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l -kynurenine: A fluorescent probe of serum albumins
BIOCHIMICA ET BIOPHYSICA ACTA
91
BBA 36252 L - K Y N U R E N I N E : A F L U O R E S C E N T P R O B E OF SERUM ALBUMINS
J O R G E E. C H U R C H I C H
Department of Biochemistry, The University of Tennessee, Knoxville, Tenn. (U.S.A.) (Received May 29th, 1972)
SUMMARY
The specific interaction of L-kynurenine with serum albumins was investigated by fluorescence spectroscopy. The addition of L-kynurenine to a solution of serum albumin induces in parallel to fluorescence enhancement a marked increase in the polarization of fluorescence of the ligand. Binding experiments conducted at constant protein concentration and varying ligand concentration reveal that the L-kynurenineprotein complex is characterized by an association constant (Ka) of 2.8. lO 5 M -1. Lkynurenine complexed to bovine serum albumin displays a m a x i m u m of emission at 435 rim, while L-kynurenine in hydrogen bonding solvents is characterized by a maxim u m of emission at 460 nm. This blue shift in the band position of the emission spect r u m is more pronounced than the corresponding shift in the absorption spectra. The possible relationship of these spectral changes to the binding of L-kynurenine to a non-polar binding site is discussed; it is proposed that during its brief fluorescence lifetime the molecule of L-kynurenine bound to the protein does not interact with the surrounding solvent.
INTRODUCTION
I t is well established that fluorescence spectroscopy is a very sensitive technique to measure the interaction between fluorescent dyes and macromolecules in solution. In 1952, Laurence I reported that the fluorescence enhancement produced by the adsorption of various dyes to the protein bovine serum albumin can be used to determine the stoichiometry of binding. Subsequently, Weber and coworkers2, ~ showed that the fluorescence properties of substituted naphthalenesulfonates are sensitive to conformational changes of the protein bovine serum albumin. A number of other studies on binding of naphthalenesulfonates to proteins have been published in the last five years, and the t e l m fluorescence probe was proposed to characterize these chromophores (see ref. 4 for general review). In spite of these studies, the mechanism of binding of natural fluorescent compounds to the selum albumins has not been extensively investigated. It is the purpose of this work to investigate whether derivatives of the amino acid L-tryptophan can be used as fluorescent probes of non-polar Biochim. Biophys. Acta, 285 (1972) 91-98
92
J. E. CHURCHICH
binding sites in serum albumins. It is reported that the binding of L-kynurenine to serum albumins is accompanied by an appreciable increase in the fuorescence intensity emitted by the ligand. The fluorescence properties (emission, polarization and fluorescence yield) of L-kynurenine complexed to serum albumins were investigated and the results compared to those of the ligand in hydrogen-bonding solvents.
METHODS Fluorescence spectra were recorded in a spectrofluorometer designed in our laboratory. Radiation from a I5o-W Xenon lamp (Hanovia) was passed through a 5oo-mm Bausch and Lomb monochromator (Blazed at 3oo nm, dispersion 3.3 nm/mm) and focused onto a thermostated cell. Fluorescence emission was observed at right angles to the exciting wavelength using a 5oo-mm Bausch and Lomb monochromator (dispersion 3.3 nm/mm) and detected by an EMI 6256S photomultiplier tube. The signal from the photomultiplier was amplified and fed to the Y-axis input of a Mosely X - Y recorder (Model I35AM), the X-axis of which was coupled to the wavelength drive of the analyzing monochromator. Calibration of both exciting light source and detector system was carried out according to procedures described in the literatureS, 6. A bandwidth of 2 nm was used in the fluorometric determinations, and quantum yields of fluorescence were calculated according to the method of Parker and Rees 7 with standards of known quantum yield (fluoresceine and quinine sulphate). Polarization of fluorescence measurements were performed in the apparatus designed in our laboratory 8. For polarization spectra measurements, the wavelength of excitation was selected by means of a quartz prism monochromator (Schoeffel, QPM 3oS). The bandwidth for excitation was 5 nm in the region 33o-4oo nm according to the calibration chart provided by the manufacturer. An analysis of the various sources of random and systematic errors shows that this apparatus is capable of measuring degree of polarization to an accuracy of I °/o for polarization of fluorescence values greater than o.I. For a complete review of the applications of this technique see ref. 9.
MATERIALS Bovine serum albumin was the Armour crystalline product. H u m a n serum albumin was purchased from Sigma and rabbit serum albumin was obtained from Mann. The enzymes alcohol dehydrogenase, lactate dehydrogenase and glutamate dehydrogenase, lysozyme ribonuclease and trypsin were purchased from Boehringer. The reagents L-kynurenine and DL-kynurenine were obtained from Calbiochem and used without further purification. The organic solvents were of spectroscopic grade (Eastman). Because of the sensitivity of serum albumins to small amounts of detergents, all glassware was soaked in water and rinsed with distilled ethanol before drying. Concentrations of kynurenine were determined by absorbance at 360 nm using the molar extinction coefficient of 515 ° . Protein concentrations were determined b y the method of Lowry et al. 1°.
Biochim. Biophys. Acta, 285 (1972) 91-98
93
L-KYNURENINE: A FLUORESCENT PROBE RESULTS
L-Kynurenine is characterized by a low fluorescence yield (q = o.oo5) when examined in aqueous solutions over a wide range of pH values; but it shows a substantial increase in fluorescence yield when dissolved in hydrogen-bonding solvents of lower dielectric constant than water. As shown in Fig. I, the fluorescence spectrum of L-kynurenine exhibits a clear maximum over the spectral region 460-47 ° rim. Further analysis of the spectroscopic properties reveals that the polarity of the environment, which is correlated with the dielectric constant and refractive index of the medium, affects the fluorescence yield of L-kynurenine. In order to evaluate the effect of polarity on the fluorescence yield, it was decided to employ a mixed solvent system in which the dielectric constant could be varied independently of the viscosity at constant temperature (25 °C). The system chosen consisted of mixtures of dioxane and water since this solvent system makes available an almost I5-fold variation in the dielectric constant without appreciable changes in the viscosity of the solution. The results of the fluorescence measurements as a function of the dielectric constant are illustrated in Fig. 2. It is immediately apparent that an increased fluorescence intensity of L-kynurenine was associated with a decrease in the dielectric constant of the solution.
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400 450 k (nm)
500 550
20 40 DIELECTRIC
60 80 CONSTANT
6
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Fig. I. A b s o r p t i o n and fluorescence spectra of L-kynurenine in the prescence of bovine s e r u m alb u m i n at p H 7.4 ((2)--(2)). A b s o r p t i o n and fluorescence spectra of L-kynurenine in p r o p a n e d i o l w a t e r (95:5, v/v), ( 0 - - 0 ) . The samples of L-kynurenine have the same a b s o r b a n c e at the exciting wavelength, A = o.I, at 36o n m for i-era cuvette. Fig. 2. Left. The variation in the fluorescence intensity emitted at 47 ° n m by L-kynurenine in d i o x a n e - w a t e r m i x t u r e s as a function of the dielectric constant. Right. The variation in the fluorescence intensity emitted at 435 n m b y L-kynurenine complexed to bovine s e r u m a l b u m i n as a function of the p H of the solution. Mixing ratio i mole of ligand : 2 moles of protein. All the samples have the same a b s o r b a n c e at the exciting wavelength (A360 am = o.I).
Interaction of L-kynurenine with serum albumins The effect of bovine serum albumin solutions on the fluorescence of L-kynurenine was examined at several pH values. In these experiments protein solutions of approximately 2 mg/ml were used as solvents ; and in order to maximize the binding of the ligand, the molar ratio of protein to L-kynurenine was of the order of 2:I. The fluorescence spectrum of L-kynurenine complexed to bovine serum albumin was then recorded and compared with the spectrum of free L-kynurenine (Fig. I). It was found that over the pH range from 7 to 9.5, the protein bovine serum albumin promotes an enhancement of the fluorescence emitted by the ligand (Table I). As may be seen from Biochim. Biophys. Acta, 285 (1972) 91-98
94
j . E . CHURCHICH
TABLE I FLUORESCENCE
PROPERTIES
OF
L-KYNURENINE
Sample
Solvent
Absorption
Fluorescence
Q*
p**
L-Kynurenine L-Kynurenine
W a t e r (pH 8.4) Propanediolw a t e r (95:5, v/v) W a t e r (pH 7.4)
360
45 °
0.005
o.o9
37 ° 365
460 435
0.05 0.05
o.40
W a t e r (pH 8.4)
365
435
o.ii
0.40
W a t e r (pH 8.4)
365
435
0-05
0.4°
W a t e r (pH 8.4)
365
435
0.04
o-40
L-Kynurenineb o v i n e s e r u m albumin**" L-Kynurenineb o v i n e s e r u m albumin*** L-Kynurenineh u m a n s e r u m albumin*** L-Kynureninerabbit serum albumin
Q u a n t u m yield of fluorescence. Polarization of fluorescence excited at 360 n m . *** Mixing ratio i mole of ligand : 2 moles of protein. *
**
Fig. 2, the fluorescence intensity of L-kynurenine in the presence of protein is quenched at p H values below 6, while at p H values ranging from 8 to 9, the fluorescence yield reached a m a x i m u m value. This fluctuation in the fluorescence yield of the ligand in the presence of protein at varying p H values was not paralleled by a shift in the band position of the emission spectrum. As shown in Table I, the m a x i m u m of emission of bound L-kynurenine remains invariant over the p H range from 7 to 8.4, while it displays a blue shift of approximately 20 nm when compared to L-kynurenine dissolved in hydrogen-bonding solvents. The fluorescence enhancement that follows the addition of increasing concentrations of L-kynurenine to a fixed concentration of protein was used to determine the affinity constant of the ligand for the macromolecule. Fig. 3 shows how the fluorescence intensity changed with increasing concentrations of L-kynurenine while keeping the protein concentration constant. The fluorescence intensitv of free (F0) and bound (Fro) L-kynurenine as well as the fluorescence observed when both flee and bound ligand are in equilibrium (F) were used to calculate the fraction of ligand bound (Eqn I)
I00
o
50 u_
I
2
3
4
5
6
[L:KYNURENINE] x 10 5 ( M )
Fig. 3- C h a n g e s in fluorescence i n t e n s i t y a t 435 n m on t i t r a t i o n of a fixed c o n c e n t r a t i o n of b o v i n e s e r u m a l b u m i n (2.5" IO -s M) w i t h increasing c o n c e n t r a t i o n s of L - k y n u r e n i n e . R e s u l t s o b t a i n e d a t p H 7.4 ( O - - O ) , p H 7.7 ( A - - ~ ) a n d p H 8. 4 ( 0 - - - 0 ) . T h e w a v e l e n g t h of e x c i t a t i o n was 365 rim.
Biochim. Biophys. Acta, 285 (1972) 91-98
L-KYNURENINE:
A FLUORESCENT PROBE
95
F -- F 0
a
(i)
Fm -- F 0
where Fm is the actual observed fluorescence when all the ligand has been absorbed. Fm was determined directly by adding increasing concentrations of protein to a fixed concentration of L-kynurenine. The average number of ligand molecules bound per mole of protein (~) was calculated for points along the titration curve by means of Eqn 2 : a[Lo3
--
(2)
[P0]
where [Lo] is the total ligand concentration and [Po] total protein concentration. The results were analyzed by Eqn 3 : - -
ILl
=
K~(n
-
~)
(3)
and the binding experiments conducted at three different pH values yield the results summarized in Table II. TABLE
II
BINDING OF L-KYNURENINE TO SERUM ALBUMINS Experiments
were conducted
a t 25 °C.
Sample
Bovine Bovine Bovine Human Rabbit
serum serum serum serum serum
albumin--Lkynurenine albumin-L-kynurenine albumin-L-kynurenine albumin-L-kynurenine albumin-L-kynurenine
pH
Association constant
7.4 7.7 8. 4 8. 4 8. 4
2.5" 2.7' 3' 2.8. 2. 7.
(K~) (M-l) lO5 IO5 lO5 lO 5 lO 5
Binding sites
(n)
I
0.98 I I
o.96
A comparison between the association constants obtained at different pH values, shows that maximum affinity of ligand for the protein bovine serum albumin is observed at pH 8. 4. Moreover the slight increase in affinity constant at alkaline pH is not accompanied by a change in the number of binding sites (n = i). At pH 8.4 the proteins human serum albumin and rabbit serum albumin show maximum affinity for L-kynurenine. The association constants determined by fluorometric titrations are of similar order of magnitude (Table II).
Radiationless energy transfer in the complex The effect of complex formation on the fluorescence spectrum of bovine serum albumin excited at 280 nm is illustrated in Fig. 4. While the fluorescence of the protein is quenched by addition of L-kynurenine, the emission of the bound ligand activated at 280 nm is larger than the emission given by free L-kynurenine dissolved in the solvent mixture propanediol-water (95:5, v/v) and excited at the same wavelength, 280 nm (Fig. 4). Since the absorption band of L-kynurenine overlaps the emission band of the protein, the quenching of protein fluorescence promoted by addition of Lkynurenine can at least partly be explained in terms of radiationless energy transfer from the tryptophyl residues of the protein to the ligand. A necessary condition for Biochim. Biophys. Acta, 2 8 5 (1972) 9 1 - 9 8
96
J. E. CHURCHICH
°5o[ tu 0 ~- 5 0 0 z
t
I
L
450
400
350
300
250
I
450
400
350
300
25O
o 5o
0 5OO
t
A(nm)
Fig. 4- Top. Fluorescence excitation s p e c t r u m of L-kynurenine (3" lO-6 M) in p r o p a n e d i o l - w a t e r (95:5, v/v) ( ~ --A) and in the presence of bovine s e r u m a l b u m i n (4-1o .6 M) at p H 8. 4 (O---Q). The emission m o n o c h r o m a t o r was set at 47 ° n m . B o t t o m . Fluorescence s p e c t r u m of bovine s e r u m a l b u m i n ( 4 . i o - 6 M ) in the absence ( © - - 0 ) and presence ( O - - O ) of L-kynurenine ( 3 - i o *M). The excitation m o n o c h r o m a t o r was set at 280 nm.
this energy transfer mechanism to be operative is extensive overlap between the emission band of the donor and the absorption band of the acceptor. This condition is met by L-kynurenine bound to the protein since the overlap integral (J(~)) yields the value IO-la cm 3. M 1, whenfD (~), the fluorescence spectrum of the donor is normalized to I, in units of quanta on a wavenumber scale, eA (~) is the molar decadic extinction coefficient of the acceptor (L-kynurenine). Substituting J (~) in Forster's n equation : 1
R0 ~ [9 ' I°-2a " /~2 . ~ D " J(g)/n*]"
(4)
one obtains the critical distance of transfer (R 0 = 45 fit) for /52 = 2/3 (orientation factor), ~D = o.48 (quantum yield of tryptophyl residues in the protein) and n 4 = 3.13 (refractive index of water). It becomes evident from these calculations that a radiationless transfer mechanism may be oparative; and that the critical distance of transfer (R 0 -- 45 fit) is compatible with the actual spatial separation between the two tryptophyl residues and the molecule of L-kynurenine.
Polarization of fluorescence The technique of polarization of fluorescence provided valuable information on the rotational mobility of L-kynurenine bound to bovine serum albumin. The polarization of fluorescence of the ligand in tile presence of protein was measured at 25 °C. The results of these measurements, together with the polarization of fluorescence values of free L-kynurenine in water and glycerol are given in Fig. 5. The effect of viscosity on the polarization of fluorescence of L-kynurenine is easily explained in terms of the rotational motion of the chromophore during the interval zM (fluorescence lifetime). The fluorescence is largely depolarized in water (p = 0.09) because the molecules of L-kynurenine assume a nearly random orientation between excitation and emission. Conversely, the polarization of fluorescence is increased in glycerol (p = 0.42 ) because the molecules of L-kynurenine do not change their orientation during the interval TM. In the absence of rotational motion and torsional vibrations, it can be shown that the polarization of fluorescence varies between the limits p = ½, Riochim. 13iophys..4cla, 285 (T972) 9 t 98
L - K Y N U R E N I N E ' A FLUORESCENT PROBE 0.5
I o
I
1
~
=
360
380
97
i
c
o
04
g 03 02 0.1
~
a I
320
340
4.00
Mnm) Fig. 5. Polarization of fluorescence versus w a v e l e n g t h of excitation. L - K y n u r e n i n e in glycerol w a t e r (98:2, v/v) ( O O), L - k y n u r e n i n e in w a t e r a t p H 8. 4 ( A - - A ) a n d L - k y n u r e n i n e in t h e presence of bovine s e r u m a l b u m i n a t p H 8. 4 (I~-~O). Mixing ratio of ligand to p r o t e i n i :2. T h e fluorescence excited over t h e spectral r a n g e 32o-36o n m a n d 360-4oo n m was isolated w i t h Corning CS-3-74 a n d CS-3-72 glass filters, respectively.
when the polarization vectors of absorption and emission are parallels ; and p -½ when the polarization vectors are perpendicular. Although these ideal limits are rarely realized, it should be noted that the polarization values corresponding to the long wavelength absorption band (36o nm) tend to approach the ideal limit p = o.5. This finding is interpreted to mean that the transition moment for the 36o nm absorption band should be most nearly parallel to the transition moment of the emission band. It should be noted that the binding of L-kynurenine to the protein (Fig. 5) causes a uniform increase in the polarization values over the entire range of wavelengths examined. Since the polarized fluorescence of the ligand bound to the protein (p o.4 o) is practically identical to that emitted by L-kynurenine in a viscous solvent (p = o.42), it seems reasonable to conclude that the rotational motion of the ligand is largely suppressed as result on noncovalent interactions with the binding site of the macromolecule. DISCUSSION
The experiments reported above have shown that the stoichiometry and affinity of binding of L-kynurenine for serum albumins can be easily determined by fluorescence spectroscopy. The most salient feature of these studies is the effect of binding on several spectroscopic properties of L-kynurenine. The addition of L-kynurenine to a solution of serum albumin induces in parallel to fluorescence enhancement, a blue shift in the band position of the emission spectrum and a concurrent increase in the polarization of fluorescence emitted by the ligand. The increase in fluorescence of bound Lkynurenine resembles the behavior of some substituted naphthalenesulfonates which are practically nonfluorescent in water, but fluoresce strongly when bound to proteins. While the naphthalene dyes are known to interact with several proteins, the binding of L-kynurenine to serum albumins is quite specific as indicated by the following lines of evidence (a) The serum albumins examined (human, bovine and rabbit, Table II) enhance the fluorescence of L-kynurenine to a greater degree than other proteins studied. The enzymes lactate dehydrogenase, alcohol dehydrogenase, and glutamate dehydrogenase failed to cause any significant change in the spectroscopic properties of the ligand. Similar negative results were obtained with enzymes of low molecular weight such as lysozyme, ribonuclease, and trypsin. (b) The binding of L-kynurenine Biochim. Biophys. Acta, 285 (1972) 91-98
98
J . E . CHURCHICH
appears to occur in a sterospecific manner since the mixing of DL-kynurenine with serum albumins (mixing ratio, I mole of DL-kynurenine :I mole of protein) induces a fluorescence enhancement at 435 nm which is 5o% of the value observed when Lkynurenine is complexed to the serum albumins under similar experimental conditions. Although this experimental evidence is indirect, it should be noted that the binding of L-tryptophan to bovine serum albumin is sterospecific as demonstrated by the equilibrium dialysis studies of McMenamy and Oncley 1~. (c) The fluorescence yield of L-kynurenine complexed to serum albumins is sensitive to small variations in the p H of the solution. The reason for the fluctuations in quantum yield are not certain, but it is possible that subtle conformational changes of the protein are responsible for this behavior. It was shown in this paper that fluorescence enhancement of Lkynurenine is also observed when this compound is dissolved in hydrogen bonding solvents of decreasing dielectric constant. An analysis of the spectroscopic properties of L-kynurenine in hydrogen-bonding solvents reveals that the polarity of the medium affects the wavelength of maximal emission and absorption, producing a batochromic shift of Io nm as the solvent polarity decreases from water to dioxan. This batochromic shift in the fluorescence spectrum is not observed when L-kynurenine interacts with the binding sites of the protein. In marked contrast to the fluorescence of L-kynurenine in hydrogen bonding solvents, the emission spectrum of the ligand complexed to the protein displays a hypsochromic shift of approximately 3o nm when compared to Lkynurenine in water. In order to understand the spectral changes induced by binding, it is important to realize that the blue shift in fluorescence is more pronounced and of opposite direction than the corresponding shift in absorption (Table I). A behavior of this kind strongly suggests a change in solute solvent stabilization in the excited state as compared to the ground state 1~. The fact that the molecule of L-kynurenine, which is firmly bound to the protein, displays a blue shift in the emission spectrum is interpreted to mean that during its excited lifetime the chromophore does not interact with the surrounding solvent. It is therefore proposed that some steric interference by amino acid residues at the binding site prevents the access of water molecules to complexed l.-kynurenine in the singlet excited state. ACKNOWLEDGEMENT
This work was supported by a grant GB 33395 from the National Science Foundation. REFERENCES i 2 3 4 5 6 7 8 9 io Ii 12 13
D. J. R. Laurence, Biochem. f . , 51 (1952 ) 168. G. W e b e r and L. Young, J. Biol. Chem., 239 (1964) 1415. E. Daniel and G. Weber, Biochemistry, 5 (1966) 1893. G. M. E d e l m a n and W. O. McClure, Ace. Chem. Res. I (1968) 65 . W. I-I. Melhuish, J. Phys. Chem., 65 (1961) 229. J. E. Churchich, J. Biol. Chem., 242 (1967) 4414 . C. A. P a r k e r and W. T. Rees, Analyst, 85 (196o) 596. J. E. Churchich, Biochim. Biophys. Aeta, 147 (1967) 32. F. Dorr, Angew. Chem. Int. Ed., 5 (1966) 478 . O. H. Lowry, N. J. Rosebrough, A. L. F a r r and R. J. Randall, J. Biol. Chem., 193 (1951) 265. T. H. Foster, Discuss. Faraday Soc., 27 (1959) 265. R. H. ~'VicMenamy and J. L. Oncley, J. Biol. Chem., 233 (1958) 1436. W. Liptay, in O. Sinanoglu, Modern Quantum Chemistry, Istanbul Lectures, P a r t II, Academic Press, New York, 1965 , p. 173.
Biochim. Biophys. Aeta, 285 (1972) 91-98
91
BBA 36252 L - K Y N U R E N I N E : A F L U O R E S C E N T P R O B E OF SERUM ALBUMINS
J O R G E E. C H U R C H I C H
Department of Biochemistry, The University of Tennessee, Knoxville, Tenn. (U.S.A.) (Received May 29th, 1972)
SUMMARY
The specific interaction of L-kynurenine with serum albumins was investigated by fluorescence spectroscopy. The addition of L-kynurenine to a solution of serum albumin induces in parallel to fluorescence enhancement a marked increase in the polarization of fluorescence of the ligand. Binding experiments conducted at constant protein concentration and varying ligand concentration reveal that the L-kynurenineprotein complex is characterized by an association constant (Ka) of 2.8. lO 5 M -1. Lkynurenine complexed to bovine serum albumin displays a m a x i m u m of emission at 435 rim, while L-kynurenine in hydrogen bonding solvents is characterized by a maxim u m of emission at 460 nm. This blue shift in the band position of the emission spect r u m is more pronounced than the corresponding shift in the absorption spectra. The possible relationship of these spectral changes to the binding of L-kynurenine to a non-polar binding site is discussed; it is proposed that during its brief fluorescence lifetime the molecule of L-kynurenine bound to the protein does not interact with the surrounding solvent.
INTRODUCTION
I t is well established that fluorescence spectroscopy is a very sensitive technique to measure the interaction between fluorescent dyes and macromolecules in solution. In 1952, Laurence I reported that the fluorescence enhancement produced by the adsorption of various dyes to the protein bovine serum albumin can be used to determine the stoichiometry of binding. Subsequently, Weber and coworkers2, ~ showed that the fluorescence properties of substituted naphthalenesulfonates are sensitive to conformational changes of the protein bovine serum albumin. A number of other studies on binding of naphthalenesulfonates to proteins have been published in the last five years, and the t e l m fluorescence probe was proposed to characterize these chromophores (see ref. 4 for general review). In spite of these studies, the mechanism of binding of natural fluorescent compounds to the selum albumins has not been extensively investigated. It is the purpose of this work to investigate whether derivatives of the amino acid L-tryptophan can be used as fluorescent probes of non-polar Biochim. Biophys. Acta, 285 (1972) 91-98
92
J. E. CHURCHICH
binding sites in serum albumins. It is reported that the binding of L-kynurenine to serum albumins is accompanied by an appreciable increase in the fuorescence intensity emitted by the ligand. The fluorescence properties (emission, polarization and fluorescence yield) of L-kynurenine complexed to serum albumins were investigated and the results compared to those of the ligand in hydrogen-bonding solvents.
METHODS Fluorescence spectra were recorded in a spectrofluorometer designed in our laboratory. Radiation from a I5o-W Xenon lamp (Hanovia) was passed through a 5oo-mm Bausch and Lomb monochromator (Blazed at 3oo nm, dispersion 3.3 nm/mm) and focused onto a thermostated cell. Fluorescence emission was observed at right angles to the exciting wavelength using a 5oo-mm Bausch and Lomb monochromator (dispersion 3.3 nm/mm) and detected by an EMI 6256S photomultiplier tube. The signal from the photomultiplier was amplified and fed to the Y-axis input of a Mosely X - Y recorder (Model I35AM), the X-axis of which was coupled to the wavelength drive of the analyzing monochromator. Calibration of both exciting light source and detector system was carried out according to procedures described in the literatureS, 6. A bandwidth of 2 nm was used in the fluorometric determinations, and quantum yields of fluorescence were calculated according to the method of Parker and Rees 7 with standards of known quantum yield (fluoresceine and quinine sulphate). Polarization of fluorescence measurements were performed in the apparatus designed in our laboratory 8. For polarization spectra measurements, the wavelength of excitation was selected by means of a quartz prism monochromator (Schoeffel, QPM 3oS). The bandwidth for excitation was 5 nm in the region 33o-4oo nm according to the calibration chart provided by the manufacturer. An analysis of the various sources of random and systematic errors shows that this apparatus is capable of measuring degree of polarization to an accuracy of I °/o for polarization of fluorescence values greater than o.I. For a complete review of the applications of this technique see ref. 9.
MATERIALS Bovine serum albumin was the Armour crystalline product. H u m a n serum albumin was purchased from Sigma and rabbit serum albumin was obtained from Mann. The enzymes alcohol dehydrogenase, lactate dehydrogenase and glutamate dehydrogenase, lysozyme ribonuclease and trypsin were purchased from Boehringer. The reagents L-kynurenine and DL-kynurenine were obtained from Calbiochem and used without further purification. The organic solvents were of spectroscopic grade (Eastman). Because of the sensitivity of serum albumins to small amounts of detergents, all glassware was soaked in water and rinsed with distilled ethanol before drying. Concentrations of kynurenine were determined by absorbance at 360 nm using the molar extinction coefficient of 515 ° . Protein concentrations were determined b y the method of Lowry et al. 1°.
Biochim. Biophys. Acta, 285 (1972) 91-98
93
L-KYNURENINE: A FLUORESCENT PROBE RESULTS
L-Kynurenine is characterized by a low fluorescence yield (q = o.oo5) when examined in aqueous solutions over a wide range of pH values; but it shows a substantial increase in fluorescence yield when dissolved in hydrogen-bonding solvents of lower dielectric constant than water. As shown in Fig. I, the fluorescence spectrum of L-kynurenine exhibits a clear maximum over the spectral region 460-47 ° rim. Further analysis of the spectroscopic properties reveals that the polarity of the environment, which is correlated with the dielectric constant and refractive index of the medium, affects the fluorescence yield of L-kynurenine. In order to evaluate the effect of polarity on the fluorescence yield, it was decided to employ a mixed solvent system in which the dielectric constant could be varied independently of the viscosity at constant temperature (25 °C). The system chosen consisted of mixtures of dioxane and water since this solvent system makes available an almost I5-fold variation in the dielectric constant without appreciable changes in the viscosity of the solution. The results of the fluorescence measurements as a function of the dielectric constant are illustrated in Fig. 2. It is immediately apparent that an increased fluorescence intensity of L-kynurenine was associated with a decrease in the dielectric constant of the solution.
0.5
' '1
. . . .
I ' ' ' ' 1
'J''
'
I
'
I
'
I
4o
0.4 Z o.3
30wz
~02 /
~o
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0.1 j 300
~
~0
50
so
g _J tu
l0
350
400 450 k (nm)
500 550
20 40 DIELECTRIC
60 80 CONSTANT
6
7
8
9
I0
PH
Fig. I. A b s o r p t i o n and fluorescence spectra of L-kynurenine in the prescence of bovine s e r u m alb u m i n at p H 7.4 ((2)--(2)). A b s o r p t i o n and fluorescence spectra of L-kynurenine in p r o p a n e d i o l w a t e r (95:5, v/v), ( 0 - - 0 ) . The samples of L-kynurenine have the same a b s o r b a n c e at the exciting wavelength, A = o.I, at 36o n m for i-era cuvette. Fig. 2. Left. The variation in the fluorescence intensity emitted at 47 ° n m by L-kynurenine in d i o x a n e - w a t e r m i x t u r e s as a function of the dielectric constant. Right. The variation in the fluorescence intensity emitted at 435 n m b y L-kynurenine complexed to bovine s e r u m a l b u m i n as a function of the p H of the solution. Mixing ratio i mole of ligand : 2 moles of protein. All the samples have the same a b s o r b a n c e at the exciting wavelength (A360 am = o.I).
Interaction of L-kynurenine with serum albumins The effect of bovine serum albumin solutions on the fluorescence of L-kynurenine was examined at several pH values. In these experiments protein solutions of approximately 2 mg/ml were used as solvents ; and in order to maximize the binding of the ligand, the molar ratio of protein to L-kynurenine was of the order of 2:I. The fluorescence spectrum of L-kynurenine complexed to bovine serum albumin was then recorded and compared with the spectrum of free L-kynurenine (Fig. I). It was found that over the pH range from 7 to 9.5, the protein bovine serum albumin promotes an enhancement of the fluorescence emitted by the ligand (Table I). As may be seen from Biochim. Biophys. Acta, 285 (1972) 91-98
94
j . E . CHURCHICH
TABLE I FLUORESCENCE
PROPERTIES
OF
L-KYNURENINE
Sample
Solvent
Absorption
Fluorescence
Q*
p**
L-Kynurenine L-Kynurenine
W a t e r (pH 8.4) Propanediolw a t e r (95:5, v/v) W a t e r (pH 7.4)
360
45 °
0.005
o.o9
37 ° 365
460 435
0.05 0.05
o.40
W a t e r (pH 8.4)
365
435
o.ii
0.40
W a t e r (pH 8.4)
365
435
0-05
0.4°
W a t e r (pH 8.4)
365
435
0.04
o-40
L-Kynurenineb o v i n e s e r u m albumin**" L-Kynurenineb o v i n e s e r u m albumin*** L-Kynurenineh u m a n s e r u m albumin*** L-Kynureninerabbit serum albumin
Q u a n t u m yield of fluorescence. Polarization of fluorescence excited at 360 n m . *** Mixing ratio i mole of ligand : 2 moles of protein. *
**
Fig. 2, the fluorescence intensity of L-kynurenine in the presence of protein is quenched at p H values below 6, while at p H values ranging from 8 to 9, the fluorescence yield reached a m a x i m u m value. This fluctuation in the fluorescence yield of the ligand in the presence of protein at varying p H values was not paralleled by a shift in the band position of the emission spectrum. As shown in Table I, the m a x i m u m of emission of bound L-kynurenine remains invariant over the p H range from 7 to 8.4, while it displays a blue shift of approximately 20 nm when compared to L-kynurenine dissolved in hydrogen-bonding solvents. The fluorescence enhancement that follows the addition of increasing concentrations of L-kynurenine to a fixed concentration of protein was used to determine the affinity constant of the ligand for the macromolecule. Fig. 3 shows how the fluorescence intensity changed with increasing concentrations of L-kynurenine while keeping the protein concentration constant. The fluorescence intensitv of free (F0) and bound (Fro) L-kynurenine as well as the fluorescence observed when both flee and bound ligand are in equilibrium (F) were used to calculate the fraction of ligand bound (Eqn I)
I00
o
50 u_
I
2
3
4
5
6
[L:KYNURENINE] x 10 5 ( M )
Fig. 3- C h a n g e s in fluorescence i n t e n s i t y a t 435 n m on t i t r a t i o n of a fixed c o n c e n t r a t i o n of b o v i n e s e r u m a l b u m i n (2.5" IO -s M) w i t h increasing c o n c e n t r a t i o n s of L - k y n u r e n i n e . R e s u l t s o b t a i n e d a t p H 7.4 ( O - - O ) , p H 7.7 ( A - - ~ ) a n d p H 8. 4 ( 0 - - - 0 ) . T h e w a v e l e n g t h of e x c i t a t i o n was 365 rim.
Biochim. Biophys. Acta, 285 (1972) 91-98
L-KYNURENINE:
A FLUORESCENT PROBE
95
F -- F 0
a
(i)
Fm -- F 0
where Fm is the actual observed fluorescence when all the ligand has been absorbed. Fm was determined directly by adding increasing concentrations of protein to a fixed concentration of L-kynurenine. The average number of ligand molecules bound per mole of protein (~) was calculated for points along the titration curve by means of Eqn 2 : a[Lo3
--
(2)
[P0]
where [Lo] is the total ligand concentration and [Po] total protein concentration. The results were analyzed by Eqn 3 : - -
ILl
=
K~(n
-
~)
(3)
and the binding experiments conducted at three different pH values yield the results summarized in Table II. TABLE
II
BINDING OF L-KYNURENINE TO SERUM ALBUMINS Experiments
were conducted
a t 25 °C.
Sample
Bovine Bovine Bovine Human Rabbit
serum serum serum serum serum
albumin--Lkynurenine albumin-L-kynurenine albumin-L-kynurenine albumin-L-kynurenine albumin-L-kynurenine
pH
Association constant
7.4 7.7 8. 4 8. 4 8. 4
2.5" 2.7' 3' 2.8. 2. 7.
(K~) (M-l) lO5 IO5 lO5 lO 5 lO 5
Binding sites
(n)
I
0.98 I I
o.96
A comparison between the association constants obtained at different pH values, shows that maximum affinity of ligand for the protein bovine serum albumin is observed at pH 8. 4. Moreover the slight increase in affinity constant at alkaline pH is not accompanied by a change in the number of binding sites (n = i). At pH 8.4 the proteins human serum albumin and rabbit serum albumin show maximum affinity for L-kynurenine. The association constants determined by fluorometric titrations are of similar order of magnitude (Table II).
Radiationless energy transfer in the complex The effect of complex formation on the fluorescence spectrum of bovine serum albumin excited at 280 nm is illustrated in Fig. 4. While the fluorescence of the protein is quenched by addition of L-kynurenine, the emission of the bound ligand activated at 280 nm is larger than the emission given by free L-kynurenine dissolved in the solvent mixture propanediol-water (95:5, v/v) and excited at the same wavelength, 280 nm (Fig. 4). Since the absorption band of L-kynurenine overlaps the emission band of the protein, the quenching of protein fluorescence promoted by addition of Lkynurenine can at least partly be explained in terms of radiationless energy transfer from the tryptophyl residues of the protein to the ligand. A necessary condition for Biochim. Biophys. Acta, 2 8 5 (1972) 9 1 - 9 8
96
J. E. CHURCHICH
°5o[ tu 0 ~- 5 0 0 z
t
I
L
450
400
350
300
250
I
450
400
350
300
25O
o 5o
0 5OO
t
A(nm)
Fig. 4- Top. Fluorescence excitation s p e c t r u m of L-kynurenine (3" lO-6 M) in p r o p a n e d i o l - w a t e r (95:5, v/v) ( ~ --A) and in the presence of bovine s e r u m a l b u m i n (4-1o .6 M) at p H 8. 4 (O---Q). The emission m o n o c h r o m a t o r was set at 47 ° n m . B o t t o m . Fluorescence s p e c t r u m of bovine s e r u m a l b u m i n ( 4 . i o - 6 M ) in the absence ( © - - 0 ) and presence ( O - - O ) of L-kynurenine ( 3 - i o *M). The excitation m o n o c h r o m a t o r was set at 280 nm.
this energy transfer mechanism to be operative is extensive overlap between the emission band of the donor and the absorption band of the acceptor. This condition is met by L-kynurenine bound to the protein since the overlap integral (J(~)) yields the value IO-la cm 3. M 1, whenfD (~), the fluorescence spectrum of the donor is normalized to I, in units of quanta on a wavenumber scale, eA (~) is the molar decadic extinction coefficient of the acceptor (L-kynurenine). Substituting J (~) in Forster's n equation : 1
R0 ~ [9 ' I°-2a " /~2 . ~ D " J(g)/n*]"
(4)
one obtains the critical distance of transfer (R 0 = 45 fit) for /52 = 2/3 (orientation factor), ~D = o.48 (quantum yield of tryptophyl residues in the protein) and n 4 = 3.13 (refractive index of water). It becomes evident from these calculations that a radiationless transfer mechanism may be oparative; and that the critical distance of transfer (R 0 -- 45 fit) is compatible with the actual spatial separation between the two tryptophyl residues and the molecule of L-kynurenine.
Polarization of fluorescence The technique of polarization of fluorescence provided valuable information on the rotational mobility of L-kynurenine bound to bovine serum albumin. The polarization of fluorescence of the ligand in tile presence of protein was measured at 25 °C. The results of these measurements, together with the polarization of fluorescence values of free L-kynurenine in water and glycerol are given in Fig. 5. The effect of viscosity on the polarization of fluorescence of L-kynurenine is easily explained in terms of the rotational motion of the chromophore during the interval zM (fluorescence lifetime). The fluorescence is largely depolarized in water (p = 0.09) because the molecules of L-kynurenine assume a nearly random orientation between excitation and emission. Conversely, the polarization of fluorescence is increased in glycerol (p = 0.42 ) because the molecules of L-kynurenine do not change their orientation during the interval TM. In the absence of rotational motion and torsional vibrations, it can be shown that the polarization of fluorescence varies between the limits p = ½, Riochim. 13iophys..4cla, 285 (T972) 9 t 98
L - K Y N U R E N I N E ' A FLUORESCENT PROBE 0.5
I o
I
1
~
=
360
380
97
i
c
o
04
g 03 02 0.1
~
a I
320
340
4.00
Mnm) Fig. 5. Polarization of fluorescence versus w a v e l e n g t h of excitation. L - K y n u r e n i n e in glycerol w a t e r (98:2, v/v) ( O O), L - k y n u r e n i n e in w a t e r a t p H 8. 4 ( A - - A ) a n d L - k y n u r e n i n e in t h e presence of bovine s e r u m a l b u m i n a t p H 8. 4 (I~-~O). Mixing ratio of ligand to p r o t e i n i :2. T h e fluorescence excited over t h e spectral r a n g e 32o-36o n m a n d 360-4oo n m was isolated w i t h Corning CS-3-74 a n d CS-3-72 glass filters, respectively.
when the polarization vectors of absorption and emission are parallels ; and p -½ when the polarization vectors are perpendicular. Although these ideal limits are rarely realized, it should be noted that the polarization values corresponding to the long wavelength absorption band (36o nm) tend to approach the ideal limit p = o.5. This finding is interpreted to mean that the transition moment for the 36o nm absorption band should be most nearly parallel to the transition moment of the emission band. It should be noted that the binding of L-kynurenine to the protein (Fig. 5) causes a uniform increase in the polarization values over the entire range of wavelengths examined. Since the polarized fluorescence of the ligand bound to the protein (p o.4 o) is practically identical to that emitted by L-kynurenine in a viscous solvent (p = o.42), it seems reasonable to conclude that the rotational motion of the ligand is largely suppressed as result on noncovalent interactions with the binding site of the macromolecule. DISCUSSION
The experiments reported above have shown that the stoichiometry and affinity of binding of L-kynurenine for serum albumins can be easily determined by fluorescence spectroscopy. The most salient feature of these studies is the effect of binding on several spectroscopic properties of L-kynurenine. The addition of L-kynurenine to a solution of serum albumin induces in parallel to fluorescence enhancement, a blue shift in the band position of the emission spectrum and a concurrent increase in the polarization of fluorescence emitted by the ligand. The increase in fluorescence of bound Lkynurenine resembles the behavior of some substituted naphthalenesulfonates which are practically nonfluorescent in water, but fluoresce strongly when bound to proteins. While the naphthalene dyes are known to interact with several proteins, the binding of L-kynurenine to serum albumins is quite specific as indicated by the following lines of evidence (a) The serum albumins examined (human, bovine and rabbit, Table II) enhance the fluorescence of L-kynurenine to a greater degree than other proteins studied. The enzymes lactate dehydrogenase, alcohol dehydrogenase, and glutamate dehydrogenase failed to cause any significant change in the spectroscopic properties of the ligand. Similar negative results were obtained with enzymes of low molecular weight such as lysozyme, ribonuclease, and trypsin. (b) The binding of L-kynurenine Biochim. Biophys. Acta, 285 (1972) 91-98
98
J . E . CHURCHICH
appears to occur in a sterospecific manner since the mixing of DL-kynurenine with serum albumins (mixing ratio, I mole of DL-kynurenine :I mole of protein) induces a fluorescence enhancement at 435 nm which is 5o% of the value observed when Lkynurenine is complexed to the serum albumins under similar experimental conditions. Although this experimental evidence is indirect, it should be noted that the binding of L-tryptophan to bovine serum albumin is sterospecific as demonstrated by the equilibrium dialysis studies of McMenamy and Oncley 1~. (c) The fluorescence yield of L-kynurenine complexed to serum albumins is sensitive to small variations in the p H of the solution. The reason for the fluctuations in quantum yield are not certain, but it is possible that subtle conformational changes of the protein are responsible for this behavior. It was shown in this paper that fluorescence enhancement of Lkynurenine is also observed when this compound is dissolved in hydrogen bonding solvents of decreasing dielectric constant. An analysis of the spectroscopic properties of L-kynurenine in hydrogen-bonding solvents reveals that the polarity of the medium affects the wavelength of maximal emission and absorption, producing a batochromic shift of Io nm as the solvent polarity decreases from water to dioxan. This batochromic shift in the fluorescence spectrum is not observed when L-kynurenine interacts with the binding sites of the protein. In marked contrast to the fluorescence of L-kynurenine in hydrogen bonding solvents, the emission spectrum of the ligand complexed to the protein displays a hypsochromic shift of approximately 3o nm when compared to Lkynurenine in water. In order to understand the spectral changes induced by binding, it is important to realize that the blue shift in fluorescence is more pronounced and of opposite direction than the corresponding shift in absorption (Table I). A behavior of this kind strongly suggests a change in solute solvent stabilization in the excited state as compared to the ground state 1~. The fact that the molecule of L-kynurenine, which is firmly bound to the protein, displays a blue shift in the emission spectrum is interpreted to mean that during its excited lifetime the chromophore does not interact with the surrounding solvent. It is therefore proposed that some steric interference by amino acid residues at the binding site prevents the access of water molecules to complexed l.-kynurenine in the singlet excited state. ACKNOWLEDGEMENT
This work was supported by a grant GB 33395 from the National Science Foundation. REFERENCES i 2 3 4 5 6 7 8 9 io Ii 12 13
D. J. R. Laurence, Biochem. f . , 51 (1952 ) 168. G. W e b e r and L. Young, J. Biol. Chem., 239 (1964) 1415. E. Daniel and G. Weber, Biochemistry, 5 (1966) 1893. G. M. E d e l m a n and W. O. McClure, Ace. Chem. Res. I (1968) 65 . W. I-I. Melhuish, J. Phys. Chem., 65 (1961) 229. J. E. Churchich, J. Biol. Chem., 242 (1967) 4414 . C. A. P a r k e r and W. T. Rees, Analyst, 85 (196o) 596. J. E. Churchich, Biochim. Biophys. Aeta, 147 (1967) 32. F. Dorr, Angew. Chem. Int. Ed., 5 (1966) 478 . O. H. Lowry, N. J. Rosebrough, A. L. F a r r and R. J. Randall, J. Biol. Chem., 193 (1951) 265. T. H. Foster, Discuss. Faraday Soc., 27 (1959) 265. R. H. ~'VicMenamy and J. L. Oncley, J. Biol. Chem., 233 (1958) 1436. W. Liptay, in O. Sinanoglu, Modern Quantum Chemistry, Istanbul Lectures, P a r t II, Academic Press, New York, 1965 , p. 173.
Biochim. Biophys. Aeta, 285 (1972) 91-98