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Photophysics in Motionally Constrained Bioenvironment: Interaction of Norharmane with Bovine Serum Albumin{ Arabinda Mallick and Nitin Chattopadhyay* Department of Chemistry, Jadavpur University, Calcutta, India Received 12 July 2004; accepted 18 November 2004

for fatty acids, hematin, bilirubin etc (5). BSA is the carrier of fatty acids, amino acids, metals, drugs and pharmaceuticals (6). X-ray diffraction studies have shown that the principal binding sites in serum albumins are located in the hydrophobic cavities (7). The molecular interactions are often monitored using different optical techniques. Fluorescence spectroscopy is perhaps the most important technique to study the interaction of probes with the proteins because of the high sensitivity of the technique and the relative ease of its use. Molecular interactions of different fluorophores such as 1-anilino-8-naphthalene sulfonate (8), diethylamino coumarin (9), 3-hydroxy flavone (10) and quercetin (11) with BSA and HSA have been studied by different authors. Norharmane (9H-pyrido[3,4-b]-indole) (NHM) (Scheme 1), which can be found in approximately 26 plant families, cells and animal tissues, belongs to the group of naturally occurring bioactive alkaloids possessing an extensive pharmacological activity, as central nervous system stimulant, hallucinogens and paralyzants of cardiac muscle (12,13). The interest on the photophysical study of NHM in different microheterogeneous environments originates principally from two aspects. The first one stems from its novel biological applications, such as photosensitizer toward a variety of systems, including bacteria, fungi, viruses etc. (14,15) and as fluorescence standard (16) for the determination of fluorescence quantum yields of different biological molecules in different environments. Photodynamic therapy (PDT), using photosensitizing chemicals combined with light to produce singlet oxygen, is a modality for cancer treatment (17). NHM has been reported to be

ABSTRACT Steady-state photophysics of norharmane (NHM), a bioactive alkaloid, has been studied in the presence of a model transport protein, bovine serum albumin (BSA). The emission spectrum undergoes a remarkable change upon addition of BSA to the aqueous solution of NHM in buffer. Addition of BSA leads to a marked increase in the fluorescence anisotropy of the neutral species of NHM, although the fluorescence anisotropy for the cationic species is almost invariant to BSA addition, suggesting that the neutral species is located in a motionally restricted environment of BSA, whereas the cationic species remains in the bulk aqueous phase. The binding constant (K) and free energy change (DG) for the probe–protein binding have been calculated from the fluorescence data. Light has been thrown on the action of urea on protein-bound NHM. The denaturation study suggests that the protein, in its native form, binds with NHM. Polarity of the microenvironment around the probe has been determined from a comparison of the fluorescence properties of the two prototropic species of NHM in water–dioxane mixture with varying composition.

INTRODUCTION Serum albumins, bovine serum albumin (BSA) and human serum albumin (HSA), are most widely studied abundant proteins in plasma (1,2). The three-dimensional structure of HSA has been resolved (3). However, regarding the structure of BSA, there are contradictory results. The most accepted one is that suggested by Brown describing this protein to have three domains, each consisting of a large double loop, a short connecting segment, a small double loop, a long connecting segment (Hinge), another large double loop and a connecting segment to the next domain (4). It is noteworthy that the presence of more than one tryptophan (Trp) residue in BSA makes the system more complex than HSA containing only one Trp residue. Serum albumins are known to bind a variety of biological probe molecules. It has a great affinity {Posted on the website on 9 December 2004 *To whom correspondence should be addressed: Department of Chemistry Jadavpur University, Raja S. C. Mullick Road, Calcutta, West Bengal 700 032, India. Fax: 91-33-24146266; e-mail: [email protected] Abbreviations: BSA, bovine serum albumin; FRET, fluorescence resonance energy transfer; HEPES, N-[2-hydroxyethyl]piperazine-N9-[2-ethanesulphonic acid]; HSA, human serum albumin; NHM, norharmane; PDT, photodynamic therapy; Trp, tryptophan.

Scheme 1. Different acid–base equilibria for NHM. CN: cation-neutral, NA: neutral-anion, ZA: zwitterion–anion and CZ: cation–zwitterion.


2005 American Society for Photobiology 0031-8655/05

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420 Arabinda Mallick and Nitin Chattopadhyay the probe is not located near Trp moiety of BSA. The determined polarity parameter (ET(30) 5 57.2) further suggests that the probe does not penetrate deep within the protein and locates itself near the protein–water interface.

MATERIALS AND METHODS

Figure 1. Emission spectra of NHM as a function of BSA concentration (kexc 5 350 nm). Curves (i)fi(vii) correspond to 0, 20, 40, 65, 90, 140, 190 lM BSA, respectively. Inset gives the variation of relative emission intensities of the cationic to neutral species as a function of BSA concentration.

quite effective in producing singlet oxygen, and it could be used as an efficient cancer cell photosensitizer (18). The extent of photodynamic action depends not only on the singlet oxygen production but also on the biodistribution of the probe molecule in the cytoplasmic and mitochondrial membranes, the retention and the nature of the binding inside the cell. Thus, the binding interaction of NHM with different biosystems is important to study the net PDT efficiency. The second aspect pertains to its unusual emission properties such as multiple fluorescence, extreme sensitivity toward microenvironment and pH. The photophysical or photochemical (or both) properties of NHM have been shown to be strongly modified by the solvents (19–21). Importance of the study of NHM probe is further emphasized from the understanding that it is formed as photoproducts from Trp in human lenses (22). On the basis of the prototropic studies, a number of acid–base equilibria have been proposed for NHM molecular system (Scheme 1), depending on the pH of the solution. Literature reports indicate that in aqueous medium between pH 1 and 10 only the cationic species emits; at pH ;12.3, emissions of the three species, viz. neutral, cation and zwitterion, have been recorded, and the anionic species starts absorbing only at pH equal to or above 14 (21,23). Studies on the proton transfer in NHM have been made in different solvents such as water, dichloromethane, chloroform, ethanol and acetonitrile–acetic acid mixture (20,21,23). Keeping in mind the wide application of the fluorescence molecules, susceptible to proton transfer reactions, as probes in the studies of biophysical interest, in this work, we have studied the fluorescence behavior of NHM in the model transport protein, BSA. A good number of biochemical and molecular biological investigations have been made using NHM (24–26). In this article, we have exploited the photophysical methods to study the interaction of NHM with the model transport protein, BSA. To the best of our knowledge, this is the first report of this sort to deal with the interaction of NHM with BSA. The study reveals that the probe is bound to BSA only by physical forces, resulting in a motional restriction on it. Despite a very good overlap between the emission band of Trp and the absorption band of NHM, lack of fluorescence resonance energy transfer (FRET) from Trp in BSA to the probe (NHM) suggests that

NHM procured from Aldrich USA (St. Louis, MO) was further purified by recrystallization from ethanol. BSA (98%, Fraction V) and N-[2hydroxyethyl]piperazine-N9-[2-ethanesulphonic acid] (HEPES) buffer from SRL (Mumbai, India) were used as received. Buffer solution (50 mM) was prepared and its pH was adjusted to 7.0. The same buffer solution was used throughout the experiment. Analytical grade urea (Loba Chemie, Mumbai, India) was used without further purification for the denaturation study. Spectroscopic grade 1,4-dioxane (Aldrich USA) was used in the polarity measurement experiments. Triply distilled water was used throughout the experiment. The fluorophore was excited at 350 nm so as to excite principally the neutral species of NHM in the ground state (21). Shimadzu MPS 2000 absorption spectrophotometer and Spex fluorolog-2 spectrofluorimeter were used for the absorption and emission spectral studies, respectively, at 298 K. The steady-state fluorescence anisotropy measurements were performed with a Hitachi spectrofluorimeter F-4010 model. Steady-state anisotropy, r, is defined by: r ¼ ðIVV
G  IVH Þ=ðIVV þ 2G  IVH Þ with G 5 IHV/ IHH where IVV and IVH are the intensities obtained with the excitation polarizer oriented vertically and the emission polarizer oriented vertically and horizontally, respectively. I terms associated with G refers to the similar parameters as mentioned above for the horizontal position of the excitation polarizer. All the anisotropy measurements were performed at room temperature (298 K). For all the experiments, concentration of NHM was ca 2 3 10
5 M and the pH was set at 7.

RESULTS AND DISCUSSION The absorption spectrum of NHM in HEPES buffer solution at pH 7.0 shows two bands with maxima at 348 and 372 nm corresponding to the neutral and cationic species, respectively (21). Addition of BSA to the aqueous buffer solution of NHM hardly changes the absorption spectrum. Room-temperature emission spectrum of NHM solution in HEPES buffer shows a single and unstructured band peaking at 450 nm, ascribed to the cationic species (21,27). Figure 1 depicts the emission spectra of NHM as a function of BSA concentration. Gradual addition of BSA in buffered solution changes the emission spectrum drastically. A new blueshifted structured emission band with peak at 380 nm develops at the cost of the emission corresponding to the cationic species at 450 nm, resulting in an isoemissive point at 410 nm. Consistent with the existing literature, the structured 380 nm band has been ascribed to the neutral species of NHM (21). A plot of the ratio of the fluorescence intensities of cation to neutral species of NHM (Fc/Fn) against the concentration of BSA shows, in a better manner, the nature of variation in the fluorescence behavior of NHM in the presence of the protein (Fig. 1 inset). The fluorometric behavior of NHM in the protein environment can be rationalized considering the prototropic character of the probe as well as the micropolarity–microrigidity around the fluorophore within the proteinous environment. A fluorometric study in a varying composition of water–dioxane mixture, to be discussed in a subsequent section, shows a similar enhancement of the emission of the neutral species of NHM and a concomitant decrease in the emission of the cationic one when the dioxane proportion is increased in the solvent mixture. This implies that the micropolarity of the environment plays a dominant

Photochemistry and Photobiology, 2005, 81 role in the prototropic behavior of NHM. Because an increase in the dioxane proportion in water–dioxane mixture lowers the polarity of the environment, the variation in the fluorescence behavior of NHM in BSA indicates that the polarity around the fluorophore bound to BSA is less than that in the bulk aqueous phase (27). It is true that the polarity in a homogeneous environment is not exactly the same as the polarity in a protein medium. However, in microheterogeneous environments such as micelles, proteins, lipids, the micropolarity is often determined and expressed in equivalent ET(30) scale comparing the fluorescence behavior of a probe in mixed solvent with that in the microheterogeneous environments (7,28–35). This polarity has been referred to as the static polarity by Kasha (33). Because the absorption spectra remains undisturbed with the addition of BSA, the change in the fluorescence properties of NHM is ascribed to the excited-state prototropic process. An enhancement in the emission of the neutral species of NHM in BSA environment indicates that the protonated state is preferred in the excited state and deprotonated state is favored in the ground state.

As mentioned above, there is hardly any change in the absorption spectrum of NHM solution upon addition of BSA. This indicates that the interaction between NHM and BSA (if any) is not sufficiently strong to put a signature on the absorption spectra. However, the fluorescence spectra clearly reflect an interaction between the two. A logical compromise of the two is the assumption that there is weak physical interaction between NHM and BSA. To see how strongly NHM binds with BSA, the binding constant value has been determined from the fluorescence intensity data by the Benesi–Hildebrand equation (36). ð1Þ

where DF 5 Fx
F0 and DFmax 5 F‘
F0, and where F0, Fx and F‘ are the fluorescence intensities of the particular species of NHM considered in the absence of protein, at an intermediate protein concentration and at a protein concentration for fluorescence saturation, respectively; K being the binding constant and [L] the protein concentration. Fluorescence anisotropy study, to be discussed in the next section, implies that the cationic species of NHM prefers to be in the aqueous phase and hardly binds with the protein. On the other hand, the neutral NHM shows a remarkable increase in the fluorescence anisotropy upon addition of BSA to the aqueous buffered solution, reflecting a binding interaction between BSA and the neutral NHM. Hence, we have only monitored the neutral fluorescence of NHM to determine the binding constant between the probe and the protein. Rearrangement of (Eq. 1) leads to the following simpler form ðF‘
F0 Þ=ðFx
F0 Þ ¼ 1 þ ðK½LÞ
1

Figure 2. Plot of (F‘
F0)/(Fx
F0) against [L]
1. For detail see text.

DG values for the binding constants of some other weakly bonding systems reported earlier (8,37).

NHM-BSA binding

1=DF ¼ 1=DFmax þ ð1=K½LÞð1=DFmax Þ

421

ð2Þ

Existence of the isoemissive point in Fig. 1 and linearity in the plot of (F‘
F0)/(Fx
F0) against [L]
1 confirms a 1:1 interaction between NHM and BSA. The binding constant value has been determined from the slope of the plot of (F‘
F0)/(Fx
F0) against [L]
1 (Fig. 2) and the value of K was determined to be 1.0 3 104 M
1 at ambient temperature (258C). From the determined K value, the free energy change for the probe–protein binding has been calculated to be DG 5
22.8 kJ mol
1 (615%) at 298 K. The estimated value lies in the range of

Fluorescence anisotropy study Measurement of fluorescence anisotropy has great importance in biochemical research because any factor that affects the shape and segmental flexibility of a molecule affects this parameter (38). Keeping in mind the wide application of this technique and also an independent evidence of NHM-BSA interaction, this technique has been exploited in this investigation. An increase in the rigidity of the surrounding environment around a fluorophore results in an increase in the fluorescence anisotropy. We have monitored the fluorescence anisotropy as a function of BSA concentration for both the fluorescence bands of NHM (at 380 and 450 nm corresponding to the neutral and the cationic species, respectively). The fluorescence anisotropy monitoring at 450 nm corresponding to cationic species does not show a significant change, indicating that the cationic species remains in the bulk aqueous phase all through and is not incorporated into the BSA environment. However, the fluorescence anisotropy value monitoring the neutral species shows a marked increase on moving from the aqueous phase to the BSA environment (Fig. 3), suggesting that the rotational diffusion of the probe molecule is restricted significantly. This observation reflects the incorporation of the neutral species of NHM into the BSA environment. We have extended the anisotropy measurements of NHM in glycerol–water mixture of different composition with an intention to see whether determination of microviscosity around the probe is feasible or not. Incidentally, the anisotropy value of NHM in 90% glycerol–water mixture is still remarkably lower than the anisotropy in the BSA environment at the saturation level. As per literature (39), the viscosity in the BSA environment is supposed to be not high enough to exceed the viscosity of a 90% glycerol– water mixture. This observation indicates the dominance of the rotational correlation time of BSA over the normal viscosity effect. Involvement of some specific interaction such as hydrogen bonding centerd around the protein and the heteroatoms present in the probe molecule, leading to some additional restriction imposed on the motion of the overall molecule or trapping of the

422 Arabinda Mallick and Nitin Chattopadhyay

Figure 3. Variation of fluorescence anisotropy (r) of NHM as a function of BSA concentration (kem 5 380 nm).

probe in some motionally constrained site (such as crevice) of the protein, might be invoked for the explanation. Effect of urea on protein-bound NHM After finding the binding interaction between NHM and BSA, we intended to observe the denaturing effect of the protein on the binding activity and on the overall photophysics of the probe. Hence, we have studied the effect of urea on the protein-bound NHM. Gradual addition of urea to the BSA-bound NHM changes the emission spectrum in a manner opposite to that observed upon gradual addition of BSA to the aqueous NHM solution. In the presence of urea, the fluorescence band of the cationic species increases at the cost of the band of the neutral species (Fig. 4). Fluorescence anisotropy study, as discussed earlier, suggests that the cationic species of NHM remains principally in the bulk aqueous phase. Thus, an enhancement in the emission of the cationic species at the cost of the emission of the neutral species with the addition of urea suggests that the fluorophore molecule is expelled from the proteinous environment to the aqueous phase.

Figure 4. Emission spectra of BSA-bound NHM as a function of added urea. Curves (i)fi(v) corresponds to 0, 2, 4, 6 and 8 M urea, respectively (kexc 5350 nm and [BSA] 5 200 lM). Inset: Variation of fluorescence anisotropy (r) of BSA-bound NHM as a function of added urea (kem 5 380 nm, [BSA] 5 200 lM).

Figure 5. Emission spectra of NHM in dioxane–water mixture under the same experimental condition (kexc 5 350 nm). Water–dioxane compositions (vol percent) of the solvent mixtures for curves (i)fi(vii) correspond, respectively, to 100:00, 80:20, 70:30, 65:35, 60:40, 55:45 and 20:80. The figure has been adapted from Ref. 27.

Inset of Fig. 4 shows the variation of the fluorescence anisotropy of the NHM-BSA system as a function of urea concentration. The inset of Fig. 4 having a pattern quite opposite to that of Fig. 3 reflects that addition of urea leads to weakening of the probe– protein interaction, resulting in the release of the probe into the bulk aqueous phase. Consistent with our earlier studies (28,29,40), we believe that urea removes some water molecules adjacent to the protein with the denaturation of the latter. The resulting destabilization of the environment leads to the ejection of the guest molecule from the proteinous environment to the bulk water phase. It is important to mention here that in the presence of 9 M urea the anisotropy value corresponds closely to the value obtained in the aqueous phase. Thus, the observation reveals that BSA binds with the fluorophore, NHM, in its native form and denaturation of BSA leads to the release of the fluorophore from the protein environment to the bulk aqueous phase. Micropolarity around the probe For a couple of decades, fluorescent probes have been serving a unique role in the determination of the microscopic polarity of the biological systems (30–32). The polarity determined through different photophysical parameters of the probe gives a relative measure of the polarity of the microenvironment. It is true that the polarity of a homogeneous environment is not the same as the polarity of a heterogeneous environment such as proteins. Kasha has referred to this polarity as static polarity (33). In microheterogeneous environments, the micropolarity is often determined and expressed in equivalent ET(30) scale comparing the fluorescence behavior of a probe in mixed solvents of known polarities with that in the microheterogeneous environments (28,29–35). In an earlier study, we have determined the polarity of the micellar microenvironments, using this bioactive molecular system (27). In this study, we have extended our effort of exploiting the fluorometric technique to determine the micropolarity around the probe in BSA environment. To determine the micropolarity in BSA environment, the fluorescence behavior of NHM within this protein environment has been compared with that in water–dioxane mixture of varying composition (Fig. 5). Figure 5 reveals that with a decrease in the water proportion in the water–dioxane

Photochemistry and Photobiology, 2005, 81

423

CONCLUSIONS This work reports the study of the interaction of NHM, a biological photosensitizer, with a model transport protein, BSA. The photophysical behavior of the probe is modified in the BSA environment compared with that in the aqueous phase. This has been exploited to determine the probe–protein binding efficiency, nature of the microenvironment and finally the micropolarity around the probe. This work further demonstrates the action of urea on BSA. Denaturation of the protein leads to the release of the fluorophore molecules from the protein to the bulk aqueous phase. Acknowledgements—Financial support from Council of Scientific and Industrial research, Government of India, is gratefully acknowledged. The authors appreciate the cooperation received from H. Chakraborty and Prof. S. Basak of Saha Institute of Nuclear Physics for the anisotropy measurements. The authors thank one of the reviewers for critical comments. Figure 6. Variation of log of fluorescence yield of neutral to cationic species of NHM in water–dioxane mixture against ET(30). Part of the figure has been adapted from Ref. 27.

REFERENCES

solvent mixture, the emission intensity of the neutral species increases at the cost of the emission corresponding to the cationic species. This denotes that as the polarity of the microenvironment around the probe is reduced the cation is destabilized, resulting in an increase in the band corresponding to the neutral species. An increase in the neutral to cation relative fluorescence yield in the BSA environment from that in the pure buffered aqueous medium indicates that the microenvironment around the probe in BSA is reasonably less polar than the bulk water. To get a qualitative measure of the polarity at the protein–fluorophore binding site, empirical solvent polarity parameter, ET(30), based on the transition energy for the solvatochromic intramolecular charge transfer absorption of the betaine dye 2,6-diphenyl-4-(2,4,6 triphenyl-1pyridino) phenolate as developed by Reichardt has been used (41,42). A linear correlation was obtained from the plot of the logarithmic ratio of the peak intensities of the neutral to the cationic species of NHM, using different compositions of water–dioxane mixture for which ET(30) values are known (Fig. 6). Comparing the value of the logarithm of the ratio of the band intensity of the neutral-cation of NHM in BSA environment, with the above correlation, we have determined the micropolarity around the probe to be 57.2 in terms of ET(30) (Fig. 6). This value represents that the micropolarity of this environment at the binding site of NHM is more than the micropolarities of cetyltrimethylammoniumbromide and triton X-100 micellar systems (ET(30) around 54, Ref. 27) determined through the similar method. That there is hardly any change in the absorption spectrum of NHM upon binding with BSA and also the determined micropolarity (ET(30) 5 57.2) suggests that the probe does not penetrate deep into the proteinous core. Rather, it binds near the protein– water interface region. Quite often, proximity of a fluorophore to the Trp moiety is determined through FRET. Because of the presence of good overlap between the emission spectrum of Trp present in BSA (donor) and the absorption spectrum of NHM (acceptor), we looked for the possibility of FRET by exciting the composite system at the excitation of Trp. We found no evidence of the process. This suggests that the fluorophore (NHM) is not located near Trp of BSA. However, the exact location of the probe within BSA is yet to be determined.

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