ࡱ> {}z5@ Fbjbj224XXV333804D5tU25:5" 6 66K8K8K8RRRRRRR$6VRXS&;G8K8&;&;S 66TCCC&; 66RC&;RCPCDVK@L 6h5 @l%x3@5L QT0UALxYAYLLYKMK88hCE9T9K8K8K8SSDBRESOLVED FLUORESCENCE EMISSION SPECTRA OF PRODAN IN WATER / ETHANOL SOLUTIONS Marija Ragu~1,2 and Jasminka Brnjas-Kraljevi1 1University of Zagreb, School of Medicine, Zagreb, Croatia, 2University of Mostar, School of Medicine, Mostar, Bosnia and Herzegovina KEY WORDS: PRODAN, ethanol, steady-state emission spectra Contact address: Jasminka Brnjas-Kraljevi1 University of Zagreb School of Medicine Department of Physics and Biophysics Salata 3 b 10 000 Zagreb / Croatia tel: * 385 1 4566 924 e-mail: kraljevi@mef.hr Abstract The fluorescence steady-state emission spectra of lipophylic fluorescence probe PRODAN in ethanol/water solvents of different concentrations (0.3, 0.9, 3 M ethanol) have been extensively studied and analytically described. After correction for background effects the complex experimental spectra were resolved into two Gaussian curves simulating two fluorescence transitions. The energy separation of (0.158 0.001) eV was independent on ethanol concentration and temperature. For the decrease in solution polarity linear increase in fluorescence intensity accompanied with blue shift for both transitions were determined. The inclination of linear function for 25C is steeper than the inclination of linear function for 37C, and it is slightly more pronounced for l1 = 519 nm (37.5% increase) than for l2 = 555 nm (32.2% increase). The linear dependences of fluorescence's intensities on PRODAN concentration for all ethanol concentrations indicate that no PRODAN self quenching takes place even at higher PRODAN concentrations. INTRODUCTION The great interest in introducing molecular probe 6-propionyl-2-dimethylaminonaphthalene, PRODAN, into investigations of membrane and biomolecular structures was supported by the strong dependence of its fluorescence behavior on the environmental polarity. This well recognized lipophylic fluorescence probe mainly was used for investigations of physical and chemical properties of membranes and biological macromolecules [1-5]. Because of 2-dimetilamino group PRODAN locates itself on the phospholipids-water interface [6]. That qualify PRODAN as a suitable reporter for the structural changes of the surface. However, for the unequivocal interpretation of the results gained by such experiments the behavior of the probe molecule in the environment with different characteristic must be known. Several detailed studies upon PRODAN fluorescence behavior [7-9] has been preformed and theory argued [10,11] since it has been designed but the unanimous interpretation is not accepted. It is agreed that the well defined dipole moment change by excitation is 8 D [12] and not 20 D as detected by first measurements [13]. The big dipole moment change introduces the pronounced solvent dipolar relaxation phenomena and consequently the existence of excited states of different energies depending on media polarity. In pure solvents it can be accurately measured by Stock shift solvent polarity dependence. The quantum yield of PRODAN fluorescence in polar environment is less than in apolar one [14,15] The complex steady state emission spectra of PRODAN in pure solvent some authors are explaining with two equivalently important excited states defined by electron distribution in the molecule: locally excited, CT, and twisted intermolecular charge transfer, TICT, state [16], with measurable differences in energy and occupancy dependent on the polarity of the solution [17]. The complex relaxation curve therefore would be expected, with more than one relaxation (life) time. But, the time dependent fluorescence measurements more than once reported one exponential curve [18]. Arguing is still going on about the mechanism of PRODAN fluorescence on the level of relaxation processes [19]. There is agreement about experimental expectation for theoretical predictions, but not in unequivocal interpretation of experimental results. Our goal was to follow the structural changes in lipoproteins induced by different environmental conditions particularly different ethanol concentrations. For that purpose we decided to use PRODAN as an extrinsic fluorescence probe dissolved in lipid moiety and amino acid tryptophane as a natural, intrinsic, fluorescence probe on the apoprotein. For our investigations PRODAN seemed as the good candidate because of its high dependence on the environmental polarity and confirmed difference in the Stock shift and quantum yield when in water and in 100% alcohol [20]. The behavior that was not in favor of the choice was that in complex lipoprotein solutions PRODAN would predominantly participate in lipid environment of the particle but would also dissolve in water solution. In both environments it is fluorescence active. The problem we had to solve, prior to the lipoprotein investigations, was to determine the reliable method for resolving the spectra in pure solvents of different ethanol concentrations and to establish the sensitivity of the method for the interpretation of detected changes in PRODAN spectra in complex biological structures. Therefore, the primary goal was not to discuss the processes of relaxation, but to find reliable procedure to interpret the spectra for environmental properties changes in structured lipoprotein solutions. MATERIALS AND METHODS The samples of fluorescent lipophylic membrane probe PRODAN were prepared in solutions of ethanol in 0.01 M phosphate buffer with 0.2 M NaCl. PRODAN dissolved in organic solvent N,N-dimethyl-formamid, DMF, was gradually added to the buffer to the final concentrations of 0.25; 0.5; 1.5; 2 and 3 mM. The ethanol concentrations in measured samples were 0.3; 0.9 and 3 M. The spectra in buffer solution without ethanol and in 100% ethanol solution were measured to support the discussion about environmental polarity importance. In the same reasoning the measurements of PRODAN dissolved in DMF, have been preformed. All the samples have been prepared immediately prior to the measurements, and thermostated for 10 minutes in the prethermostated cell compartment. PRODAN probe was used as was purchased from "Molecular Probe" (Eugen, OR). All other chemicals were purchased from "Kemika" (Zagreb, Croatia) and were p.a. grade. The steady-state fluorescence spectra were recorded on Carry Eclipse, Varian Spectrofluorimeter at the Laboratory for Microwave Spectroscopy, R. Boakovi Institute, Zagreb, Croatia. The wavelength of excitation was 360 nm for all measured samples. The emission spectra were recorded in the interval 370 nm  700 nm. Excitation and emission monochromator band passes were kept constant at 5 nm. The amplifier voltage in all measurements was 650 V except for absolute ethanol measurements where 600 V was applied. Absorption values for all samples at wavelength 360 nm, checked on Carry-Varian Spectrophotometer, were lower than 0.05 indicating that no correction for inner filter effect is necessary. The temperature of the sample compartment was kept constant within 0.1C by circulating water bath. Because of further investigations the measurements at two temperatures 25C and 37C have been preformed. The refractive indexes of ethanol solutions have been determined by standard refractometer, Zeis Opton equipped with Na-lamp l = 550 nm, at temperature of 25C. The dielectric responses of all investigated solutions were measured by usage of home-made capacitive chamber in conjunction with Agilent 4294A precision impedance analyzer from 40 to 100 MHz at temperature of 25C at the Institute of Physics, Zagreb, (by mr.sc. S. Dolanski Babi). SPECTRA ANALYSIS The PRODAN emission spectra were corrected for the background contribution, particularly the Raman band at 410 nm, Fig.1. Namely, by excitation width 360 nm in water solutions the Raman bend at 410 nm was measurable. Therefore the spectra of adequate solvents without PRODAN were subtracted from experimental PRODAN solution spectra. For the subtraction of the spectra a simple program, part of the Microcal Origin 6.1 professional, was used. The PRODAN steady-state emission spectra retained their complex structure after correction. Therefore the model of two fluorescence transitions was applied in further analysis of those spectra. The experimental data have been fitted with the sum of two Gaussian functions:  EMBED Equation.3  (1) where A1, A2 and lc1, lc2 are maximal fluorescence intensities and corresponding wavelengths of the two transitions respectively. w1 and w2 are related to curves half-width w(1) and w(2) by  EMBED Equation.3  (2) The fitting procedure was performed by adjusted computer program Microcal Origin 6.1 Professional. The polarity of alcohol solutions were calculated for all used solvents according to the Eq. (3) [14] after the refractive indexes and dielectric constants have been determined  EMBED Equation.3  (3) The results of the calculations are presented in Table I. Table I. Polarity of ethanol/water solvents cethanol / MneD f01.3323 77.85 0.32000.31.3331 76.03 0.31950.91.3346 74.68 0.318831.3418 69.87 0.315317.71.3604 24.33 0.2887 RESULTS AND DISSCUSSION In this work we investigate the sensitivity of PRODAN fluorescence emission spectra parameters to the micro environmental polarity in solutions of different ethanol concentrations. Steady-state emission spectra have been recorded as a function of ethanol and PRODAN concentrations at 25C and 37C. Recorded spectra corrected for the background contribution (Fig.2A) were theoretically simulated applying the Eq.(1). In buffer solutions there are two well resolved florescence transitions at l1 = 519 nm and at l2 = 555 nm. Both show the intensity dependence on ethanol concentration (Fig.2B). Two arguments for two distinct fluorescence transitions can be offered. The energy difference between them being (0.158 0.001) eV would allow either explanation. So small differences in fluorescence emission energies could be defined either by distinctive excitation states of PRODAN molecule or by two vibrational levels of the ground state mostly probable for PRODAN to relaxate into. The energy of excited states will depend on the polarity of the environmental solution, what has already been argued by several authors [9] while proposing the solvent mediated relaxation for PRODAN molecules [4, 18]. That consequently means that the abundance of excited states would express sensitivity on the polarity of the solution, while such dependence would not be expected for ground state structure. The differences in the behavior would be detected as changes in intensities and in wavelengths of the two transitions dependent on the change in the polarity of the solution. This expected difference in the behavior could therefore be the probe for the environmental polarity in the structured biological solutions. At first, the influence of ethanol concentration on PRODAN fluorescence intensity has been determined. The consequence of increased ethanol concentration is decrease in the solution polarity, as presented in Table I. Therefore, the intensities of two transitions are presented as the function of solvent polarity, Df (Fig.3). For both maxima the intensities are linearly decreasing with increasing polarity. In the concentration range up to 3 M ethanol, which is concentration of interest in biological investigations, the decrease in solution polarity is only 2 %. However, this small change induced the increase in intensity of about 50 %. At the same time the increase rate is temperature dependent. The inclination of linear function for 25C is steeper than the inclination of linear function for 37C, and it is slightly more pronounced for l1 = 519 nm (37.5% increase) than for l2 = 555 nm (32.2% increase). The maxima wavelengths (wave numbers) of both transitions are alcohol concentration dependent. Fig. 4 represents the Stock shift for both maxima as the function of polarity at two measured temperatures. The dependence on temperature is negligible as the two linear functions representing the dependence of Dn on polarity are nearly identical. Both transitions express a blue shift of (2270 20) cm-1. That means, that energy difference between two transitions stays unchanged by polarity changes indicating that ethanol is not introducing a structural changes in PRODAN molecule. PRODAN structure is not affected by change in solution polarity, what is very important when this dye is introduced into biological molecules with high nonpolar regions. Further, it goes in favor of the prediction that in this case PRODAN fluorescence emission is mainly governed by relaxation over solution molecules and not over intramolecular charge transfer [9]. That indicates that Stock shifts in this case could be a reliable parameter for determination of ethanol induced changes in lipoproteins. To define the sensitivity of the method we have measured the steady-state spectra for solutions of variable PRODAN concentrations: 0.25, 0.5, 1.5, 2 and 3 mM in 0.3, 0.9 and 3 M ethanol solutions (Fig.5). The maxima wavelengths for both transitions are PRODAN concentration independent as it would be expected. The slope of linear functions, describing the dependence of fluorescence emission intensity upon the PRODAN concentration, increased with increasing ethanol concentration (decreasing polarity). Linearity of the dependence could be rationalized that even in the solution of highest PRODAN concentration there is no PRODAN / PRODAN dipolar relaxation process (self quenching does not occur). This conclusion is very important when using the dye in biological molecules investigation. Because of lipophylicity of the dye it will participate more in the hydrophobic environment of the biological molecules increasing the concentration in the membrane in comparison to the one in water environment [19]. For conclusions driven from the changes in spectra intensities to be relevant it is absolutely necessary to avoid self quenching of PRODAN molecules. There is measurable increase in the slope of linear functions with increase in ethanol concentration. Solvent molecules are more involved in relaxation processes in solutions of lower polarity. The half-width of the spectral curves, w(1) = (57.2 0.9) nm and w(2) = (81 3) nm are PRODAN concentration as well as ethanol concentration independent in the range of measured ethanol concentrations. The influence of the ethanol on the line-width of the steady-state spectra of PRODAN is neglectable. There is only 7% decrease from pure water to 100% ethanol solution. The decrease of 0.24% for 3 M ethanol solution allows us to consider it constant for that range of solution polarity differences. The line-width of measured steady-state emission spectra are large what is to be expected when the spectral relaxation is the process of emission [14]. The increase of half-width is the consequence of nanosecond relaxation because in that case the emission is from the partly relaxed state [21]. The reasoning is connected with entropy factor of the relaxation processes. The conclusion is that changes in solution polarity neither promote no hinder motions either of PRODAN itself or of the small molecules it relaxes to. CONCLUSION It was the purpose of this research to quantitative steady-state emission spectra of PRODAN and second to provide reliable parameters for the spectroscopic behavior of PRODAN dissolved in solvents of varying ethanol concentrations. 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The wavelengths and half-width of both transitions are PRODAN concentration independent indicating that by applying PRODAN in investigations of membranes even the expected increase in concentration would not induce the self quenching behavior. The linear dependence of fluorescence intensity with increasing PRODAN concentration is relationship, which supports this conclusion. The noticed increase in the rate of fluorescence intensity within the more concentrated ethanol solutions is easily rationalized with increased hydrophobicity if the process of emission is governed by interaction with solute molecules. Namely, it is known that the transfer of energy from the excited PRODAN to adjacent solvent molecules due to the alignment of the solvent dipoles is diminished in hydrophobic environment, the excited state energy is not changed and the energy of transition has smaller Stock shift. The energy factor defining the fluorescence emission is very dependent on solution polarity, and presents the reliable parameter for following the changes in properties of PRODAN environment. The independence of energy of transitions separation upon ethanol concentration, polarity of surrounding, indicates that the two transitions are the consequence of PRODAN relaxation in two distinct vibrational levels of ground state. The decrease of polarity induces the blue shift of transitions wavelengths because the relaxation process for fluorescence emission is primarily governed by solute molecules. The decrease of polarity means the decrease in dipole interactions of media molecules with PRODAN influencing its excitation state energy [22]. We have reached our aim. The linearity in dependence of all measured parameters with the increase of ethanol concentration (decrease of solution polarity) would justify the subtraction procedure we propose. The measured spectra of PRODAN in buffer / ethanol solutions should be corrected for PRODAN concentration in water environment of HDL /buffer/ethanol samples. The subtraction of these spectra from the spectra measured in HDL sample would give the spectra of PRODAN in the HDL particle. Further investigation of ethanol influences on HDL structure could be followed be identifying changes in the spectra of PRODAN in HDL. ACKNOWLEDGMENT This research was supported by grant from Ministry of Science, Education and Sport of Republic Croatia No. 018043. REFERENCES R.B Mcgregor and G. Weber (1986) Nature 319, 70-73. P.L.G. Chong (1988) Biochemistry 27, 399-404. R. Hutterer, A.B.J Parusel and M. Hof (1998) J Fluoresc. 8, 389-393. R. Hutterer, F.W. Schneider W.Th. Hermens, R. Wagenvoord and M. Hof (1998) Biochim.Biophysc. Acta 1414, 155-164. E.K. Krasnowska, L.A. Bagatolli, E. Gratton and T. Parasassi (2001) Biochem. Biophys. Acta 1511, 330-340. T. Parasassi, E.K. Krasnowska, L. Bagatolli and E. Gratton (1998) J. Fluoresc. 8, 365-373. P. Ilich and F.G. Prendergast (1989) J. Phys. Chem. 93, 4441- 4447. J. Catalan, P. Perez, J. Laynez and F. Garcia (1991) J. Fluoresc. 1, 215-224. A. Parusel (1998) J. Chem. Soc. Faraday Trans. 94, 2923-2927. A.B.J. Parusel, W. Nowak, S. Grimme and G. Khler (1998) J. Phys. Chem. 102, 7149-7156. A. B. J. Parusel, F. W. Schneider and G. Khler (1997) J. Mol Struct (Theochem) 398-399, 341-346. A. Balter, W. Nowak, W. Pawelkiewicz and A. Kowalczyk (1988) Chem. Phys. Lett. 143, 565-570. G. Weber and F. J. Farris (1979) Biochemistry 18, 3075-3078. J. R. Lakowicz (1999) Principles of Fluorescence Spectroscopy, 2.nd ed. Kulwer Academic /Plenum Press, NY K.A. Al-Hassan and M. Khanfer (1998) J. Fluoresc. 8, 139-152. A. Kawsk (1999) Naturforsch. 54a, 379-381 B. Sengupta, J. Guharay and P. K. Sengupta (2000) Spectrochimica Acta part A 56, 1433-1441. C.E. Bunker, T.L. Bowen and Y-P. Sun (1993) Photochem Photobiol 58, 499-505. E. Krasnowska, E. Gratton and T. Parasassi (1998) Biophysical J. 74, 1984-1993. R. Hutterer and M. Hof (2002) Z.Phys.Chem. 216, 1-14. A. Samanta and R. W. Fessenden (2000) J. Phys. Chem A 104, 8972-8975. D. Toptygin (2003) J. Fluoresc. 13, 201-219 Fig. 1. Correction of experimental spectra for background contribution, lex = 360 nm, amplifier voltage V = 650 V, excitation and emission bandpasses 5nm A) the experimental spectra of PRODAN in ethanol / water solution B) experimental spectra for ethanol / water solution without PRODAN C) corrected steady state PRODAN fluorescence spectra are the basis for further analyses. Fig. 2. Steady state fluorescence spectra of PRODAN in ethanol solutions. A) theoretical simulations, Eq.(1) of corrected experimental data for fluorescence emission of 0.5 mM PRODAN in 0M (solid), 0.3M (dash), 0.9M (dot) and 3M (dash dot) ethanol solutions at 25oC and 37oC B) presentation of resolved Gaussian curves achieved by theoretical analysis of data in Fig 2A.( the same kind of presentation is preserved) Fig. 3. PRODAN fluorescence emission intensities of two theoretically obtained transitions, lem=519 nm (%,%) and lem = 555 nm (,%), in dependence on solvent polarity, f, at 25 oC (%,) and 37 oC (%,%). Fig. 4. 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Fig. 5. PRODAN fluorescence emission intensities of two theoretically obtained transitions, A) lem = 519 nm a $,.0 (<݄|hqhMz hRhf+h]^hf+CJaJ h]^hf+Uh[vhf+H*h&hf+OJQJ h~hf+hH,hf+5 hqhf+ hf+5hMzhf+5h]hf+H*hf+CJaJhjhf+CJaJ hf+H*hf+ h&hf+/nd B) lem = 555 nm, dependence on PRODAN concentrations in 0 M (%), 0.3 M (%), 0.9 M (%) and 3 M (%) ethanol solutions. 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