Static and Dynamic Interaction of a Naturally Occurring Photochromic

J. Phys. Chem. B 2008, 112, 16793–16801

16793

Static and Dynamic Interaction of a Naturally Occurring Photochromic Molecule with Bovine Serum Albumin Studied by UV-Visible Absorption and Fluorescence Spectroscopy Pier Luigi Gentili,* Fausto Ortica, and Gianna Favaro Dipartimento di Chimica, UniVersita` di Perugia, 06123 Perugia, Italy ReceiVed: July 05, 2008; ReVised Manuscript ReceiVed: October 21, 2008

In this work, the interaction of a naturally occurring chromene, flindersine (FL), and bovine serum albumin (BSA) has been investigated by UV-vis absorption and fluorescence spectroscopy, time-resolved lifetime measurements, steady state photochemistry, and semiempirical calculations. The interplay of FL with tryptophan (Trp) has been studied in parallel. The interaction of FL with BSA causes fluorescence quenching of BSA through both static and dynamic quenching mechanisms. FL binds BSA with a stoichiometry that varies from 1.09:1 to 0.80:1 as the temperature increases from 293 to 308 K. The reaction is characterized by negative enthalpy (∆H° ) -193 kJ mol-1) and negative entropy (∆S° ) -550 J K-1 mol-1), indicating that the predominant forces in the FL-BSA complex are hydrogen bonding and van der Waals forces. The binding distance between the protein and the photochrome was calculated as 2.5 nm, according to the Foerster theory on resonance energy transfer. The effect of FL concentration on the BSA fluorescence was analyzed according to the maximum entropy method. FL also quenches the emission of Trp with a mechanism that, based on the experimental evidence, excludes both static and dynamic effects. An alternative relaxation pathway, consisting in an electron transfer from a prefluorescent state of Trp to FL, is put forward. The photobehavior of FL is affected by the interplay with BSA but not with Trp. When FL is complexed with BSA, it becomes a more fluorescent and more reactive species. Semiempirical calculations of the lowest optically active electronic transitions of hypothetical FL photoproducts suggest the most likely structure for the photoproduct. 1. Introduction As active components of cells, proteins control the chemical reactions occurring in the cell and receive signals from outside of the cell. Their shape determines whether they can go into cells, passing through biological membranes. They form scaffoldings that allow other molecules to be linked specifically and tightly. These properties can be exploited for drug delivery. Therefore, interactions of proteins, such as bovine (BSA) or human (HSA) serum albumins with biological molecules, drugs, and dyes1-18 have been a subject widely investigated in recent years. From these studies, BSA and HSA were found to have a high affinity toward extremely different kinds of molecules, so they can serve as solubilizers and carriers for these materials in living organisms. Notably, they play an irreplaceable role in transportation of drugs to their targets. Therefore, BSA and HSA have been used as model proteins for a great variety of biophysical and physicochemical studies. In this work, the interaction of BSA with a naturally occurring photochromic compound, flindersine (FL, Scheme 1) is investigated by absorption spectroscopy and steady state and timeresolved fluorescence. The system flindersine-tryptophan has been also studied in parallel for the purpose of comparison. FL exhibits excitation wavelength dependence of fluorescence and photochemistry19 and possesses two close-lying electronic excited states, having n,π* and π,π* character, respectively, whose coupling can be tuned by changing the solvent proticity and the temperature.20 FL is a secondary metabolite synthesized by plants, such as Flindersia australis, belonging to the family of Rutaceae. It plays a role in antimicrobial activity and can potentially find use in photochemotherapy.21 Therefore, it is * Corresponding author. Phone: +39 075 5855576; fax: +39 075 5855598; e-mail: [email protected].

SCHEME 1: Flindersine (FL)

worthwhile investigating its capability of linking to a protein such as BSA. 2. Experimental Section 2.1. Materials. Bovine serum albumin (BSA, min. 99%, from Sigma) and L-tryptophan (Trp, min. 99%, from Carlo Erba) were used as received. Flindersine (FL, from John Morgan, Forest Products Research Laboratory, England) was used without further purification since it appeared pure on HPLC analysis. BSA, Trp, and FL were dissolved in phosphate buffer solutions (PBS) at pH ) 7.2 with an ionic strength I ) 0.18 mol dm-3 in the presence of 0.15 mol dm-3 of NaCl. Freshly prepared BSA and Trp solutions were used for all measurements. 2.2. Apparatus and Methods. 2.2.1. Steady State Absorption Measurements. The absorption spectra at room temperature were recorded on a PerkinElmer Lambda 800 spectrophotometer or a Perkin-Elmer Lambda 5 spectrophotometer. A Hewlett-Packard 8453 diode array spectrophotometer was also used. To control the temperature, an Oxford Instruments cryostat (precision ( 1 K, accuracy ( 0.2 K) was used in the photoreaction quantum yield determinations, and a water thermal bath was used in the fluorescence quenching experiments. 2.2.2. Photochemical Quantum Yields. The reaction quantum yield of FL was determined at 283 K by exciting at 342

10.1021/jp805922g CCC: $40.75  2008 American Chemical Society Published on Web 12/05/2008

16794 J. Phys. Chem. B, Vol. 112, No. 51, 2008

Gentili et al.

nm. Experiments of irradiation at 275 nm were also carried out. A 125 W Xe lamp, coupled with a Jobin-Yvon monochromator (16 nm band-pass), was used to irradiate 1 cm3 of solution in a fluorimetric cell with 1 cm path at a right angle with respect to the monitoring beam of the spectrophotometer. The radiation intensity was determined using potassium ferrioxalate actinometry. The emitted photon density was on the order of 7 × 10-7 moles of photons dm-3 s-1 at 342 nm and 8 × 10-8 moles of photons dm-3 s-1 at 275 nm. The FL concentration was nearly 7 × 10-5 mol dm-3. The decrease of FL concentration was ′ /dt), at followed from the decrease of its absorbance, -(dAFL two analytical wavelengths, 348 and 365 nm. The quantum yield, ΦPC, was determined from the relationship shown in eq 1,

ΦPC

( )

′ dAFL dt ) ′ εFLI0(1 - 10-AFL)

-

(1)

′ is the FL where I0 is the intensity of the incident light, AFL ′ is the molar absorbance at the excitation wavelength, and εFL absorption coefficient of FL at the analysis wavelength. The reaction was followed up to 2% transformation, in order to ignore the contribution of the photoproduct at the analysis wavelength. 2.2.3. Emission Measurements. Corrected emission spectra were recorded using a Spex Fluorolog - 21680/1 spectrofluorimeter, controlled by the Spex DM 3000F spectroscopy software. The fluorescence quantum yields, ΦF, were determined by measuring and comparing corrected areas under the spectra of the standard (anthracene in ethanol, A ) 0.05 at 366 nm, ΦF ) 0.2722) and the sample. The fluorescence quenching experiments as a function of the temperature were performed using a Varian Eclipse fluorimeter equipped with a quartz optical fiber. The optical fiber was immersed into the solutions in the test tube whose temperature was regulated through a water thermal bath. Data of fluorescence quenching were corrected for trivial absorption of the exciting and emitted light by the quencher.23 Excitation and emission wavelengths were selected in order to minimize corrections. The fluorescence lifetimes were measured with a timecorrelated single-photon counting fluorometer (Edinburgh Instruments 199S). A LED centered at 265 nm, having repetition rate of 700 kHz and pulse fwhm (full-width-half-maximum) of 560 ps, was used as irradiation source. The experimental decay function F(t), eq 2, is a convolution of the instrument response function R(t) and the fluorescence decay function I(t):

F(t) ) R(t) X I(t)

(2)

The deconvoluted fluorescence decay curves, I(t), were analyzed by two methods: the nonlinear least-squares method implemented into the IBH decay analysis software and the maximum entropy method (MEM) using the MemExp software available online.24 In the case of the nonlinear least-squares method, the deconvoluted I(t) data were fitted by a polyexponential function (eq 3),

I(t) )

∑ Aie- τ

t i

i

Figure 1. (A) Absorption (solid line) and fluorescence (dashed-dot line) spectra of BSA (red) and FL (black) in PBS (pH ) 7.2); (B) absorption (solid line) and fluorescence (dashed-dot line) spectra of Trp (red) and FL (black) in PBS (pH ) 7.2).

(3)

where τi are lifetimes and Ai are their relative weights, with i variable from 1 to 3. The best-fit function was chosen considering the magnitude of χ2 and the shape of the autocorrelation function of the weighted residuals. Using the MEM method, the fluorescence decay function, I(t), was fitted by the relationship given in eq 4, where g(log τ) and h(log τ) are the lifetime distributions that correspond to decay and rise kinetics,

I(t) ) D0

t

∫-∞+∞ (g(log τ) - h(log τ))e- τ d log τ + 3

∑ (bk - ck)(t/tmax)k

(4)

k)0

respectively, and the polynomial accounts for the baseline. The fit procedure entails the maximization of the function Q defined in eq 5,

Q ) S - λχ2 - RI

(5)

where S is entropy, I is a normalization factor, and λ and R are Lagrange multipliers. In the definition of χ2, the standard errors in the measured data were assumed to be Gaussian type. The choice of the best-fit function is made by considering the magnitude of χ2 and of the correlation length of the residuals. 2.2.4. Theoretical Calculations. The semiempirical PM3 method was used to compute fully optimized ground-state geometries and ZINDO/S to simulate the absorption spectra. 3. Results and Discussion 3.1. Absorption and Fluorescence Spectra of BSA, FL, and Trp. The absorption and fluorescence spectra of BSA and Trp in an aqueous buffered solution (pH ) 7.2) at room temperature are compared with those of FL in Figure 1A and 1B, respectively. The absorption spectrum of BSA is characterized by a band at 277 nm and a more intense absorption at shorter wavelengths. The fluorescence of BSA shows a maximum at 336 nm, with a Stokes shift of about 6350 cm-1. The BSA fluorescence largely overlaps the absorption profile of the lowest energy electronic transition of FL (λmax ) 342 nm). Trp shows quite similar spectral features as BSA, with an absorption peak at 278 nm and a fluorescence band having a maximum at 351 nm. The similarity indicates that the Trp moieties are mostly responsible for the lowest energy transition

Interaction of a Photochrome with Bovine Serum Albumin

J. Phys. Chem. B, Vol. 112, No. 51, 2008 16795

TABLE 1: Absorption and Fluorescence Properties in Buffered Water (pH ) 7.2) Compared with Some Literature Values for BSA, Trp, and FL at Room Temperature absorption BSA Trp FL a

fluorescence

λmax/nm

max/dm3 mol-1cm-1

λmax/nm

ΦF (λexc/nm)

lifetime/ns

277 278 343

39300 5200 9200

336 351 434

0.1 (275) 0.12 (275) (0.18)a 0.005 (330) 0.004 (310)

5.34 (5)a 2.64 (3.13; 0.51)b 1.0

Reference 26. b Reference 27.

of BSA, which contains two tryptophan residues: Trp-212, which is located within a hydrophobic pocket of the protein, and Trp-134, on the surface of the molecule.25 The large Stokes shifts (∆ν > 6000 cm-1) indicate that excitation into the Franck-Condon state of both biomolecules is followed by very rapid solvent reorganization around a structure having notably different dipole moment compared to the ground state. Also, fluorescence quantum yields and lifetimes of BSA and Trp are of the same order of magnitude. The absorption and emission characteristics of the three molecules are reported in Table 1. Comparison with some literature data shows an excellent agreement for BSA.26 In the case of Trp, for which we observed a single lifetime, a double exponential decay is reported in the literature,27 with relative intensities dependent on the pH and the buffer chemical composition. The most consistent interpretation was that the fluorescence of tryptophan originates from at least two conformers. However, at pH > 7 a single exponential decay was detected,28 as confirmed by us. The analysis of the fluorescence decay kinetics (Figure 2, inset), through the maximum entropy method (Figure 2), reveals that, in the case of the macromolecular BSA, a large set of conformers gives rise to a distribution of lifetimes that can be fitted by a Gaussian function centered at 5.69 ns and having a fwhm of 1.60 ns, whereas in the case of the free Trp molecule a delta function having a maximum at 2.58 ns is the best representation of the distribution of the limited number of rotamers that the amino acid assumes at room temperature. The spectral properties of BSA and Trp in relation to those of FL are ideal for an efficient FRET (fluorescence resonance energy transfer) wherein the amminoacid molecules (Trp or BSA) act as donors and FL as acceptor. The application of the Foerster theory29 demonstrates that the critical transfer radius (R0, eq 6, wherein the orientation factor κ2 is taken as 2/3, N is the Avogadro number, n is the refractive index, FBSA(νj) is the fluorescence intensity of BSA at νj and εFL(νj) is the molar adsorption coefficient of FL at the same νj) to FL is 2.5 nm for BSA and 2.6 nm for Trp.

R60 )

9000(ln 10)κ2ΦBSA 4

4

128π Nn

∫0∞

FBSA(ν)εFL(ν) ν4

dν

evidence for the occurrence of an interaction in the ground and/ or in the excited state. Also, a temperature effect on these properties can be a reliable probe in order to thoroughly analyze the thermodynamic aspects of the interplay. On the other hand, additional information could be obtained from observations of spectral (both absorption and fluorescence) and photochemical changes of FL upon additions of BSA. The absorption spectrum of FL undergoes a slight modification in the presence of BSA (see arrows in Figure 3A), whereas it does not change at all when FL interacts with Trp (Figure 3B). 3.2.1. Effect of FL on BSA Fluorescence. The addition of FL to a solution of BSA induces quenching of the protein fluorescence but no changes in spectral shape (Figure 4). This behavior indicates that the chemical microenvironment of the fluorescent amino acid residues in the protein, tryptophan and tyrosine, does not change appreciably. Even the fluorescence anisotropy of BSA does not undergo modifications by addition of FL. For λexc ) 275 nm, r ≈ 0.1 in the absence of FL as well as when the nBSA/nFL ratio is 1.5 or 0.1.

Figure 2. Fluorescence lifetime distributions for Trp (red) and BSA (black) according to the maximum entropy method. Inset: decay kinetics of BSA (black) and Trp (red) fluorescence.

(6)

These values are close to those found for the interaction of several other molecules with serum albumins (HSA or BSA).5,7,10,12,14,16,17,30 3.2. Interactions of BSA-FL and Trp-FL. Several kinds of measurements were carried out on the BSA-FL system (and in parallel on the Trp-FL system) to recognize whether some interaction occurred and to understand the nature of such interaction. Given the large Stokes shift and consistent emission quantum yield, the fluorescence of BSA is the property most suitable to be investigated. Intensity, spectral shape and changes in lifetime of BSA fluorescence upon addition of FL can give

Figure 3. Absorption spectrum of FL upon addition of (A): BSA with a molar ratio nBSA/nFL varying from 0 to 1.2 and (B): Trp with molar ratio nTrp/nFL varying from 0 to 6.6.


Gentili et al. TABLE 2: BSAa Fluorescence Lifetimesb as a Function of FL Concentration at Room Temperature

Figure 4. Quenching of BSA (5.12 × 10-7 mol dm-3) fluorescence observed by increasing FL concentration (0-4.65 × 10-6 mol dm-3). The arrow highlights the effect of FL addition.

[FL](mol dm-3)

τ1 (ns)

0 1.25 × 10-6 1.86 × 10-6 2.46 × 10-6 3.00 × 10-6 3.62 × 10-6 4.18 × 10-6 4.73 × 10-6 5.28 × 10-6 5.81 × 10-6 6.33 × 10-6 6.85 × 10-6 7.35 × 10-6 7.85 × 10-6 8.33 × 10-6 8.81 × 10-6 9.28 × 10-6 9.75 × 10-6 1.02 × 10-5 1.11 × 10-5

5.34 5.19 5.14 5.19 5.10 5.02 4.95 4.95 4.84 4.86 4.74 4.69 4.64 4.55 4.51 4.40 4.36 4.39 4.34 4.26

A1 (%)

98.5 98.05 97.5 97.06 96.55 96.81 96.22 95.19 94.68 92.60 91.65 89.98 90.13 89.22 87.10 88.03 88.65

τ2 (ns)

0.77 ( 0.18 0.66 ( 0.15 0.62 ( 0.11 0.66 ( 0.10 0.65 ( 0.08 0.67 ( 0.09 0.67 ( 0.09 0.71 ( 0.07 0.73 ( 0.07 0.81 ( 0.06 0.73 ( 0.05 0.81 ( 0.05 0.79 ( 0.06 0.80 ( 0.05 0.88 ( 0.04 0.84 ( 0.05 0.74 ( 0.04

A2 (%)

χ2

1.52 1.95 2.50 2.94 3.45 3.19 3.78 4.81 5.32 7.40 8.35 10.02 9.87 10.78 12.90 11.93 11.35

1.60 1.51 1.99 1.66 1.65 1.44 1.53 1.47 1.49 1.65 1.40 1.44 1.37 1.35 1.34 1.36 1.28 1.13 1.22 1.32

[BSA] ) 3.31 × 10-7 mol dm-3 in PBS. b Uncertainties on τ1 are within (0.02 ns for all measurements, whereas those on τ2 depend on its relative contribution. a

Figure 5. Stern-Volmer plot for the quenching of BSA (5.12 × 10-7 mol dm-3 in PBS) fluorescence by FL (λexc ) 275 nm and λem ) 335 nm).

The quenching ratios of the BSA fluorescence, I0/I (where I0 and I are the emission intensities in the absence and in the presence of FL, respectively), were plotted against the FL concentration, according to the Stern-Volmer equation (see Figure 5). The plot shows an upward curvature and is well-fitted by the function of eq 7,

I0/I ) 1 + K[FL] + K′[FL]2

(7)

suggesting the coexistence of both static and dynamic quenching. The coefficients K and K′ should correspond to the sum and product of the dynamic (KD) and static (KS) quenching constants, respectively. The upward curvature is determined by their relative importance. If the curvature is not so much accentuated, as found in the BSA-FL system, it means that they differ by a considerable amount. The occurrence of dynamic quenching can be recognized by measurements of fluorescence lifetime of BSA as a function of FL concentration. To detect a shortening of BSA lifetime, significantly large nFL/nBSA molar ratios have to be reached. Table 2 reports the results of the nonlinear leastsquares analysis of the fluorescence decay kinetics recorded at room temperature. At the smallest FL additions, the data are well-fitted by monoexponential decay functions. When the number of FL moles added is almost 10 times that of BSA moles, the decay kinetics become biexponential due to appearance of a shorter component. The long component (τ1) is progressively shortened by addition of FL and its relative weight

Figure 6. Stern-Volmer plot for the dynamic quenching of BSA (3.31 × 10-7 mol dm-3) fluorescence by FL, according to eq 8, correlation coefficient r ) 0.988.

(A1%) decreases, whereas the shorter component (τ2) remains substantially constant and its relative weight (A2%) progressively increases. The dynamic contribution to the quenching mechanism can be quantified by applying the Stern-Volmer eq 8.

τ01 ) 1 + KD[FL] τ1

(8)

From the slope of the linear plot obtained, Figure 6, the dynamic constant, KD ) (2.49 ( 0.06) × 104 dm3 mol-1, is obtained and the quenching rate constant results kq ) KD/τ01 ) (4.7 ( 0.1) × 1012 dm3 mol-1 s-1. The high value of the quenching rate constant, larger than the limit imposed by the viscosity, 2 × 1010 dm3 mol-1 s-1,32 indicates that the quenching occurs via Coulombic resonance interaction, which is not controlled by diffusion. Quenching constants exceeding the diffusion-controlled value were generally found for the interaction of serum albumins with other molecules.1,3,4,6,7,12,18,30-33 The results of the analysis of the fluorescence decay kinetics by the maximum entropy method (MEM) are portrayed, for some representative data, in Figure 7.



Figure 8. (A) Examples of linear Stern-Volmer trends of fluorescence at low quencher (FL) concentrations and at different temperatures (squares: 293 K; circles: 308 K). (B) Application of eq 10 to the fluorescence quenching data at 293 K (squares) and 308 K (circles).

TABLE 3: Logarithm of the Association Constant (KS) and Stoichiometric Coefficient (nS) for the Interaction of FL and BSA at Different Temperatures (T)

Figure 7. Lifetime distribution functions for BSA fluorescence excited at 265 nm with increasing the nFL/nBSA ratio: 0 (A), 5.8 (B), 9.7 (C), 15.4 (D), 19.3 (E), 25 (F), 34.7 (G), and 40.5 (H).

MEM analysis reveals that in any case there exists a distribution of conformers for BSA. In the absence of FL, the distribution is broad (fwhm ) 1.59 ns) and is centered at 5.69 ns. Upon addition of FL, it becomes gradually narrower and progressively shifts to shorter lifetimes. A second distinct distribution appears that moves to shorter lifetimes and becomes narrower at increasing FL amounts. The close proximity of FL molecules reduces the excited-state lifetime of BSA and its ability to relax to a broad distribution of conformations. This phenomenology was also revealed in the case of HSA/quercetin interaction.14 The above results on dynamic interaction were exploited to separate dynamic and static quenching in eq 7. Static quenching is described by the general chemical equation (9). In this equation, ns represents the number of FL molecules associated with each protein.

BSA + nsFL ) BSA(FL)ns

(9)

From the quenching of the fluorescence of BSA by FL, the thermodynamic constant (KS) and stoichiometry (ns) of the binding process can be determined, according to eq 10.34

(

log

)

I0 - 1 ) log KS + ns log[FL] I

T (K)

log KS

ns

293.0 295.1 298.3 301.1 303.5 305.3 308.4

5.9 ( 0.3 5.21 ( 0.30 5.20 ( 0.35 4.43 ( 0.17 4.66 ( 0.20 4.14 ( 0.23 4.11 ( 0.24

1.09 ( 0.05 0.99 ( 0.05 0.98 ( 0.06 0.86 ( 0.03 0.88 ( 0.03 0.83 ( 0.04 0.80 ( 0.04

comparing the plots of Figures 5 and 6, it is observed that the dynamic quenching becomes appreciable only for [FL] > 3 × 10-6 mol dm-3 (molar ratio nFL/nBSA > 7). Therefore, below this concentration value, the quenching can be considered purely static. By increasing the temperature, the trend of the quenching plots changes and the upward curvature becomes unappreciable. This indicates that the relative contribution of the two quenching mechanisms is temperature dependent. In agreement with the expectation that the constant for the static association decreases with increasing temperature, static interaction is assumed to be the temperature-dependent process. Some examples of linear SV trends at different temperatures and for [FL] < 3 × 10-6 mol dm-3 (molar ratio nFL/nBSA < 7) are shown in Figure 8A. On the basis of such kind of plots, eq 10 was applied to the data collected at different temperatures in a limited range of FL concentration. Some graphical examples of this treatment are depicted in Figure 8B; all numerical results are summarized in Table 3. The great value of the binding constant, KS, entails a strong affinity of FL to BSA. It is rather sensitive toward the temperature; in fact, KS decreases almost 2 orders of magnitude (from 7.9 × 105 to 1.3 × 104 dm3 mol-1) when the temperature is increased by roughly 15 K. The data in Table 3 allow the thermodynamic parameters ∆H° and ∆S° of the association process to be determined, according to the van’t Hoff equation (11).

(10)

To isolate the contribution of static from dynamic quenching, only the initial part of the quenching plot, which is mainly influenced by the first-order concentration dependence and therefore is linear, was utilized. In fact, as can be deduced by

ln KS )

∆S° ∆H° R RT

(11)

The linear fit of ln KS as a function of the reciprocal absolute temperature is portrayed in Figure 9, from where ∆S° ) -(550 ( 100) J K-1 mol-1 and ∆H° ) -(193 ( 31) kJ mol-1 are


Figure 9. van’t Hoff diagram wherefrom the thermodynamic parameters for the association reaction between FL and BSA were obtained.

Figure 10. Quenching of Trp (1.72 × 10-5 mol dm-3) fluorescence by FL at nFL/nTrp ratio varying from 0 to 1.9. Inset: treatment of the quenching data according to eq 10, correlation coefficient: r ) 0.997.

obtained from the intercept and slope, respectively. Hence, ∆G° ) -29 kJ mol-1 at 298 K. The interplay of FL with BSA is exothermic and causes a reduction of entropy. In most of literature cases, less negative3-7,10-12,16,30,34,35 or even positive1,9,31 ∆H° and positive ∆S° values1,3-7,9-11,16,30,31 were found. Only a limited number of works on similar subjects report negative ∆S° values.11,12,33,35 From the extent and the sign of the variation of enthalpy and entropy, determined in this work, it can be inferred that straight after the first addition of FL to a solution of BSA in PBS, the photochromic molecules tightly bind to the aminoacidic cavities through van der Waals forces and/or hydrogen bonding.36 Because the binding of FL to BSA effectively quenches the protein fluorescence, it can be envisaged that it involves the hydrophobic pocket of domain II wherein Trp 212 is located.1 Most likely, the FL molecular dimensions and the presence of the lactame group, able to dock to both hydrogen donors and acceptors, can explain its peculiar binding thermodynamic parameters with respect to the vast majority of the compounds so far investigated in their interplay with albumin proteins. The dimensions of FL, relative to those peculiar of the host aminoacidic cavity, rule the stoichiometry of the complexation reaction. The value of the coefficient ns is around 1. Really, its value is slightly larger than 1 at the lowest temperature (ns ) 1.09 ( 0.05 at 293.0 K) and decreases monotonically by heating (ns ) 0.80 ( 0.04 at 308.4 K). 3.2.2. Effect of FL on Trp Fluorescence. To shed light on the peculiar properties of the interaction between FL and BSA, it is useful to also investigate the interplay between FL and the free Trp amino acid. FL is able to effectively quench not only the emission of BSA, but also that of Trp, as it can be inferred looking at the

Gentili et al.

Figure 11. Effect of BSA additions on the fluorescence spectrum of a 7.5 × 10-6 mol dm-3 FL solution, excited at 318 nm in buffered water at pH ) 7.2, at room temperature.

Figure 12. Spectral evolution upon irradiation at 342 nm of a PBS solution with [FL] ) 6.7 × 10-5 mol dm-3 and [BSA] ) 1.1 × 10-4 mol dm-3 at 283 K. Inset: kinetic evolution at 365 and 310 nm.

SCHEME 2: Electrocyclization reaction of FL upon UV irradiation and thermal back-reaction

spectra of Figure 10, recorded at increasing amounts of FL by exciting Trp at 275 nm. By applying eq 10 to the corrected fluorescence data, the constant and stoichiometry of the quenching process can be determined. The stoichiometry results are 1:1, since n ) (1.05 ( 0.03), and the constant is K ) (3.8 ( 1.0) × 104 dm3 mol-1 at room temperature. This value is about 1 order of magnitude lower than that determined for the BSA-FL system, indicating that the environment provided by BSA favors the interaction of the Trp residues in the protein compared with the free amino acid. Because the fluorescence lifetime of Trp is unaffected by FL, at least up to a molar ratio nFL/nTpr ) 2, only static quenching is effective in the TrpsFL system. However, K is insensitive to temperature changes in the range 296-313 K, which is probably related to a very low reaction enthalpy. Excited-state electron transfer from the indole ring to an electrophilic acceptor such as FL could be the mechanism of quenching of Trp by FL, as it has been proposed in ref 37. The electron transfer can occur from a prefluorescent state of Trp, whereby the quenching process does not affect the emission lifetime and results are independent of the temperature.



TABLE 4: Wavelengths of the Optically Active Electronic Transitions at the Lowest Energies for FL, its Tautomer, and the Most Likely Photoproducts

3.2.3. Effect of BSA on the Photoresponse of FL. The reciprocal influence between FL and BSA affects their photobehaviors. In fact, we have ascertained above that FL quenches the emission of BSA. On the other hand, we will see now how BSA influences the photoresponse of FL when FL and BSA are tightly bound. When excitation is selectively performed on FL, the linkage with BSA affects the rate of the processes involved in the relaxation dynamics of FL. Both the intensity and lifetime of FL emission increase, and the emission maximum progressively shifts to the red by adding BSA. With the maximum BSA addition to a 7.5 × 10-6 mol dm-3 FL solution (corresponding to a molar ratio nBSA/nFL ) 4.3), the fluorescence quantum yield of FL is approximately doubled and the lifetime increases from (1.0 ( 0.2) ns to (3.6 ( 0.2) ns. The emission maximum shifts

from 400 to 430 nm (see Figure 11). This behavior, along with the absorption change observed (Figure 3), indicates that the microenvironment created by the macromolecule induces some changes in the ground as well in the excited-state of FL. No similar effect on FL fluorescence was observed in the presence of Trp. FL is known to be a photochromic compound by virtue of an electrocyclization reaction involving the single C(2)-O bond (see Scheme 2) of the pyran moiety.19 In this work, the photochromism of FL is investigated in PBS at pH ) 7.2, where the molecule is surrounded just by the solvent, and in a 1.1 × 10-4 mol dm-3 BSA buffered solution, where it is embedded in the macromolecule. Upon prolonged irradiation (λexc ) 342 nm), the absorption spectrum of FL in PBS, in both the absence and presence of BSA, evolves to the

16800 J. Phys. Chem. B, Vol. 112, No. 51, 2008 same photoproduct with the absorption maximum shifted to shorter wavelength (λmax ) 324 nm) with respect to that of FL (see Figure 12). The photoprocess is faster with BSA. The photoreaction quantum yield (ΦPC) is found to be 0.01 for the free FL in PBS and is 0.04 when FL is bound to BSA. When the light stimulation is removed, the photoproduct, very slowly, spontaneously evolves to the starting molecule. The thermal back-reaction at 283 K is slow when FL is freely dissolved in PBS (k∆ ) 6.2 × 10-4 s-1), but it is even slower when the photoproduct of FL is hindered by the tight microenvironment generated by the protein (k∆ ) 2.2 × 10-4 s-1). The comparison with previous photochemical results, obtained in 3-methylpentane,19,20 indicates that the photoproduct here observed has a different absorption spectrum, hypsochromically shifted toward the UV region. This behavior can be understood by taking into account the differences in the molecular environment. In a hydrocarbon solvent, the FL molecules could be intramolecularly hydrogen-bonded, easily leading to intramolecular proton transfer with formation of the lactime tautomer (-CO-NH T -COHdN-). In a polar medium, H-bonding with the solvent impedes tautomerization. Therefore, it is likely that the photoproduct derives from the keto form in water and from the tautomer in a hydrocarbon solvent. To have an idea of what compound is accumulated upon irradiation, we simulated the main optically active electronic transitions for plausible photoproducts, taking into account the possibility of the tautomeric equilibrium lactame-lactime for the -CO-NH group (Table 4). The intense transitions located around 360 nm for both open forms derived from the lactime tautomers exclude them as plausible photoproducts. Moreover, since the compound produced by UV irradiation thermally reverts to FL in the dark, the photoproduct most likely to be formed could be the FL transoid-cis isomer, since the back-process requires rotation around a single bond, whereas back-conversion of the transoidtrans isomer would imply rotation around a double bond. This latter requires a larger energy and, therefore, it generally does not occur thermally but photochemically. This assignment is also in agreement with other literature studies on structurally related photochromic compounds.38-41 The role played by BSA in accelerating the photoreaction is that of creating a hydrogenbonding microenvironment, as also supported by the thermodynamics of the association process, which favors the light induced opening and stabilizes the FL molecule in its keto form. No photochemistry was observed for FL when irradiation (λexc ) 275 nm) was carried out on either the BSA or Trp absorption bands. 4. Summary and Conclusions In this paper, it is shown that FL has a high binding affinity to BSA; the interplay affects the behavior of both the protein and the photochrome. Fluorescence of BSA, which is due mainly to the Trp residues in the protein, is quenched by FL in both static and dynamic ways. The ground-state association process is characterized by a large equilibrium constant, which is very sensitive to the temperature due to the consistent negative ∆H° value. The association process is accompanied by a marked decrease in entropy, which indicates that hydrogen bonds and van der Waals interactions play the most important roles in stabilizing the complex. BSA excited-state relaxes through two different distributions of conformers, likely corresponding to different locations of Trp in the protein scaffolding. FL experiences a hydrogen-bonding microenvironment in the protein cavities, which is reflected in the physical and chemical

Gentili et al. relaxations of its excited state. Compared with the free FL molecule, absorption and especially fluorescence spectral features change; furthermore, fluorescence yield and lifetime are enhanced. Photochemistry of FL occurs faster, but the thermal back reaction is slowed down when FL is embedded in the macromolecule. A sort of “photocatalytic binding site” is created by the protein around the FL guest. The interplay between FL and the free Trp is of different phenomenology since FL quenches Trp fluorescence, but the process is insensitive to temperature and does not alter the luminescence lifetime of the amino acid. Finally, the FL photobehavior is not influenced by the presence of Trp. Acknowledgment. This research was funded by the Italian “Ministero per l’Universita` e la Ricerca Scientifica e Tecnologica” and the University of Perugia in the framework of a PRIN-2006 Project (“Photophysics and photochemistry of chromogenic compounds for technological applications”). The authors are grateful to Professor P. J. Steinbach for valuable suggestions in using MemExp. References and Notes (1) Wei, Y.-L.; Li, J.-Q.; Dong, C.; Shuang, S.-M.; Liu, D.-S.; Huie, C. W. Talanta 2006, 70, 377–382. (2) Chakrabarty, A.; Mallick, A.; Haldar, B.; Das, P.; Chattopadhyay, N. Biomacromolecules 2007, 8, 920–927. (3) Cui, F.-L.; Wang, J.-L.; Cui, Y.-R.; Li, J.-P. Anal. Chim. Acta 2006, 571, 175–183. (4) Kandagal, P. B.; Seetharamappa, J.; Ashoka, S.; Shaikh, S. M. T.; Manjunatha, D. H Int. J. Biol. Macromol 2006, 39, 234–239. (5) Hu, Y.-J.; Liu, Y.; Sun, T.-Q.; Bai, A.-M.; Lu¨, J.-Q.; Pi, Z.-B Int. J. Biol. Macromol. 2006, 39, 280–285. (6) Lu, J.-Q.; Jin, F.; Sun, T.-Q.; Zhou, X.-W. Int. J. Biol. Macromol. 2007, 40, 299–304. (7) Bian, H.; Li, M.; Yu, Q.; Chen, Z.; Tian, J.; Liang, H Int. J.Biol. Macromol. 2006, 39, 291–297. (8) Bose, B.; Dube, A. J. Photochem. Photobiol. B: Biol. 2006, 86, 49–55. (9) Sun, S.-F.; Zhou, B.; Hou, H.-N.; Liu, Y.; Xiang, G.-Y. Int. J. Biol. Macromol. 2006, 39, 197–200. (10) Kanti Maiti, T.; Ghosh, K. S.; Samanta, A.; Dasgupta, S. J. Photochem. Photobiol. A: Chem. 2008, 194, 297–307. (11) Banerjee, T.; Singh, S. K.; Kishore, N. J. Phys. Chem. B 2006, 110, 24147–24156. (12) Shaikh, S. M. T.; Seetharamappa, J.; Kandagal, P. B.; Manjunatha, D. H.; Ashoka, S. Dyes Pigments 2007, 74, 665–671. (13) Qu, L. B.; Wang, L.; Yang, R.; Chen, X. L.; Li, P. Yao Xue Xue Bao 2006, 41, 352–357. (14) Roliski, O. J.; Martin, A.; Birch, D. J. S. J. Biomed. Opt. 2007, 12, 34013. (15) Dangles, O.; Dufour, C.; Bret, S. J. Chem. Soc., Perkin Trans. 1999, 2, 737–744. (16) Bi, S.; Ding, L.; Tian, Y.; Song, D.; Zhou, X.; Liu, X.; Zhang, H. J. Mol. Struct. 2004, 703, 37–45. (17) Mishra, B.; Barik, A.; Priyadarsini, K. I.; Mohan, H. J. Chem. Sci. 2005, 117, 641–647. (18) Papadopoulou, A.; Green, R. J.; Frazier, R. A. J. Agric. Food Chem. 2005, 53, 158–163. (19) Becker, R. S.; Pelliccioli, A. P.; Romani, A.; Favaro, G. J. Am. Chem. Soc. 1999, 121, 2104–2109. (20) Gentili, P. L.; Ortica, F.; Romani, A.; Favaro, G. J. Phys. Chem. A 2007, 111, 193–200. (21) Hanawa, F.; Fokialakis, N.; Skaltsounis, A. L. Planta Med. 2004, 70, 531–535. (22) Melhuish, W. H. J. Phys. Chem. 1961, 65, 229–235. (23) Favaro, G.; Masetti, F.; Ramadori, R. J. Photochem. 1979, 10, 349– 357. (24) (a) Steinbach, P. J.; Ionescu, R.; Matthews, C. R Biophys. J. 2002, 82, 2244–2255. (b) Steinbach, P. J J. Chem. Inf. Comput. Sci. 2002, 42, 1476–1478. (25) Peter, T., Jr AdV. Protein Chem. 1985, 37, 161–245. (26) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; 1st Edition, Plenum Press: New York, 1983. (27) Szabo, A. G.; Rayner, D. M. J. Am. Chem. Soc. 1980, 102, 554– 563.

Interaction of a Photochrome with Bovine Serum Albumin (28) Gudgin, E.; Lopez-Delgado, R.; Ware, W. R. Can. J. Chem. 1981, 59, 1037–1044. (29) Foerster, T. Disc. Faraday Soc. 1959, 27, 7–17. (30) Yuan, J.-L.; lv, Z.; Liu, Z.-G.; Hu, Z.; Zou, G.-L. J. Photochem. Photobiol. A: Chem. 2007, 191, 104–113. (31) Wang, Y.-Q.; Zhang, H.-M.; Zhang, G.-C; Tao, W.-H.; Tang, S.H. J. Mol. Struct. 2007, 830, 40–45. (32) Lakowicz, J. R.; Weber, G. Biochemistry 1973, 12, 4161–4170. (33) Barik, A.; Mishra, B.; Kunwar, A.; Priyadarsini, K. I. Chem. Phys. Lett. 2007, 436, 239–243. (34) Feng, X.-Z; Lin, Z.; Yang, L.-J.; Wang, C.; Bai, C-l. Talanta 1998, 47, 1223–1229. (35) Zhang, Y.-Z; Zhou, B.; Liu, Y.-X.; Zhou, C.-X.; Ding, X.-L.; Liu, Y. J. Fluoresc. 2008, 18, 109–118.

J. Phys. Chem. B, Vol. 112, No. 51, 2008 16801 (36) Ross, P. D.; Subramanian, S. Biochemistry 1981, 20, 3096–3102. (37) Chen, Y.; Barkley, M. D. Biochemistry 1998, 37, 9976–9982. (38) Gartner, C. A.; Wen, B.; Wan, J.; Becker, R. S.; Jones, G., II; Gygi, S. P.; Nelson, S. D. Biochemistry 2005, 44, 1846–1855. (39) Favaro, G.; Mazzucato, U.; Ottavi, G.; Becker, R. S. Mol. Cryst. Liq. Cryst. 1997, 298, 137–144. (40) Favaro, G.; Romani, A.; Ortica, F. Photochem. Photobiol. Sci. 2003, 2, 1032–1037. (41) Ortica, F.; Smimmo, P.; Favaro, G.; Mazzucato, U.; Delbaere, S.; Venec, D.; Vermeersch, G.; Frigoli, M.; Moustrou, C.; Samat, A. Photochem. Photobiol. Sc. 2004, 3, 878–885.

JP805922G

Static and Dynamic Interaction of a Naturally Occurring Photochromic

Recommend Documents