14514
J. Phys. Chem. 1996, 100, 14514-14519
Excited State Proton Transfer Reaction as a Probe for the Microenvironment of a Binding Site of Bovine Serum Albumin: Effect of Urea Ranjan Das, Sivaprasad Mitra, Debnarayan Nath, and Samaresh Mukherjee* Department of Physical Chemistry, Indian Association for the CultiVation of Science, JadaVpur, Calcutta-7000 32, India ReceiVed: August 2, 1995; In Final Form: May 8, 1996X
The dynamics of proton dissociation from 2-naphthol 6,8-disulfonate (NSOH) in its first excited singlet state has been studied in the microenvironment of the binding sites of a protein, bovine serum albumin (BSA), using time-resolved fluorimetry. In concentrated salt solution the dissociation is slowed down as an exponential function of activity of water in the solution. This kinetic parameter has been used to probe the microenvironment of the binding sites of bovine serum albumin at which NSOH is bound. The dissociation of the proton in water is a very fast reaction, k′12 ) 7.2 × 109 s-1, but upon binding to BSA the rate of proton dissociation is slowed down significantly to 2.0 × 109 s-1. This slow dissociation rate constant suggests a strong interaction of the water molecules with the inner walls of the cavity. On addition of urea k12 increases to 2.5 × 109 s-1 because of increased availability of water molecules to hydrate the dissociated proton. The ability of the water molecules to hydrate the dissociated proton in the site is equivalent to a homogeneous salt solution with activity, a(H2O) = 0.67, but in the presence of urea, the activity of the water molecules in the binding site is 0.78.
1. Introduction Protein-bound fluorescent probes have been used extensively for elucidating information regarding environment of their binding sites.1-3 Dodiuk et al.2 inserted 8-anilinonaphthalene 1-sulfonate and 6-anilinonaphthalene 2-sulfonate derivatives in the heme binding site of apomyoglobin and obtained ET (30) values of 34 mol and 42 kcal/mol, respectively, for the same site’s polarity from these two fluorophores. Due to this discrepancy, they concluded that a fluorescent probe is an unreliable tool for measuring polarity of the protein-binding site. However, elucidation of more conclusive information about the properties of the microenvironment is obtainable from measurements of a well-defined probing reaction such as proton transfer of photoacids. Photoacids are those compounds that are stronger acids in their first excited singlet state than in the ground state (pK* < pK0).4 Both steady state and time-resolved measurements have been employed to determine the rate of proton dissociation from these compounds in homogeneous environments5,6 and protein.7 The currently accumulated theories on the rate of proton transfer and its diffusion are suitable for deriving specific information about the environment where the reaction takes place. Thus, parameters such as activity of water, density of immobile binding sites, viscosity of the solvent, or electrostatic interaction can be measured. Therefore, the advantage of a short observation period is self-evident with the presence of such opportunities. Nowadays the advent of ultrafast laser spectroscopic techniques enable us to choose a very short time frame of observation. If we limit our monitoring to a very short time scale, after a proton has been released in a defined site, the physical information obtained by the analysis will reflect only the space that the proton could probe during this observation period. Thus the temporal resolution is transformed into spatial resolution. Under proper conditions, the microspace of the specific site of a protein can be studied, totally insensitive to the huge bulk volume. X
Abstract published in AdVance ACS Abstracts, July 15, 1996.
S0022-3654(95)02204-0 CCC: $12.00
In the present study we therefore employ time-resolved nanoand picosecond fluorimetric techniques to investigate the properties of the binding sites of bovine serum albumin as well as the change in the microenvironmental property of the same, in the presence of a well-known protein denaturant, urea, using proton dissociation reaction of 2-naphthol 6,8-disulfonate (NSOH) in the excited state as a probe. Two processes take place subsequent to excitation of the photoacid (NSOH): (i) dissociation of a proton from the bound fluorophore followed by its diffusion in the cavity or microspace of the binding site; (ii) the escape of the proton from the binding site to the bulk. These two steps are common for all enzymes participating in energy-coupled proton translocation. 2. Experimental Section 2-Naphthol 6,8-disulfonate (potasium salt) (NSOH) was obtained from (BDH, U.K). It was vacuum sublimed and recrystallized twice from ethanol. It showed a single TLC spot. Triply distilled, deionized water was used throughout this study. Bovine serum albumin (BSA) (99%) was obtained from Sigma Chemical Co. and used as received. Ultrapure recrystallized urea was obtained from Spectrochem Pvt. Ltd., Bombay, and used as received. All the solutions were prepared in a phosphate buffer (pH 6). Sample concentrations were maintained at 5 × 10-5 mol dm-3. Absorption and emission spectra were recorded in JASCO 7850 and Perkin Elmer MPF 44B spectrophotometers, respectively. In fluorescence studies the samples were excited at 330 nm to avoid emission due to tryptophan. The time-resolved fluorescence decays were recorded in nanoand picosecond setups. The nanosecond setup is either a Model 199 spectrofluorimeter from Edinburgh Instruments, U.K., or a nanosecond spectrometer from Applied Photophysics. Ltd., U.K., in which the sample is excited by a pulsed nitrogen lamp (fwhm ≈ 1.2-1.6 ns). In the picosecond setup the sample is excited by the second harmonic of a Coherent CW mode-locked Antares Nd:YAG laser (76S). The emission is collected at magic angle (54.7°) polarization by a Hamamatsu MCP PM tube (2809U). The full width at half-maximum (fwhm) of the © 1996 American Chemical Society
Microenvironment of a Binding Site of BSA
J. Phys. Chem., Vol. 100, No. 34, 1996 14515
Figure 1. Emission spectra of NSOH (in quartz cell of 1 cm optical path length): (A) 50 µM NSOH in water at pH 6; (B) 50 µM NSOH in 10 µM BSA; (C) 50 µM NSOH in 60 µM BSA + 2.9 M urea; and (D) 50 µM NSOH in 60 µM BSA.
SCHEME 1 k ′12
NSOH* + H2O
k′21
kr hν1
NSOH + H2O
(NSO–)* + H+(H2O) k ′r hν2
k1
NSO– + H3O+
k –1
instrument response function is about 55 ps. Deconvolutions of the fluorescence decays were made using global lifetime analysis software (Photon Technology International Inc., Version VI.1). The goodness of the exponential fit to the decays was judged by several parameters, e.g. reduced chi-square (χ2), Durbin-Watson parameter (DWP), and residual and auto correlation functions. In all the cases reported here, χ2 ) 1.01.2 and DWP ) 1.8-2.1 3. Results and Discussion 3.1. Steady State Emission Spectra. Figure 1 shows the emission spectrum of 2-naphthol 6,8-disulfonate (NSOH) in a buffer of pH 6. At this pH, the ground state is fully protonated (pK° ) 8.33) but not so the first excited singlet state (pK*< 0.33).4 The excited molecule dissociates and a very strong fluorescence band at 460 nm due to NSO-* ion is observed with very little fluorescence at 385 nm. This 385 nm band is attributed to the neutral form (NSOH*) fluorescence, as shown in Scheme 1. Upon binding to bovine serum albumin (BSA), significant enhancement in neutral form fluorescence at 385 nm with a concomitant decrease in intensity of the 460 nm band is observed. However, in the presence of 3 M urea, the intensity of the 460 nm band is found to increase (Figure 1), with a consequent decrease of the neutral form fluorescence. These results are explained on the basis of availability or unavailability of water molecules8-10 needed to accept the proton. On ligation to the binding sites of BSA, water molecules are removed from the immediate neighborhood of NSOH. As a result the concentration of water molecules (proton acceptors) required to hydrate the dissociated proton from the bound fluorophore decreases, diminishing the probability of successful proton transfer from the bound ligand. This fact accounts for the enhanced fluorescence of the 385 nm band. In the presence of urea, the number of bound fluorophores decreases, as is
Figure 2. Scatchard Plot: (A, top) Binding of NSOH to BSA; (B, bottom) Binding of NSOH to BSA in the presence of 3 M urea; [NSOH]f ) concentration of free NSOH molecules, [NSOH]b ) concentration of bound NSOH molecules, and [BSA] ) concentration of bovine serum albumin.
evident from Scatchard plots in Figure 2. The analysis of the results of fluorimetric titration of BSA by NSOH by a Scatchard plot11 corresponds to a binding of NSOH at three independent and identical sites of BSA; that is, NSOH is bound to BSA with a stoichiometry of 3:1 (Figure 2a). However, in the presence of urea it decreases to 1 (Figure 2b); that is, NSOH is bound to BSA with a stoichiometry of 1:1. Recent experimental12 and computer simulation studies13,14 reveal that urea displaces water molecules around the hydrophobic group and thus changes the solvation of protein. This leads to removal of some of the bound NSOH molecules from the binding sites of protein to the bulk. In consequence the number of free NSOH molecules increases and a greater number of free NSOH molecules undergo successful proton transfer, resulting in an enhanced anionic emission. Another plausible explanation is that urea induces some structural changes in the binding site of BSA, leading to availability of an increased number of water molecules in the cavity of the binding site, which in turn enhances the probability of successful proton transfer. 3.2. Time-Resolved Emission Spectra. Figure 3 displays the fluorescence time profile of the neutral form of NSOH molecules in water. The emission decays in a single exponent with a time constant of 137 ps (Table 1). This observation is consistent with the analytical expression for the decay of neutral form fluorescence in bulk media given in eq 1 derived through proposed Scheme 1. The time constant for the decay of the neutral form (τ1 ) 137 ps, Table 1) is similar to the time constant for the appearance of the anionic form (τ1 ) 140 ps, rise time for the anion). This time constant of 137 ps in water
14516 J. Phys. Chem., Vol. 100, No. 34, 1996
Das et al.
TABLE 1: Lifetime Values (τ1, τ2) for Various Systemsa system
emission wavelength, nm
time resolution, ps/ch
τ1, ns
385 385 460 385 460 385 460 460 385 385 460 460 385
168 14 14 168 168 28 28 168 168 28 28 168 168
8.7 0.13 0.14 8.6 12.8 0.4 0.41 12.7 5.3 0.35 0.34 12.7 4.7
50 µM NSOH in 2 M HCl 50 µM NSOH in water
50 µM NSOH in 6 µM BSA
5 µM NSOH in 60 µM BSA + 2.9 M urea a
τ2, ns
A1
A2
4.0
-1.86
1.86
5.28 3.93
0.87 -1.3
0.13 1.3
4.8 4.4
0.92 -1.27
0.08 1.27
τi ) lifetime; Ai ) pre-exponential factor. Lifetimes with a negative pre-exponential factor correspond to rise times.
Figure 3. Fluorescence time profile of 50 µM NSOH in water (L ) apparatus response function): (A) emission wavelength ) 460 nm; (B) emission wavelength ) 385 nm. Time resolution per channel ) 14 ps, Dotted points correspond to raw data; solid line gives the best fit.
Upon binding to BSA, the time constants become significantly longer (Table 1 and Figure 4). The same time constants of 410 ( 10 ps are measured for both the appearance of the anionic form and decay of the neutral one (Table 1). The fluorescence time profile in Figure 4 also reveals that after the fast decay, lasting about 0.6 ns, a slower decay process takes place; that is, the decay of NSOH* emission is a biexponential function. The rapid phase of the NSOH* decay represents a proton dissociation in the binding site accompanied by a rapid recombination reaction. The end of the rapid phase corresponds with the time when the velocities of dissociation and recombination are equal. During the slow phase, the photoacid and its deprotonated form in the cavity are in equilibrium, but the escape of the proton from the cavity shifts the NSOH* population toward NSO-*, as shown in Scheme 2. In the presence of urea, faster time constants (τdecay ) 350 ps, τrise ) 345 ps) are observed (Table 1), and again the decay is a biexponential one. 3.3. Kinetics of Proton Dissociation in Pure Water and in the Binding Site of BSA. (a) Proton Dissociation in Pure Water. The dissociation of an excited NSOH molecule in pure water is given in Scheme 1, where kr, k′r are fluorescence decay rate constants for free NSOH* and NSO-*, respectively. k′12 and k′21[H]+ are dissociation and recombination rate constants for the forward and backward reactions, and k1, k-1 are the ground state rate constants. If we designate the population of NSOH in the excited state as N, then the decay dynamics of the population N at pH 6 is well described by the expression
N(t) ) Ni exp(-k′t)
(1)
k′ ) kr + k′12
(2)
where
Figure 4. Fluorescence time profile of 50 µM NSOH in 60 µM BSA (L ) apparatus response function): (A) emission wavelength ) 460 nm, (B) emission wavelength ) 385 nm. Time resolution per channel ) 28 ps. Dotted points correspond to raw data; solid line gives the best fit.
corresponds to rapid dissociation of the proton from the free fluorophore in water.
(b) Proton dissociation in the binding site of BSA. The kinetics of proton dissociation in the binding site of BSA is given in Scheme 2. Immediately after excitation, there is only one excited species bound to BSA. Let us designate the population number of this bound NSOH* as N1. The lifetime of this bound fluorophore is determined by three rate constants: the fluorescence decay
SCHEME 2 k12
NSOH* + (H2O)cav
kf
NSOH + hν1
k21
(NSO–)* + H+(H2O)cav k ′f
NSO– + hν2
kesc
(NSO–)* + H+(H2O)bulk k ′f
NSO– + hν2
Microenvironment of a Binding Site of BSA
J. Phys. Chem., Vol. 100, No. 34, 1996 14517
TABLE 2: Dissociation (k12) and Recombination (k21[H+]) Rate Constants system
k12, s-1
k21[H+], s-1
50 µM NSOH in water 50 µM NSOH in 80 µM BSA 50 µM NSOH in 80 µM BSA + 2.9 M urea
7.2 × 109 2.0 × 109 2.5 × 109
3.4 × 108 2.5 × 108
(kf), proton dissociation (k12), and recombination (k21[H+]). Our detailed analysis based on the dynamics of the population N1 is given by expression
N1(t) ) A1 exp(-γ1t) + A2 exp(-γ2t)
(3)
where
γ1,2 )
(kt + k2) - [(kt + k2)2 - 4(ktk2 - k12k21[H+])]1/2 2 kt ) kf + k12
(4)
k2 ) k21[H+] + kesc + k′f
(5)
where k′f and kesc are the fluorescence decay rate constant of the bound anion and the rate constant of proton escape from the cavity to the bulk, respectively. The values of dissociation and recombination rate constants for various compositions are listed in Table 2 and are explained on the basis of properties of water molecules in sections 3.5 and 3.6. 3.4. Escape of the Proton from the Binding Site: Factors Affecting Rate Constant of Proton Escape (kesc). On excitation of a bound NSOH molecule, similar time constants of 410 ( 10 ps were measured for both the appearance of the NSO-* form and decay of the NSOH* form (Table 1). This portion of the decay and rise in the fluorescence time profile (Figure 4) represents occurrence of proton dissociation in the cavity of the binding site. About 0.6 ns after the pulse, the coevolution of the two forms is lost and the decay of bound NSOH* assumes a slower phase with τ ) 5.28 ns. This time constant is shorter than the radiative plus nonradiative decay time (τf ) 8.6 ns, Table 1) of the neutral form of free NSOH*. This observation indicates that a parallel mechanism is also operative that consumes the NSOH* form. The dissociated proton is ejected into a small cavity in the binding site of BSA of which a substantial space is occupied by the deprotonated photoacid. In consequence, the probabilities of reprotonation are very high and within a short time frame the velocity of recombination equals that of dissociation, leading to an apparent equilibrium. Rapid transition between NSO-* and NSOH*, in the site, couples the lifetimes of the two forms, and both should decay with the same time constant. The experimental findings do not agree with this expectation (Table 1), and about 0.6 ns after the pulse the NSOH* population decays with a longer lifetime of 5.28 ns (Table 1). The corresponding evolution of NSO-* assumes a steady state and much later an exponential decay with τNSO-*(BSA) ) 12.7 ns. This independent decay implies that the proton that converts the deprotonated form to neutral form is no longer present in the site. As long as the proton is inside the cavity of the binding site, it effectively recombines with NSO-*, but once it escapes out of the site to the bulk (pH 6), the probability of reprotonation of NSO-* before it decays to the ground state is nil. Thus NSOH*, in a site that has lost its proton, will decay only in the anionic form. The parallel reaction, which consumes NSOH* and builds up NSO-* population, is the escape of the proton from the
binding site and can be expressed by the following relation:
kapp ) kf + kesc
(6)
where kapp ) (τapp)-1, τapp ) fluorescence decay time of bound NSOH*, kf ) (τf)-1, where τf ) fluorescence decay time of free NSOH*, and kesc) rate constant of proton escape. Thus knowing the values of τapp and τf from Table 1, kesc can be calculated. In BSA, kesc ) (5.28)-1 - (8.6)-1 ) 0.7 × 108 s-1, but in the presence of 3 M urea, kesc ) (4.8)-1 - (8.6)-1 ) 0.9 × 108 s-1. Recently, the escape of a proton out of a proteinous cavity has been treated in a precise manner by Shimoni and coworkers.15 According to them, the escape of a proton out of a proteinous cavity to the bulk is dependent on two parameters: (i) the nature of opening of the microscopic cavity and (ii) the electrostatic potential in the Coulomb cage of the bound anion which attracts the proton to the interior of the cavity. The Coulomb cage is defined as the space around an ion in which the electrostatic potential is larger than the thermal energy, where the Coulomb cage radius is given by the expression
|Z1Z2|e2 Re ) kBT
(7)
where Z1, Z2 are tha valences of the interacting ions, e is the electronic charge, is the dielectric constant of the medium, and kB is the Boltzmann constant. A narrow opening slows the emergence of the dissociated proton to the bulk, resulting in a long escaping time (τesc) for the proton. The electrostatic potential at a water/protein interface is affected by the dielectric discontinuity at the boundary.16 The effective dielectric constant of a small cavity varies with its shape and the nature of the low-dielectric boundary. In consequence, the electrostatic interaction on the surface of the protein is intensified and can be expressed by a local, position dependent, effective dielectric constant.17 At the water/protein interface, the effective dielectric constant is smaller than that of bulk water;15,18 for example, the effective dielectric constant (eff) of bulk water is 78, whereas in a small cavity like the heme-binding site of apomyoglobin it is 8. So, the electric potential in the heme-binding site can be 10 times as large as that in bulk water. This increase in electric potential expands the Coulomb cage of the bound anion to few tens of angstrom units. Thus, the emergence of the proton from the electrostatic grip of a protein-bound charge will be slower, delaying the escape of the proton from the cavity to the bulk. So the long escaping time (τesc) of 13.6 ns of the dissociated proton of NSOH from the microscopic cavity of BSA to the bulk water is consistent with a narrow opening of the cavity and strong electrostatic potential in the Coulomb cage of the BSA-bound NSO-*. This strong electric potential originates due to a less polar (lower eff) interior of the cavity than the bulk water. 3.5. Properties of the Water Molecules in the Binding Site of Bovine Serum Albumin and Estimation of IntraCavity Activity. NSOH in its excited state does not transfer a proton to an organic solvent. It exhibits a single emission at 380-385 nm in ethanol, methanol, propanol, DMSO etc., which is attributed to neutral form fluorescence. Thus, proton dissociation from bound NSOH reveals the presence of water molecules in the cavity of the binding site. The rate constant of proton dissociation is a function of the proton hydrating properties of the solvent.8-10 According to Robinson9,10 and others,19,20 the stabilization of the proton
14518 J. Phys. Chem., Vol. 100, No. 34, 1996 increases with the number of water molecules forming the H+(H2O)n complex. The number of hydration molecules in pure water does not exceed 10, as at n g 10 the gain in hydration energy is identical with the energy required to remove a water molecule from the bulk. When the activity of water in a homogeneous solution is lowered by addition of strong electrolytes, the rate of dissociation becomes much lower. There is an exponential relationship between the rate of dissociation and activity of water in the solution;21 kdis ) k0dis (aH2O)n, where kdis) measured rate, k0dis ) rate in pure water. The slope of the exponential function for 8-hydroxypyrene 1,3,6-trisulfonate (n ) 6.9) was found to be independent of the nature of the electrolyte (LiBr, LiCl, KCl, MgCl2, etc.).22 This exponential relationship correlates well with the idea that a proton transfer takes place between a proton donor and a proton accepting cluster. This proton-accepting cluster is constituted of water molecules interconnected by proper hydrogen bonds to stabilize a dissociated proton by hydration. In concentrated salt solution, the water molecules are preferably oriented around the hydrated ions in their primary and secondary solvation shells. Due to this high prevalence of oriented water molecules, availability of the number of water molecules in the proton-accepting cluster decreases and the size of the accepting cluster in concentrated salt solution with reduced water activity is smaller (n < 10). This incomplete hydration will lower the stabilization energy. This thermodynamic consideration is reflected in the kinetic results. Under such limiting conditions, the encounter between the photoacid and proton-accepting cluster determines the rate of proton dissociation. The same phenomenon is observed in the microspace of a binding site of BSA. Due to the high surface/ volume ratio, the orientation of water molecules is influenced by interaction with the inner wall of the cavity. Thus, in homogeneous salt solution and in the cavity of a binding site of BSA, the proton transfer process is inhibited by the inadequacy of the water molecules of the environment to hydrate the proton. Therefore, the rate of proton dissociation for NSOH decreases from 7.2 × 109 s-1 in pure water to 2.0 × 109 s-1 in the binding site of BSA. Thus, the rate of proton dissociation in the microspace of the binding site of BSA, k12 ) 2.0 × 109 s-1, is indicative of a dissociation taking place in an environment with proton-hydrating capacity equivalent to that of concentrated salt solution with water activity of aH2O ) 0.67. This value of equivalent water activity is estimated from the plot of dissociation rate constant vs a(H2O) for 2-naphthol 6,8 disulfonate (NSOH) from ref 8. In the presence of urea, the dissociation rate constant in the binding site of BSA increases from 2.0 × 109 to 2.5 × 109 s-1 (Table 2). This increased value of the dissociation rate constant is found to be equivalent to a homogeneous salt solution with a(H O) = 0.78. This result indicates that there is an alteration 2 in the property of water molecules in the cavity of a binding site in the presence of urea. The increased value of a(H2O) in the presence of urea suggests an increase in the number of properly oriented water molecules in the binding site to stabilize the dissociated proton by hydration, hence decreasing interaction of the water molecules with the inner surface of the cavity. This reasoning accounts for the enhancement in k12 in the cavity of a binding site of BSA in the presence of urea. 3.6. Kinetics of Proton Recombination in the Cavity of a Binding Site. The volume of the cavity of the binding site of BSA is large enough to incorporate 50-60 water molecules inside it. Now, a single proton/60 water molecules is equivalent to [H+] = 1 M. As long as the proton is inside the cavity, its local concentration is very high. This high local concentration
Das et al. and slow escape of the proton (kesc ) 0.73 × 108 s-1, τesc ) 13.6 ns) accounts for the enhanced fluorescence of the neutral form relative to that in pure water. The high local concentration and long escaping time of the proton from the cavity to the bulk increase the number of recombinations of the NSO-*/H+ ion pair inside the cavity, leading to an increased proton recombination rate, k21[H+], relative to that in pure water (Table 2). Similar results were also observed for pyranine in lysozyme by Yam and co-workers.23 In the presence of urea k21[H+] decreases relative to that in the binding site of BSA because of a decrease in the escape time (τesc) 10.8 ns), which in turn decreases the number of recombinations. This is also evident from the ratio of intracavity reactions (krec/kesc), i.e the ratio of escaping time (τesc) to recombination time (τrec). The number of recombinations should increase with increase in the τesc/τrec ratio, where τesc is the time constant for proton escape from the cavity to the bulk and τrec is the time constant for recombination of the NSO-*/H+ ion pair ()(krec)-1. Thus, the τesc/τrec ratio is 4.62 and 2.7 for NSOH in the binding site of BSA in the absence and presence of urea, respectively. This ratio (τesc/τrec) modifies an important time parameter, dwell time, τdwell.24 τdwell is defined as the time frame where the proton is held within the cavity: when τesc > τrec, the dwell time of the proton inside the cavity is prolonged,24 and this longer τdwell ensures a greater number of recombinations. τdwell increases with an increase in the τesc/ τrec ratio. 4. Conclusions The steady state and time-resolved fluorescence properties of the bound fluorophore (NSOH) are quite different from those of the free fluorophore. NSOH binds to BSA with a stoichiometry of 3:1, but in the presence of urea the stoichiometry of binding changes to 1:1. Time-resolved fluorimetric analysis shows a decrease in the proton dissociation rate of NSOH on going from pure water to the binding site of BSA. This observation reveals that the water molecules form a matrix in the microspace of the cavity differing in properties from bulk water; it exhibits reduced activity equivalent to a homogeneous salt solution with a(H2O) ) 0.67. This reduced activity in the binding site of BSA is compatible with enhanced ordering of the water by the protein resulting from strong interaction of the water molecules with the inner walls of the cavity. In the presence of urea, the activity of water molecules in the binding site is 0.78. The enhancement of neutral form fluorescence of bound NSOH is assigned as due to several reasons: (i) unavailability of properly oriented water molecules to accept the dissociated proton in the binding site; (ii) high local concentration of proton inside the cavity; (iii) slow rate of proton escape (kesc) from the cavity to the bulk; and (iv) high τesc/τrec ratio, which leads to an increased number of NSO-*/H+ recombinations and, hence, also to an increased rate of recombination, k21[H+]. In the presence of urea, the τesc/τrec ratio decreases, and as a result, k21[H+] decreases. The slow rate of proton escape (kesc) from the microspace of the cavity to the bulk is explained as due to a narrow “cavity” opening and strong electrostatic potential in the Coulomb cage of the bound NSO-*. Acknowledgment. The authors R.D. and S.M. are grateful to CSIR and UGC, respectively, for providing them with the research fellowships. We express our gratitude to Dr. Kankan Bhattacharyya of our department for providing us with his picosecond TCSPC setup procured through a grant from DST, Govt. of India. We are also grateful to Dr. Samita Basu of SINP, Calcutta, for her generous help in measurement of
Microenvironment of a Binding Site of BSA nanosecond lifetimes. One of the authors, R.D., is grateful to Mr. Swarup Chakrabarty of IICB, Calcutta, for helpful discussions. Thanks are due to Ms. Maitreyi Nandi of SINP for preparation of the manuscript in Microsoft Word version. References and Notes (1) Azzi, A. Q. ReV. Biophys. 1975, 8, 237. (2) Dodiuk, H.; Kanety, H.; Kosower, E. M. J. Phys. Chem. 1979, 83, 515. (3) Ireland, J. F.; Wyatt, P. A. H. AdV. Phys. Org. Chem. 1976, 12, 131. (4) Arnaut, L. J.; Formosinho, A. J. J. Photochem. Photobiol, A 1993, 75, 48. (5) Huppert, D.; Gutman, M.; Kaufmann, K. J. AdV. Chem. Phys. 1981, 47, 643. (6) Huppert, D.; Kolodney, E. Chem. Phys. 1982, 63, 401. (7) Loken, M. R.; Mayer, J. W.; Gohlke, J.; Brand, C. Biochemistry 1972, 11, 4779. (8) Huppert, D.; Kolodney, E.; Gutman, M.; Nachliel, E. J. Am. Chem. Soc. 1982, 104, 6949. (9) Robinson, G. W.; Thistlethwaite, P.; Lee, J. J. Phys. Chem. 1986, 90, 4224. (10) Krishnan, R.; Fillingim, T. J.; Lee, J.; Robinson, G. W. J. Am. Chem. Soc. 1990, 112, 1353.
J. Phys. Chem., Vol. 100, No. 34, 1996 14519 (11) Scatchard, E. Ann. N.Y. Acad. Sci. 1949, 51, 660. (12) Breslow, R.; Guo, T. Proc. Natl. Acad. Sci. U.S.A., 1990, 87, 167. (13) Kuharsky, R. A.; Rossky, P. J. J. Am. Chem. Soc. 1984, 106, 5786. (14) Kuharsky, R. A.; Rossky, P. J. J. Am. Chem. Soc. 1984, 106, 5794. (15) Shimoni, E.; Tsfadia, Y.; Nachliel, E.; Gutman, M. Biophys. J. 1993, 64, 472. (16) Mathias, R. T.; Baldo, G. J.; Manivannan, K.; Mclaughlin, S. In Electrified Interfaces in Physics, Chemistry and Biology; Guidelli, R., Ed.; Kluwer: London, 1992; p 473. (17) Gilson, M. K.; Rashin, A.; Fine, R.; Honig, B. J. Mol. Biol. 1985, 183, 503. (18) Gutman, M.; Tsfadia, Y.; Masad, A.; Nachliel, E. Biochim. Biophys. Acta 1992, 141, 1109. (19) Searny, J. O.; Fern, J. B. J. Chem. Phys. 1974, 61, 5282. (20) Cunningham, J. A.; Payzant, D.; Kebarle, P. J. Am. Chem. Soc. 1972, 94, 7627. (21) Lee, J. J. Am. Chem. Soc. 1989, 111, 427. (22) Gutman, M.; Huppert, D.; Nachliel, E. Eur. J. Biochem. 1982, 121, 637. (23) Yam, R.; Nachliel, E.; Kiryati, S.; Gutman, M.; Huppert, D. Biophys. J. 1991, 59, 4. (24) Gutman, M.; Nachliel, E. Biophys. Acta 1990, 1015, 391.
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