex between fluorophores and a quencher [37]. It is necessary to know quenching procedure and type for researching the quenching mechanism. Therefore, we use the binding constants' dependence on temperature to elucidate the quenching mechanism. The effect of NR on BHb fluorescence intensity is shown in Fig. 3. As the data show, the fluorescence intensity of BHb decreased regularly with the increasing concentration of NR without changing the emission maximum and shape of the peaks, which indicated that NR interacted with BHb. In order to confirm the quenching mechanism, the fluorescence quenching data were analyzed by the Stern–Volmer equation [38]: F0 ¼ 1 þ kq τ0 ½Q  ¼ 1 þ K SV ½Q  F

ð1Þ

3.3. Binding constant For static quenching process, the fluorescence data was further examined by using modified Stern–Volmer equation [42].

F0 1 1 1 ¼ þ F 0 − F f a f a K a ½Q 

ð2Þ

where Ka is the modified Stern–Volmer association constant for the accessible fluorophores, and fa is the fraction of accessible fluorescence. The modified Stern–Volmer equation was applied to determine Ka by a linear regression of F0 / (F0 − F) versus 1/[Q] (Fig. 5), the results were listed in Table 2. The value of Ka obtained is of the order of 104, indicating that there is a strong interaction between NR and BHb. The decreasing trend of Ka with increasing temperature is in accordance with KSV's dependence on temperature, which coincides with the static type of quenching mechanism. 3.4. Thermodynamic parameters and binding forces

where F0 and F are the fluorescence intensities before and after the addition of the quencher respectively. kq, KSV, [Q], and τ0 are the quenching rate constant of the bimolecular, the Stern–Volmer dynamic quenching constant, the concentration of the quencher and the average lifetime of the Hb without quencher (τ0 = 10−8 s [39]) respectively. As a rule, the maximum scatter collision quenching constant, kq of various quenching with the biopolymer was 2.0 × 1010 L · mol−1 · S−1 [40]. And the KSV values decrease with an increase in temperature for static

There are essentially four types of non-covalent interactions between small molecules and biological macro molecules, including hydrogen bonds, hydrophobic forces, electrostatic forces, and van der Waals interactions [41]. The thermodynamic parameters of binding reaction are the main evidence for confirming the binding force. If enthalpy change (ΔH0) does not vary significantly over the temperature range studied, then the thermodynamic parameters of ΔH0, entropy change

Table 1 Stern–Volmer quenching constants of the system of BHb–NR. T (K) 7.40

307 322

KSV (L · mol−1)

Stern–Volmer equation 4

F0/F = 0.9964 + 2.650 × 10 [Q] F0/F = 0.9895 + 2.318 × 104 [Q]

4

2.650 × 10 2.318 × 104

kq (L · mol−1 · S−1)

R

SD

2.650 × 1012 2.318 × 1012

0.9990 0.9991

0.0111 0.00

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Table 2 Thermodynamic parameters of BHb–NR interaction at pH 7.40. T (K)

R 10−4Ka (L · mol−1)

307 2.067 322 1.681

SD

ΔH0 ΔG0 ΔS0 (kJ · mol−1) (kJ · mol−1) (J · mol–1 · K−1)

0.9997 0.0677 −11.33 0.9997 0.0857

−25.36 −26.05

45.71 45.73

(ΔS0) and free energy change (ΔG0) can be determined by the van't Hoff equation and thermodynamic equation:

ln


 ðK a Þ2 ΔH 1 1 ¼ − ðK a Þ1 R T1 T2

ð3Þ

ΔG0 ¼ −RT ln K a

ð4Þ

ΔH 0 −ΔG0 T

ð5Þ

ΔS0 ¼

where Ka is the binding constant at the corresponding temperature T, T is absolute temperature and R is the gas constant. The values of ΔH0, ΔS0, and ΔG0 are listed in Table 2, the negative ΔG0 and the negative ΔH0 indicate that the binding process of NR to BHb is spontaneous and the formation of BHb–NR complex is exothermic reaction. According the views of Ross [41], if ΔH0 ≈ 0, ΔS0 N 0, the main force is electrostatic effect; if ΔH0 N 0, ΔS0 N 0, hydrophobic interaction plays a major role in the reaction; if ΔH0 b 0, ΔS0 b 0, the main forces are van der Waals and hydrogen bond interactions [41]. Thus it is difficult to interpret the thermodynamic parameters of BHb–NR interaction with a single intermolecular force. Therefore, the binding of NR to BHb might involve hydrophobic interaction strongly as evidenced by the positive values of ΔS0 and the electrostatic interaction can also not be excluded. In addition, Zhang [43] reported that NR amino surface is in protonation state. And Zhu [44] reported that the electrostatic attraction between electronegative gold nanoparticles and protonation amino of NR make gold nanoparticles fixed on the gold substrate surface easily. At pH 7.4 solutions, the hemoglobin (isoelectric point pI = 6.8) [45] bears negative charge because of the ionization of amino acid residues. So the binding of NR and BHb by electrostatic force became very easy. The hydrophobic and electrostatic interaction both exist in the binding of NR and BHb. The increase of entropy might be based on the destruction of the iceberg structure induced by the hydrophobic interaction.

Fig. 6. Effect of NR on BHb RLS spectra. (a) c(BHb) = 2.0 μM, (b) c(BHb) = 2.0 μM, c(NR) = 16.0 μM,(c) c(NR) = 16.0 μM.

Fig. 7. Overlap of the fluorescence emission of BHb (a) with the absorption spectra of NR (b). c(BHb) = 2.0 μM, c(NR) = 2.0 μM.

3.5. Characteristics of the RLS spectra The RLS spectra of BHb, BHb–NR complex, are recorded synchronously scanning from 200 to 700 nm with Δλ = 0 nm. The results are shown in Fig. 6. A remarkably increased RLS was observed with the addition of trace amount of NR to BHb solution. The production

Fig. 8. Synchronous fluorescence spectra of BHb (T = 307 K, pH = 7.40), spectra (1–7): c(BHb) = 2.0 μM, c(NR): 0, 4.0, 8.0, 12.0, 16.0, 20.0, 24.0 μM, respectively. (A) Δλ = 15 nm and (B) Δλ = 60 nm.

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of RLS is correlated with the formation of certain aggregate and the RLS intensity is dominated primarily by the particle dimension of the formed aggregate in solution [46]. According to these points, it can be conclude that the added NR may interact with BHb in solution, forming a new BHb–NR complex that could be expected to be an aggregate. Since the increased light scattering signal occurred under the given conditions, the size of BHb–NR particles may be bigger than that of BHb. 3.6. Energy transfer between NR and BHb According to Förster's non-radioactive energy transfer theory (FRET) [38,47], FRET is a special type of dynamic quenching mechanism where distance-dependent interaction between the electronic excited states of two fluorophores exists in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. Energy transfer is likely to happen under the following conditions: (1) the relative orientation of the donor and acceptor dipoles, (2) the extent of overlap of fluorescence emission spectrum of the donor with the absorption spectrum of the acceptor, and (3) the distance between the donor and the acceptor is less than 7 nm. Here the donor and acceptor were BHb and NR, respectively. The energy transfer efficiency E is defined as the following equation Eq. (6). E ¼ 1−

F R6 ¼ 6 0 F 0 R0 þ r 6

ð6Þ

where r is the distance from the ligand to the tryptophan residue of the protein, and R0 is the Förster critical distance, at which 50% of the

excitation energy is transferred to the acceptor [47]. It can be calculated from donor emission and acceptor absorption spectra using the Förster formula Eq. (7). R60 ¼ 8:79  10‐25 K 2 N−4 φ J

ð7Þ

where K2 spatial orientation factor of the dipole and K2 = 2/3 for random orientation as in fluid solution; N the average refractive index of medium; φ the fluorescence quantum yield of the donor; J the effect of the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor (Fig. 7), which could be calculated by Eq. (8) Z J¼

∞ 0

F ðλÞεðλÞλ4 dλ Z ∞ F ðλÞdλ

ð8Þ

0

where F(λ) is the corrected fluorescence intensity of the donor in the wavelength range λ to λ + Δλ; ε(λ) is the extinction coefficient of the acceptor at λ. In the present case, N = 1.36, F = 0.06 [48], according to Eqs. (6)–(8), we calculate that J = 2.61 × 10−15 cm3 · L · mol−1, E = 0.033, R0 = 1.73 nm, and r = 3.04 nm. Obviously, the donor to acceptor distance r is less than 7 nm, which indicates that the energy transfer from BHb to NR occurs with high possibility [49].

Fig. 9. The three-dimensional projections (A) and the corresponding contour spectra (B) of BHb (A-1 and B-1) and NR–BHb (A-2 and B-2). c(BHb): 2.0 μM; c(NR): 16.0 μM.

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Fig. 10. The binding mode of NR–BHb. Panel (A) shows docked NR into BHb, BHb is displayed in solid ribbon colored by amino acid chain, NR is shown using red ball and stick model. Panel (B) depicts the amino acid residues involved in binding of NR. Amino acid residues are displayed in stick, NR is shown using brown ball and stick model.

3.7. Conformation investigation In order to acquire further knowledge about the conformational change of BHb induced by NR, we measured synchronous and threedimensional fluorescence spectra of BHb with addition of NR. First introduced by Lloyd [50] in 1971, synchronous fluorescence spectroscopy technique involves simultaneous scanning of the excitation and emission monochromators while maintaining a constant wavelength interval between them. According to the theory of Miller [51], when the D-value (Δλ) between excitation and emission wavelength was stabilized at 15 or 60 nm, the synchronous fluorescence gives the characteristic information of Tyr residues or Trp residues. The effect of NR on BHb synchronous fluorescence spectra is shown in Fig. 8. It can be seen that red shift is observed for the emission maximum of Tyr and Trp residues, which indicates that the NR molecular is close enough to Tyr and Trp residues during the binding process. The conformation of BHb is changed. This implies that NR could enter into the hydrophobic pock at the α1β2 interface of BHb. The three-dimensional fluorescence spectra is also used to study the configuration change of proteins induced by small molecular. The threedimensional fluorescence spectra are shown in Fig. 9. The peak (λex = 285 nm, λem = 330 nm) mainly displays the spectral feature of Tyr and Trp residues. When protein is excited at 285 nm, it primarily discloses the intrinsic fluorescence of Tyr and Trp residues, and the phenylalanine (Phe) residue fluorescence can be negligible [52]. After the addition of NR, the fluorescence intensity decreased obviously. The phenomena revealed that the binding site was near Trp and Tyr residues.

3.8. Molecular modeling In order to further survey the binding site of NR–BHb complex, molecular modeling simulations were conducted, and the best docking energy result is displayed in Fig. 10. NR enters into the hydrophobic pock at the α1β2 interface of BHb. The result suggests that the interaction between BHb and NR is dominated by hydrophobic force, which is in agreement with the thermodynamic analysis. The free energy change ΔG0 for the binding of NR to BHb was −26.07 kJ · mol−1. The result is close to that obtained by the above mentioned experimental method. And as can be seen in Fig. 10(B), the binding site were obtained as α1-137Thr, α1-138Ser, α1-140Tyr, α1-141Arg, α2-127Lys, β2-36Pro,

β2-37Trp. Residues α-140Tyr and β-37Trp are in close proximity of NR, which provides a good structural basis to explain the efficient quenching of BHb emission in the presence of NR. At the same time, it is consistent with the synchronous and three-dimensional fluorescence results. 4. Conclusions The present study has provided insight into the interaction between neutral red and bovine hemoglobin. The results showed that neutral red could bind to bovine hemoglobin by a static quenching and form a new complex which becomes unstable with the rising temperature. Hydrophobic and electrostatic interactions played a major role in stabilizing the complex. The results of synchronous and three-dimensional fluorescence spectra indicated that the environments of Trp and Tyr residues were altered by NR. These results were confirmed by molecular docking study. The binding studies of NR with BHb not only showed toxicological importance but also provided information about the interactions of BHb with dyes. Acknowledgments This work was supported by 2013 Key Science and Technology Plan Project of Henan Province (132102110051), 2013 Science and Technology Plan Project of Henan Province (132102310123), Henan Province Health Department general project (201403046), and Youth Innovation Fund of the first affiliated hospital of Zhengzhou University. References [1] R.E. Hirsch, R.S. Zukin, R.L. Nagel, Intrinsic fluorescence emission of intact oxy hemoglobins, Biochem. Biophys. Res. Commun. 93 (2) (1980) 432–439. [2] M.M. Cox, G.N. Phillips Jr., Handbook of Proteins: Structure, Function and Methods, vol. 2, John Wiley & Sons Ltd., West Sussex, 2007. [3] S.Y. Wang, X.L. Xu, Q.L. Liu, Y.S. Xie, The Application of fluorescence spectroscopy in the study on protein conformation, Prog. Chem. 13 (2001) 257–260. [4] M.L. Kornguth, C.M. Kunin, Binding of antibiotics to the human intracellular erythrocyte proteins hemoglobin and carbonic anhydase, J. Infect. Dis. 133 (1976) 185–193. [5] M.F. Perutz, M.G. Rossmann, A.F. Cullis, H. Muirhead, G. Will, A.C.T. North, Structure of haemoglobin: a three-dimensional Fourier synthesis at 5.5-A resolution obtained by X-ray analysis, Nature 185 (1960) 416–422. [6] T. Peters, All About Albumin: Biochemistry, Genetics and Medical Applications, Academic Press, San Diego, CA, 1996.

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