- Email: [email protected]
Spectroscopic study of molecular associations between FMN and β-carbolines
Spectrochimicn Aca. Vol. 49A. No. I?, pp. 1793-1799. Printed in Great Britain
1993
0584-s39/93 @ 1993
s6.M + 0.00
Pergamon Press Ltd
Spectroscopic study of molecular associations between FMN and fi-carbolines ARMANDO
CODOAER,* PIEDAD MEDINA,ENRIQUEJOVER
and JESUSA.SANCHEZ
Departamento de Quimica Fisica, Facultad de Quimicas, Universidad de Valencia, Doctor Moliner no. 50, 46100 Burjassot, Valencia, Spain (Received 7 October 1992; in final form 4 January 1993; accepted 13 January 1993) Abstract-The spectrophotometric and thermodynamic properties of molecular complexes of flavin mononucleotide (FMN) (riboflavin 5’-phosphate) with some B-carboline derivatives have been investigated in aqueous solution. The molecular associations have been examined by means of electronic absorption spectra, since in each a new charge-transfer band has been located, and also the variation of the fluorescence emission of FMN on the solutions has been observed. The formation constants for the molecular complexes were determined from absorption data using the Foster-Hammick-Wardley method. The quenching phenomenon observed in FMN fluorescence ‘is related to the concentration of the B-carboline derivatives, allowing the calculation of the quenching constants for FMN-/?-carboline complexes. Thermodynamic parameters have been determined from the values of association constants for the molecular complexes at various temperatures. The influence of substituents in the B-carboline molecule on the stability of the complexes formed was also investigated.
of flavins with numerous compounds of biochemical interest have been observed [l-11]. Intermolecular associations of different flavins (FAD, RFN) with both n and x electron donors have been studied [12-181. In recent investigations [19-231, we have shown that a series of molecular complexes is formed between FAD, RFN and several derivatives of /l-carboline (9H-3, 46-pyrido indole) and their thermodynamic properties, as well as the equilibrium constants, were obtained by spectroscopic methods. The object of the present work was to determine the occurence of complexes between FMN and some /?-carboline derivatives, listed in Table 1, as well as the effect on such complexes of the substitution of &carboline. As shown in previous papers [19-231, the interaction between a donor D and an acceptor A can be studied using the Foster-Hammick-Wardley [24] equation to calculate the equilibrium constant K DA and molar extinction coefficient rrc for AD complex formation from absorption spectroscopic data. The ability of certain molecules and ions to quench the fluorescence of other molecules has been much studied during the last few years [25]. Charge-transfer complex formation is usually accompanied by a reduction in the fluorescence intensity of the components, and the association of molecules in their ground states can also affect classical energy transfer processes. Quenching can also occur as a result of the formation of a nonfluorescent ground state complex between the fluorophore and quencher. When this complex absorbs light it immediately returns to the ground state without emission of a photon. From the dependence of the fluorescence intensity inhibition on quencher concentration and using the STERN-V• LMER equation [26] the values of the quenching fluorescence constant, K, for these molecular associations was determined. Quenching data are frequently presented as a plot of I,,/Z against [O],, because &,/I is expected to be linearly dependent upon the concentration of quencher. A linear Stern-Volmer plot is generally indicative of a single class of fluorophores, all equally accessible to the quencher. However, it is important to recognize that observation of a linear Stern-Volmer plot does not prove a relationship between the collisional (dynamic) and static quenching. In general, static and dynamic quenching can be distinguished by their differing dependence on temperature. COMPLEXES
l
Author to whom correspondence should be addressed. 1793
1794
ARUANW CODORERet al.
We have carried out spectrophotometric measurements of the absorption complex band in aqueous solutions at several temperatures in phosphate buffer at pH 6 in order to calculate the equilibrium constants of molecular complexes formed between FMN and the /?-carboline derivatives, and from the results some thermodynamic parameters have been computed. The apparent constants for the quenching of the fluorescence of FMN by the formation of these molecular complexes and thermodynamic parameters have also been studied. The aims of the present study are to obtain information about this interaction and to point out the influence of the substituents of /3-carboline on the stability of the molecular complexes studied, such as the nature of the flavin considered. A comparison is made between the association constants obtained by means of electronic absorption and fluorescence spectroscopy. EXPERIMENTAL
Materials The /I-carboline derivatives listed in Table 1 and FMN were supplied by Sigma Chemical Co. All were purified by sublimation twice under reduced pressure. All solutions were made in phosphate buffer at pH 6, except those for Harman which were prepared at pH 5.5, in which the ionic strength was maintained constant at 0.2 M. It is assumed that, at the working concentration levels, variations in ionic strength are irrelevant.
Measurements The details of the apparatus used to obtain the absorption and emission spectra have been previously reported [22]. Absorption spectra were recorded for a solution of the electron acceptor @MN) in the same solvent used to make the solution of the complex. At each temperature, measurements were performed with different donor concentrations at constant acceptor concentration. The exciting wavelength was set at 465 nm near an optimum for absorption of light by FMN, but where no significant absorption by the ~-carboline derivatives existed. The fluorescence spectra of FMN in the presence of /I-carboline derivatives in water was measured between 470-7OOnm. Quenching of FMN fluorescence with increasing concentration of a fi-carboline derivative in water was followed at 530 nm. The concentration range of donor (/Lcarbolines) employed in all cases was 1 x lo-‘-2 x low3 M. The concentration of acceptor (FMN) was constant and of the order of 1 x 10e4 M. The temperature range studied was 5-45°C. RESULTS AND DISCUSSION
Absorption spectroscopy The Foster-Hammick-Wardley equation can be applied by taking into account the following assumptions: (i) only one complex with defined stoichiometry is formed from interaction between D and A; (ii) the complex DA absorbs at a wavelength where A and D are completely transparent; (iii) in all cases [D]O+[A]O and [A],-, is not necessarily constant; (iv) the complex is of the simple 1: 1 type; (v) Beer’s law is obeyed for the complex; (vi) a single value of the wavelength at which the maximum appears is obtained when the ionic strength, H+ concentration, and temperature remain constant; and (vii) the buffer solutions do not interact with donor and acceptor. If Al[Dlo is plotted against A, a straight line with slope -KuA and y-intercept KDA*eTC can be obtained. Typical examples of this are shown in Fig. 1 for the FMN-harm01 complex at various temperatures. As expected from the Foster-Hammick-Wardley equation, straight lines were obtained. Similar plots for the remaining complexes between FMN and &carboline derivatives have been performed and, using the leastsquares method, values of K DA and &rc and their corresponding standard error at the 95% confidence level are gathered in Table 2.
Molecular associations between FMN and /?-carbolines
1795
Fig. 1. Plots of A/[D],, versus A for Fh4N-harmol at various temperatures 5°C (D), 15°C (O), 25°C (A) and 45°C (0).
Figure 2 shows the absorption spectra bands of mixed solutions of FMN and varying concentration of harm01 in water at 25°C. The negative bands of the spectra are due to the fact that the FMN concentration in the sample cell is lower than in the reference cell, because a quantity of FMN has been involved in the complex formation in the sample cell. The presence of two characteristic isosbestic points is clear. The corresponding Foster-Hammick-Wardley plots and absorption spectra of the remaining complexes of FMN with the #I-carboline derivatives listed in Table 1, are similar to Figs 1 and 2 and are therefore not included. The computed values of K nA at four different temperatures (5, 15,25 and 45°C) were used to determine the thermodynamic parameters. Using the Van? Hoff equation, a plot of In Kn,, against l/T was made to determine the change in enthalpy /AH” and the values
I
I
1
400
450
500
550
h (nm) Fig. 2. Absorption spectra of FMN-harmol
solutions. Concentration of FMN: 1 X lO-4 M. Concentration of harmol: (1) 1 X 10m2; (2) 8 X lo-‘; (3) 6 X IO-‘; (4) 4.8 X IO-‘; (5) 4 X lo-‘; (6) 3.2 x lo-‘; and (7) 2 x IO-’ M.
ARMANDO CODOAER et al.
1796
Table 1. j3-Carboline derivatives Compound
Formula
Salt
RI
R2
Norharmane
H
H
HCI
Harmane
H
CH,
HCI
Harmol
OH
CH,
Harmine
OCH3
CH-(
HCl HCI
Laboratory
Sigma Sigma Sigma
2-Methylharmine
of AC“ and As” were estimated by the usual method. The results are listed in Table 2 with their corresponding standard errors. The results can be summarized by the following facts: (i) a new band appears at a wavelength where A and D are completely transparent; (ii) the presence of two isosbestic points in the absorption spectra is definite; (iii) the equilibrium constants decrease as the temperature increases; (iv) AH” is < -30KJ mol-‘; and (v) the Foster-Hammick-Wardley relationship is fulfilled. Therefore, all the complexes studied in the present work are charge-transfer complexes with 1: 1 stoichiometry as established elsewhere [ 181. Whereas most experimental work [27] establishes that there is a close correspondence between the value of &A and A,,, for charge-transfer complexes, in which an increase in the stability of the complex involves an increase in A,,,,,, we show in Table 2 that this is not true for the complexes between FMN and /I-carboline derivatives, as well as for, the remaining flavins (FAD, RFN) [19,20,22,23]. This can be understood from the point of view that flavins usually have a strong absorption band near the maximum of the absorption band of the complex DA. Therefore, when the complex is formed progressively by adding aliquot portions of donor, the acceptor concentration in the reference cell is increased with respect to that in the sample cell, making the determination of the true A,,,,, of the complex band difficult [19-231. Moreover, the shape of the charge-transfer band is usually broad, and as shown in Fig. 2, the band of the complex between FMN and harm01 is sharp, corresponding to the presence of the negative band absorption characteristic of FMN. Table 2. Equilibrium
constants
and thermodynamic &carbolines;
parameters
absorption
of molecular pH 6
complexes
of FMN
with
spectroscopy
Equilibrium constant KDA(1 mol-‘) -A.G” (KJ mol-‘)
-AH” (KJ mol-‘)
As” (J Km’ mol-‘)
349f28 213+15 434+27
11.87kO.19 15.12+0.14 15.15+0.17 16.81 kO.16
49.7f0.9 13.5f2.2 29.8kl.l 28.6+ 1.4
131.2+_4.1 -5.4f8.1 49.7 f 4.3 4O.Of 5.2
289 f 21
15.23+0.18
18.9fl.l
ETC
cm-‘)
5°C
15°C
220 245 192 168
295 f 19 785 k 29 104.5 f 88 2157f103
151 f 12 466+21 702f27 1161+74
771 f 47
677 + 55
DOllOr
a
Norharmane HalTllallC Harmol Harmine 2-Methyl harmine
494 495 4% 495
2035 2680 1675 1455
495
1885+230
mrr
(I mol-’ + + + k
25°C
45°C
70+6 427k24 391+28 833f:67 439 + 30
12.7f 4.3
Molecular associations between FMN and B-carbolines
500
520
540
560
580
1797
600
h (nm)
Fig. 3. Quenching of FMN by added harmol. Concentration of FMN: (1) 1 x 10m4M. Concentration of harmol: (2) 10 x lo-‘; (3) 8 X 10m3; (4) 6 X lo-‘; (5) 4.8 X lo-‘; (6) 4 X 10e3; (7) 3.2 x 10m3M; and (8) 2 x 10S3M.
An important feature, which arises from Table 2, is the considerably higher values of the equilibrium constant for some /I-carboline derivative adducts (harmane, harm01 and harmine), than the one obtained for norharmane. This can be explained in terms of the different polarity of the substituent group in the indole nucleus (the order of polarity is H < OH < OCHJ as shown in a study of the dipole moment in dioxane solution [28]. The presence of the pyridine nitrogen confers stability on the complexes between @carboline derivatives and flavins: compare the magnitudes of KDAfor indole derivatives (in Ref. [21]). The values obtained for the 2-methylharmine-FMN complex allow us to assert that the most important contribution to the stability of the complex is the nature of the planar tricyclic conjugated structure of the p-carboline, since in the case of 2methylharmine the nitrogen of the pyridine nucleus has been blocked by a methyl group. This fact is obviously closely related to the inhibition of the MAO enzyme as we and other authors argued previously [28]. Fluorescence
spectroscopy
The progressive changes of the fluorescence spectra of FMN caused by adding harm01 are shown in Fig. 3. From the values of the relative fluorescence intensities versus [D],,,
t?ooo I . 0.002 I,I. 0.004 0.006 1, 0.008 1 * 0.010 0, 0.012 t [Dl,, Fig. 4. Plots of &/I versus [D],, for the FMN-harmol complex at 5°C (m); 15°C (0); 25°C (A); and 45°C (0).
ARMANDO CODORER et al.
1798
Table 3. Quenching constants and thermodynamic parameters of molecular complexes of FMN with ,9carbolines: emission spectroscopy I,, of FMN 465 nm; ,I,, of FMN 525 nm Quenching constant K (I mol-‘)
Donor Norharmane Harmane Harm01 Harmine 2-Methyl harmine
5°C
15°C
25°C
45°C
-AC” (KJ mol-‘)
-AH” (KJ mol-‘)
421+ 10 704 + 28 1250 + 19 1940 f 33
397+9 624 f 28 894 f 15 1296+10
258+5 521 f 23 708f7 914+9
206+3 345 + 14 457+4 51056
14.01+ 0.06 15.35f0.11 16.27+0.04 16.97+0&t
14.2kl.4 13.4kO.7 18.220.5 24.4kO.4
567+6
317+9
15.66+0.04
21.4kO.3
1020f9
739+4
-As” (J K-’ mol-‘) 0.725.1 -6.7k2.6 6.6kl.8 25.3kl.4 19.4+ 1.2
good straight lines have been obtained (Fig. 4), and the values of K can be calculated at each of the temperatures by applying the Stern-Volmer relation. Results as for Figs 3 and 4 have been obtained for other complexes formed between FMN and B-carboline derivatives. The corresponding K values derived from them are in Table 3. In all cases an increase of temperature leads to a decrease of the slope of the Stern-Volmer relation. This phenomenon could be attributed to typical static quenching. Thus, the K values obtained are similar to those found from absorption spectroscopy. Thus, the apparent constant is an association constant [29]. The variation of the apparent constant for complexes of FMN with /3-carboline derivatives for substituents R, and R2 given in Table 3 confirm the above arguments based on the absorption method. The variation of the values of AC”, AH” and AS“ listed in Table 3 are in good agreement with the variation expected on the basis of the absorption results. Acknowledgements-We
thank the DGICYT projects nos OP90-0042 and PB91-0808 for support of this work.
REFERENCES [1] H. A. Harbury and K. A. Foley, Proc. Nafn Acad. Sci. 44, 662 (1958). H. A. Harbury, K. F. Lanove, P. A. Loach and R. M. Amick, Proc. Natn Acad. Sci. 45, 1708 (1959). L. D. Wright and D. B. McCormick, Experientia 20, 501 (1964). M. A. Slifkin, Biochim. Biophys. Acfu, 103, 365 (1965). H. Weil-Malherbe, Biochem. J. 40, 363 (1946). G. Weber, Biochem. J. 47, 114 (1950). D. B. McCormick, H. C. Li and R. E. Mackenzie, Spectrochim. Acta, 23A, 2353 (1967). [8] K. Ulbrych and J. Siodmak, Zesr. Nuuk. Akad. Roln. Tech. Otsztynei. Technol. Zywn 17, 55 (1982). [9] K. Matsui, K. Sugimoto and S. Kasai, J. Biochem. 91, 1357 (1982). [lo] S. E. Silva and M. A. Edwards, Bol. Sot. Chit. Quim. 27, 194 (1982). [11] Y. Byung, L. Sang Jong, L. Seung Jin and C. Hym Ho, J. Pharm. Sci. 72,592 (1983). [12] K. Burton, Biochem. J. 48, 458 (1951). [13] K. Yagi, T. Ozawa and K. Okada, Biochim. Biophys. Actu, 35, 102 (1959). (141 K. Yagi and T. Ozawa, Btbchim. Biophys. Acta 42, 381 (1960). [ 151 K. Yagi, T. Ozawa and T. Nagatsu, Nature 184, 892 (1959). 1161 K. Yagi, F. Tanaka, N. Nakasahima and K. Yoshihara, J. Biol. Chem. 258, 3799 (1983). [17] G. Williamson, P. C. Engel, J. P. Mizzer, C. Thorpe and Massey, J. Biol. Chem. 257, 4314 (1982). [ 181 M. Slifkin, Charge Transfer lnfeructions of Biomolecules, Chap. 7. Academic Press. London (1971). [19] A. CodoAer. I. S. Monzo, F. Tomas and R. Valero, Spectrochim. Actu 42A, 765 (1986). [20] A. Codoirer, I. S. Monzo, F. Tomas and R. Valero, Spectrochim. Actu 43A, 1379 (1987). [21] A. Codoher. 1. S. Monzo, P. Medina and F. Tomas, Spectrochim. Acru 43A, 389 (1987). [22] A. Codofier, 1. S. Monzo, C. Ortiz and A. Olba, J. Chem. Sot., Perkin Truns II, 107 (1989). (231 A. Codofier, P. Medina, C. Ortiz and E. Jover, Spectrochim. Actu 49A, 321 (1993). [2] [3] [4] [5] [6] [7]
Molecular associations between FMN and /?-carbolines [24] [25] [26] (27) [28] [29]
1799
R. Folster, D. L. Hammick and A. A. Wardley, 1. Am. Chem. Sot., 3817 (1953). K. M. C. Davis, Molecular Association (Edited by R. Foster). Academic Press, New York (1975). 0. Stern and M. Volmer, Phys. Z. 29, 183 (1919). G. Briegleb, Electronen-Donator-Kompfexe. Springer, Berlin (1961). I. S. Monzo, A. Codoiier, P. Medina. A. Olba and R. Valero, /. C/rem. Sot., Perkin Truns II, 261(1988). J. R. Lakowin, Principles of Fluorescence Spectroscopy, Chap. 9. Plenum Press, New York (1983).
1993
0584-s39/93 @ 1993
s6.M + 0.00
Pergamon Press Ltd
Spectroscopic study of molecular associations between FMN and fi-carbolines ARMANDO
CODOAER,* PIEDAD MEDINA,ENRIQUEJOVER
and JESUSA.SANCHEZ
Departamento de Quimica Fisica, Facultad de Quimicas, Universidad de Valencia, Doctor Moliner no. 50, 46100 Burjassot, Valencia, Spain (Received 7 October 1992; in final form 4 January 1993; accepted 13 January 1993) Abstract-The spectrophotometric and thermodynamic properties of molecular complexes of flavin mononucleotide (FMN) (riboflavin 5’-phosphate) with some B-carboline derivatives have been investigated in aqueous solution. The molecular associations have been examined by means of electronic absorption spectra, since in each a new charge-transfer band has been located, and also the variation of the fluorescence emission of FMN on the solutions has been observed. The formation constants for the molecular complexes were determined from absorption data using the Foster-Hammick-Wardley method. The quenching phenomenon observed in FMN fluorescence ‘is related to the concentration of the B-carboline derivatives, allowing the calculation of the quenching constants for FMN-/?-carboline complexes. Thermodynamic parameters have been determined from the values of association constants for the molecular complexes at various temperatures. The influence of substituents in the B-carboline molecule on the stability of the complexes formed was also investigated.
of flavins with numerous compounds of biochemical interest have been observed [l-11]. Intermolecular associations of different flavins (FAD, RFN) with both n and x electron donors have been studied [12-181. In recent investigations [19-231, we have shown that a series of molecular complexes is formed between FAD, RFN and several derivatives of /l-carboline (9H-3, 46-pyrido indole) and their thermodynamic properties, as well as the equilibrium constants, were obtained by spectroscopic methods. The object of the present work was to determine the occurence of complexes between FMN and some /?-carboline derivatives, listed in Table 1, as well as the effect on such complexes of the substitution of &carboline. As shown in previous papers [19-231, the interaction between a donor D and an acceptor A can be studied using the Foster-Hammick-Wardley [24] equation to calculate the equilibrium constant K DA and molar extinction coefficient rrc for AD complex formation from absorption spectroscopic data. The ability of certain molecules and ions to quench the fluorescence of other molecules has been much studied during the last few years [25]. Charge-transfer complex formation is usually accompanied by a reduction in the fluorescence intensity of the components, and the association of molecules in their ground states can also affect classical energy transfer processes. Quenching can also occur as a result of the formation of a nonfluorescent ground state complex between the fluorophore and quencher. When this complex absorbs light it immediately returns to the ground state without emission of a photon. From the dependence of the fluorescence intensity inhibition on quencher concentration and using the STERN-V• LMER equation [26] the values of the quenching fluorescence constant, K, for these molecular associations was determined. Quenching data are frequently presented as a plot of I,,/Z against [O],, because &,/I is expected to be linearly dependent upon the concentration of quencher. A linear Stern-Volmer plot is generally indicative of a single class of fluorophores, all equally accessible to the quencher. However, it is important to recognize that observation of a linear Stern-Volmer plot does not prove a relationship between the collisional (dynamic) and static quenching. In general, static and dynamic quenching can be distinguished by their differing dependence on temperature. COMPLEXES
l
Author to whom correspondence should be addressed. 1793
1794
ARUANW CODORERet al.
We have carried out spectrophotometric measurements of the absorption complex band in aqueous solutions at several temperatures in phosphate buffer at pH 6 in order to calculate the equilibrium constants of molecular complexes formed between FMN and the /?-carboline derivatives, and from the results some thermodynamic parameters have been computed. The apparent constants for the quenching of the fluorescence of FMN by the formation of these molecular complexes and thermodynamic parameters have also been studied. The aims of the present study are to obtain information about this interaction and to point out the influence of the substituents of /3-carboline on the stability of the molecular complexes studied, such as the nature of the flavin considered. A comparison is made between the association constants obtained by means of electronic absorption and fluorescence spectroscopy. EXPERIMENTAL
Materials The /I-carboline derivatives listed in Table 1 and FMN were supplied by Sigma Chemical Co. All were purified by sublimation twice under reduced pressure. All solutions were made in phosphate buffer at pH 6, except those for Harman which were prepared at pH 5.5, in which the ionic strength was maintained constant at 0.2 M. It is assumed that, at the working concentration levels, variations in ionic strength are irrelevant.
Measurements The details of the apparatus used to obtain the absorption and emission spectra have been previously reported [22]. Absorption spectra were recorded for a solution of the electron acceptor @MN) in the same solvent used to make the solution of the complex. At each temperature, measurements were performed with different donor concentrations at constant acceptor concentration. The exciting wavelength was set at 465 nm near an optimum for absorption of light by FMN, but where no significant absorption by the ~-carboline derivatives existed. The fluorescence spectra of FMN in the presence of /I-carboline derivatives in water was measured between 470-7OOnm. Quenching of FMN fluorescence with increasing concentration of a fi-carboline derivative in water was followed at 530 nm. The concentration range of donor (/Lcarbolines) employed in all cases was 1 x lo-‘-2 x low3 M. The concentration of acceptor (FMN) was constant and of the order of 1 x 10e4 M. The temperature range studied was 5-45°C. RESULTS AND DISCUSSION
Absorption spectroscopy The Foster-Hammick-Wardley equation can be applied by taking into account the following assumptions: (i) only one complex with defined stoichiometry is formed from interaction between D and A; (ii) the complex DA absorbs at a wavelength where A and D are completely transparent; (iii) in all cases [D]O+[A]O and [A],-, is not necessarily constant; (iv) the complex is of the simple 1: 1 type; (v) Beer’s law is obeyed for the complex; (vi) a single value of the wavelength at which the maximum appears is obtained when the ionic strength, H+ concentration, and temperature remain constant; and (vii) the buffer solutions do not interact with donor and acceptor. If Al[Dlo is plotted against A, a straight line with slope -KuA and y-intercept KDA*eTC can be obtained. Typical examples of this are shown in Fig. 1 for the FMN-harm01 complex at various temperatures. As expected from the Foster-Hammick-Wardley equation, straight lines were obtained. Similar plots for the remaining complexes between FMN and &carboline derivatives have been performed and, using the leastsquares method, values of K DA and &rc and their corresponding standard error at the 95% confidence level are gathered in Table 2.
Molecular associations between FMN and /?-carbolines
1795
Fig. 1. Plots of A/[D],, versus A for Fh4N-harmol at various temperatures 5°C (D), 15°C (O), 25°C (A) and 45°C (0).
Figure 2 shows the absorption spectra bands of mixed solutions of FMN and varying concentration of harm01 in water at 25°C. The negative bands of the spectra are due to the fact that the FMN concentration in the sample cell is lower than in the reference cell, because a quantity of FMN has been involved in the complex formation in the sample cell. The presence of two characteristic isosbestic points is clear. The corresponding Foster-Hammick-Wardley plots and absorption spectra of the remaining complexes of FMN with the #I-carboline derivatives listed in Table 1, are similar to Figs 1 and 2 and are therefore not included. The computed values of K nA at four different temperatures (5, 15,25 and 45°C) were used to determine the thermodynamic parameters. Using the Van? Hoff equation, a plot of In Kn,, against l/T was made to determine the change in enthalpy /AH” and the values
I
I
1
400
450
500
550
h (nm) Fig. 2. Absorption spectra of FMN-harmol
solutions. Concentration of FMN: 1 X lO-4 M. Concentration of harmol: (1) 1 X 10m2; (2) 8 X lo-‘; (3) 6 X IO-‘; (4) 4.8 X IO-‘; (5) 4 X lo-‘; (6) 3.2 x lo-‘; and (7) 2 x IO-’ M.
ARMANDO CODOAER et al.
1796
Table 1. j3-Carboline derivatives Compound
Formula
Salt
RI
R2
Norharmane
H
H
HCI
Harmane
H
CH,
HCI
Harmol
OH
CH,
Harmine
OCH3
CH-(
HCl HCI
Laboratory
Sigma Sigma Sigma
2-Methylharmine
of AC“ and As” were estimated by the usual method. The results are listed in Table 2 with their corresponding standard errors. The results can be summarized by the following facts: (i) a new band appears at a wavelength where A and D are completely transparent; (ii) the presence of two isosbestic points in the absorption spectra is definite; (iii) the equilibrium constants decrease as the temperature increases; (iv) AH” is < -30KJ mol-‘; and (v) the Foster-Hammick-Wardley relationship is fulfilled. Therefore, all the complexes studied in the present work are charge-transfer complexes with 1: 1 stoichiometry as established elsewhere [ 181. Whereas most experimental work [27] establishes that there is a close correspondence between the value of &A and A,,, for charge-transfer complexes, in which an increase in the stability of the complex involves an increase in A,,,,,, we show in Table 2 that this is not true for the complexes between FMN and /I-carboline derivatives, as well as for, the remaining flavins (FAD, RFN) [19,20,22,23]. This can be understood from the point of view that flavins usually have a strong absorption band near the maximum of the absorption band of the complex DA. Therefore, when the complex is formed progressively by adding aliquot portions of donor, the acceptor concentration in the reference cell is increased with respect to that in the sample cell, making the determination of the true A,,,,, of the complex band difficult [19-231. Moreover, the shape of the charge-transfer band is usually broad, and as shown in Fig. 2, the band of the complex between FMN and harm01 is sharp, corresponding to the presence of the negative band absorption characteristic of FMN. Table 2. Equilibrium
constants
and thermodynamic &carbolines;
parameters
absorption
of molecular pH 6
complexes
of FMN
with
spectroscopy
Equilibrium constant KDA(1 mol-‘) -A.G” (KJ mol-‘)
-AH” (KJ mol-‘)
As” (J Km’ mol-‘)
349f28 213+15 434+27
11.87kO.19 15.12+0.14 15.15+0.17 16.81 kO.16
49.7f0.9 13.5f2.2 29.8kl.l 28.6+ 1.4
131.2+_4.1 -5.4f8.1 49.7 f 4.3 4O.Of 5.2
289 f 21
15.23+0.18
18.9fl.l
ETC
cm-‘)
5°C
15°C
220 245 192 168
295 f 19 785 k 29 104.5 f 88 2157f103
151 f 12 466+21 702f27 1161+74
771 f 47
677 + 55
DOllOr
a
Norharmane HalTllallC Harmol Harmine 2-Methyl harmine
494 495 4% 495
2035 2680 1675 1455
495
1885+230
mrr
(I mol-’ + + + k
25°C
45°C
70+6 427k24 391+28 833f:67 439 + 30
12.7f 4.3
Molecular associations between FMN and B-carbolines
500
520
540
560
580
1797
600
h (nm)
Fig. 3. Quenching of FMN by added harmol. Concentration of FMN: (1) 1 x 10m4M. Concentration of harmol: (2) 10 x lo-‘; (3) 8 X 10m3; (4) 6 X lo-‘; (5) 4.8 X lo-‘; (6) 4 X 10e3; (7) 3.2 x 10m3M; and (8) 2 x 10S3M.
An important feature, which arises from Table 2, is the considerably higher values of the equilibrium constant for some /I-carboline derivative adducts (harmane, harm01 and harmine), than the one obtained for norharmane. This can be explained in terms of the different polarity of the substituent group in the indole nucleus (the order of polarity is H < OH < OCHJ as shown in a study of the dipole moment in dioxane solution [28]. The presence of the pyridine nitrogen confers stability on the complexes between @carboline derivatives and flavins: compare the magnitudes of KDAfor indole derivatives (in Ref. [21]). The values obtained for the 2-methylharmine-FMN complex allow us to assert that the most important contribution to the stability of the complex is the nature of the planar tricyclic conjugated structure of the p-carboline, since in the case of 2methylharmine the nitrogen of the pyridine nucleus has been blocked by a methyl group. This fact is obviously closely related to the inhibition of the MAO enzyme as we and other authors argued previously [28]. Fluorescence
spectroscopy
The progressive changes of the fluorescence spectra of FMN caused by adding harm01 are shown in Fig. 3. From the values of the relative fluorescence intensities versus [D],,,
t?ooo I . 0.002 I,I. 0.004 0.006 1, 0.008 1 * 0.010 0, 0.012 t [Dl,, Fig. 4. Plots of &/I versus [D],, for the FMN-harmol complex at 5°C (m); 15°C (0); 25°C (A); and 45°C (0).
ARMANDO CODORER et al.
1798
Table 3. Quenching constants and thermodynamic parameters of molecular complexes of FMN with ,9carbolines: emission spectroscopy I,, of FMN 465 nm; ,I,, of FMN 525 nm Quenching constant K (I mol-‘)
Donor Norharmane Harmane Harm01 Harmine 2-Methyl harmine
5°C
15°C
25°C
45°C
-AC” (KJ mol-‘)
-AH” (KJ mol-‘)
421+ 10 704 + 28 1250 + 19 1940 f 33
397+9 624 f 28 894 f 15 1296+10
258+5 521 f 23 708f7 914+9
206+3 345 + 14 457+4 51056
14.01+ 0.06 15.35f0.11 16.27+0.04 16.97+0&t
14.2kl.4 13.4kO.7 18.220.5 24.4kO.4
567+6
317+9
15.66+0.04
21.4kO.3
1020f9
739+4
-As” (J K-’ mol-‘) 0.725.1 -6.7k2.6 6.6kl.8 25.3kl.4 19.4+ 1.2
good straight lines have been obtained (Fig. 4), and the values of K can be calculated at each of the temperatures by applying the Stern-Volmer relation. Results as for Figs 3 and 4 have been obtained for other complexes formed between FMN and B-carboline derivatives. The corresponding K values derived from them are in Table 3. In all cases an increase of temperature leads to a decrease of the slope of the Stern-Volmer relation. This phenomenon could be attributed to typical static quenching. Thus, the K values obtained are similar to those found from absorption spectroscopy. Thus, the apparent constant is an association constant [29]. The variation of the apparent constant for complexes of FMN with /3-carboline derivatives for substituents R, and R2 given in Table 3 confirm the above arguments based on the absorption method. The variation of the values of AC”, AH” and AS“ listed in Table 3 are in good agreement with the variation expected on the basis of the absorption results. Acknowledgements-We
thank the DGICYT projects nos OP90-0042 and PB91-0808 for support of this work.
REFERENCES [1] H. A. Harbury and K. A. Foley, Proc. Nafn Acad. Sci. 44, 662 (1958). H. A. Harbury, K. F. Lanove, P. A. Loach and R. M. Amick, Proc. Natn Acad. Sci. 45, 1708 (1959). L. D. Wright and D. B. McCormick, Experientia 20, 501 (1964). M. A. Slifkin, Biochim. Biophys. Acfu, 103, 365 (1965). H. Weil-Malherbe, Biochem. J. 40, 363 (1946). G. Weber, Biochem. J. 47, 114 (1950). D. B. McCormick, H. C. Li and R. E. Mackenzie, Spectrochim. Acta, 23A, 2353 (1967). [8] K. Ulbrych and J. Siodmak, Zesr. Nuuk. Akad. Roln. Tech. Otsztynei. Technol. Zywn 17, 55 (1982). [9] K. Matsui, K. Sugimoto and S. Kasai, J. Biochem. 91, 1357 (1982). [lo] S. E. Silva and M. A. Edwards, Bol. Sot. Chit. Quim. 27, 194 (1982). [11] Y. Byung, L. Sang Jong, L. Seung Jin and C. Hym Ho, J. Pharm. Sci. 72,592 (1983). [12] K. Burton, Biochem. J. 48, 458 (1951). [13] K. Yagi, T. Ozawa and K. Okada, Biochim. Biophys. Actu, 35, 102 (1959). (141 K. Yagi and T. Ozawa, Btbchim. Biophys. Acta 42, 381 (1960). [ 151 K. Yagi, T. Ozawa and T. Nagatsu, Nature 184, 892 (1959). 1161 K. Yagi, F. Tanaka, N. Nakasahima and K. Yoshihara, J. Biol. Chem. 258, 3799 (1983). [17] G. Williamson, P. C. Engel, J. P. Mizzer, C. Thorpe and Massey, J. Biol. Chem. 257, 4314 (1982). [ 181 M. Slifkin, Charge Transfer lnfeructions of Biomolecules, Chap. 7. Academic Press. London (1971). [19] A. CodoAer. I. S. Monzo, F. Tomas and R. Valero, Spectrochim. Actu 42A, 765 (1986). [20] A. Codoirer, I. S. Monzo, F. Tomas and R. Valero, Spectrochim. Actu 43A, 1379 (1987). [21] A. Codoher. 1. S. Monzo, P. Medina and F. Tomas, Spectrochim. Acru 43A, 389 (1987). [22] A. Codofier, 1. S. Monzo, C. Ortiz and A. Olba, J. Chem. Sot., Perkin Truns II, 107 (1989). (231 A. Codofier, P. Medina, C. Ortiz and E. Jover, Spectrochim. Actu 49A, 321 (1993). [2] [3] [4] [5] [6] [7]
Molecular associations between FMN and /?-carbolines [24] [25] [26] (27) [28] [29]
1799
R. Folster, D. L. Hammick and A. A. Wardley, 1. Am. Chem. Sot., 3817 (1953). K. M. C. Davis, Molecular Association (Edited by R. Foster). Academic Press, New York (1975). 0. Stern and M. Volmer, Phys. Z. 29, 183 (1919). G. Briegleb, Electronen-Donator-Kompfexe. Springer, Berlin (1961). I. S. Monzo, A. Codoiier, P. Medina. A. Olba and R. Valero, /. C/rem. Sot., Perkin Truns II, 261(1988). J. R. Lakowin, Principles of Fluorescence Spectroscopy, Chap. 9. Plenum Press, New York (1983).