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Spectroscopic investigations of interaction of fluorescent nanomarkers of fluorescein family with human serum albumin at different values of pH
Current Applied Physics 11 (2011) 1126e1132
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Spectroscopic investigations of interaction of fluorescent nanomarkers of fluorescein family with human serum albumin at different values of pH I.M. Vlasova*, E.M. Bukharova, A.A. Kuleshova, A.M. Saletsky Physical Department, M.V. Lomonosov Moscow State University, Leninskie Gori, 119991 Moscow, Russia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 May 2010 Received in revised form 6 February 2011 Accepted 8 February 2011 Available online 24 February 2011
This work is dedicated to investigation of interaction of nanomarkers of fluorescein family (initial composition e fluorescein and its halogens-derivatives e erythrosin and eosin) with human serum albumin (HSA) at different values of pH. Were detected differences in polarization of fluorescence of nanomarkers, in parameters of rotational diffusion of nanomarkers, in binding mechanisms of nanomarkers to HSA, in change of secondary structure of HSA at binding of nanomarkers, in effective Stern eVolmer constants of quenching of fluorescence of nanomarkers, which are determinated by value of electronegativity of atoms of nanomarkers. The increase of electronegativity of atoms in nanomarkers leads to increase of polarization of its fluorescence, to decrease of its coefficient of rotational diffusion, to increase of its time of rotational relaxation, to increase of Einstein’s effective radius of nanomarkers, to decrease of effective SterneVolmer constants of quenching of fluorescence of nanomarkers in range from pI of HSA (4.7) to physiological pH (7.4). Ó 2011 Published by Elsevier B.V.
Keywords: Nanomarker Fluorescein Erythrosin Eosin Fluorescent analysis Raman spectroscopy Human serum albumin Binding centers of protein
1. Introduction The analysis of the mechanism of binding of biomolecules with different nanoligands is extremely interesting as from the point of view of a biomedicine and pharmaceuticals, so from the point of view of bionanotechnology: for example, at researches of influence of different pathogenic factors on organisms, at creation of new drugs and their tests etc. The human serum albumin (HSA) is the globular protein of human blood plasma (66.4 kDa, isoelectric point pI 4.7). Unique property of molecule of HSA to bind wide range of organic and inorganic ligands determines one of fundamental functions of this protein e the transport of physiological metabolites. The structural mobility of HSA molecule, provided by unique loop folding of one polypeptide chain of 585 amino acidic residues, ensures the interaction of HSA molecule with ligands [1]. Secondary structure of HSA consists from a-helix segments and segments of random clew: for example, at physiological value of pH about 50e67% of amino acidic residues of HSA are folded in the ahelix. Nowadays the domain model of HSA tertiary structure is
* Corresponding author. Fax: þ7 495 9391489. E-mail address: [email protected] (I.M. Vlasova). 1567-1739/$ e see front matter Ó 2011 Published by Elsevier B.V. doi:10.1016/j.cap.2011.02.004
accepted, according to which the molecule of HSA consists of 3 practically identical domains, each of them in turn consists of 3 loops or subdomains (2 large loops and 1 small loop). The internal zone of each domain consists of hydrophobic amino acid residues, and the external zone of each domain consists of hydrophilic amino acid residues. The domains are placed at some angles to each other, and their mutual relationship is described by model of “heart”. The mechanism of ligand binding to HSA molecule is determined by availability of binding Centers on protein. Six main binding Centers of HSA are singled out [1]: Center I and II e for binding of small organic molecules, Centers III and IV e for binding of long fatty acid chains, Center V e for binding of ligands with free SH-group, Center VI e for binding of ions of metals. Some binding reactions are provided by electrostatic interactions, other e by covalent interactions. The method of fluorescent nanomarkers plays the large role in studying of physical and chemical properties of binding centers of HSA (in particular, in blood plasma) [2e5]. Now mechanisms of interaction of fluorescent nanomarkers with HSA are widely investigated [6e10]. For research of HSA in vitro in blood plasma the anionic at physiological pH nanomarkers are used, such as nanomarkers of fluorescein family e initial composition fluorescein and its halogen-derivatives (brominated derivative eosin and iodinated derivative erythrosin).
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For practical use of nanomarkers of fluorescein family in HSA solutions it is interest to investigate the interactions of nanomarkers with HSA e to define the polarization of fluorescence of nanomarkers in HSA solutions, the parameters of rotational diffusion of nanomarkers in HSA solutions, the effective SterneVolmer constants of quenching of fluorescence of nanomarkers, the binding mechanisms of nanomarkers to HSA and sites on the protein, responsible for this binding, the changes in HSA structure at binding of nanomarkers at various values of pH. In the given work these researches are carried out by spectroscopic methods e Raman spectroscopy and fluorescent analysis. The fluorescent analysis has high sensitivity to changes of environment of nanomarkers at binding with protein [6,10]. The Near-Infrared Raman spectroscopy allows one to investigate the mechanism of binding of these nanomarkers of fluorescein family with proteins, avoiding a fluorescence of nanomarkers [11e13]. 2. Materials and methods 2.1. Fluorescent spectroscopy in investigations of polarization of fluorescence and rotational diffusion of nanomarkers of fluorescein family in HSA solutions at different pH The following buffer solutions were prepared: 0.1 M CH3COOH e KOH (pH 3.0e5.0) and 0.1 M KH2PO4 e 0.1 M NaOH (pH 6.0e8.0). For investigation of polarization of fluorescence of nanomarkers on base of buffer solutions were prepared solutions of nanomarkers (3 mM fluorescein; 30 mM erythrosin; 30 mM eosin) without or with addition of HSA (150 mM) at different values of pH (3.5e8.0). For research of rotational diffusion of nanomarkers by polarized fluorescence method on the base of buffer solutions the following solutions were prepared: 30 mM nanomarker (fluorescein, erythrosin, eosin) without or with HSA (150 mM) and with addition of various concentration of sucrose (10e200 mM) at different values of pH (3.5e8.0). Studies of polarized fluorescence of nanomarkers were made on spectrofluorimeter LS 55 (Perkin Elmer) at room temperature. Fluorescence of nanomarkers in solutions was excited with the following wavelengths: 1) fluorescein e lexcfl ¼ 440 nm; 2) erythrosin e lexcer ¼ 530 nm; 3) eosin e lexceo ¼ 520 nm. During measurements of polarized fluorescence were registered Ik and It e intensity of fluorescence, measured through a polarizer, the electrical axis of which one is directed parallel or perpendicular to polarization of exciting light. The investigation of polarization of fluorescence of nanomarkers in HSA solutions allows one to judge about local environment of nanomarkers at binding to HSA in dependence on value of pH and on electronegativity of atoms of structural formulas of nanomarkers. 2.2. Raman spectroscopy in investigation of interaction of nanomarkers of fluorescein family with HSA and in determination of changes of secondary structure of HSA at this interaction at different pH
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The measurements of the following Raman spectrograms were made: 1. Buffer solutions (pH 3.5e8.0) without protein and without nanomarker. 2. Buffer solutions (pH 3.5e8.0) with addition of nanomarker (5 mN), but without protein. 3. Buffer solutions (pH 3.5e8.0) with addition of HSA (150 mN), but without nanomarker. 4. Buffer solutions (pH 3.5e8.0), containing both nanomarker (5 mN), and HSA (150 mN). Using these measured Raman spectrograms of solutions for each nanomarker the two difference spectra were composed:
D1 ¼ spectrogramsð3Þ spectrogramsð1Þ; D2 ¼ spectrogramsð4Þ spectrogramsð2Þ: Owing to the composition of D1 the contribution of low molecular weight components of buffer solutions and contribution of water are subtracted. The remaining Raman peaks correspond to chemical bonds in native molecules of HSA. Owing to the composition of D2 the contribution of low molecular weight components of buffer solutions and contribution of water are subtracted, the contribution of the nanomarker, not bound to protein, also is subtracted. The remaining Raman peaks correspond to chemical bonds in HSA molecules after binding of nanomarker and also chemical bonds, which have arisen from nanomarker binding to protein. For research of interaction of nanomarkers of fluorescein family with HSA the Raman peaks, corresponding to chemical bonds between nanomarker (fluorescein, eosin, erythrosin) and HSA, were chosen for investigation at various values of pH (3.5e8.0). For studying of conformational rearrangements in secondary structure of HSA at binding of nanomarkers of fluorescein family at different pH (3.5e8.0) the spectral bands of Amide-I and Amide-III are investigated. 2.3. Fluorescent spectroscopy in researches of effective SterneVolmer constants of quenching of fluorescence of nanomarkers in HSA solution at different pH On base of mention above buffer solutions were prepared the solutions of nanomarkers (3 mM fluorescein, 30 mM eosin, 30 mM erythrosin) with addition of different concentrations of HSA (0e150 mM) at different pH (3.5e8.0). Studies of fluorescence of nanomarkers in solutions with different concentration of HSA were made on spectrofluorimeter LS 55 (Perkin Elmer) at room temperature. Fluorescence of nanomarkers in solutions was excited with the following wavelengths: 1) fluorescein e lexcfl ¼ 440 nm; 2) erythrosin e lexcer ¼ 530 nm; 3) eosin e lexceo ¼ 520 nm. 3. Results and discussion
The HSA solutions were obtained by dilution of protein up to concentration 150 mN in mention above buffer solutions. The 5 mN nanomarker (fluorescein, eosin, erythrosin) was added in HSA solutions at different pH (3.5e8.0). The investigations were carried with the help of Raman spectrometer with Fourier-Transformation EQUINOX 55 (Bruker) with FRA-106 with InGaAs detector, wavelength of excitation light was 1064 nm (NdYAG laser). The laser power was 450 mW. The spectral range was 400e3000 cm1, the spectral resolution was 2 cm1. The samples of solutions were placed in the quartz cell at room temperature.
3.1. The polarized fluorescence of nanomarkers of fluorescein family in HSA solutions at different pH For an estimation of influence of HSA on polarization of fluorescence of three nanomarkers of fluorescein family the comparative analysis of degree of polarization of fluorescence (P) of nanomarkers is carried out, both in solutions without protein, and in solutions with HSA, at different values of pH (Fig. 1). The degree of polarization of fluorescence of nanomarkers was calculated by
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excited molecules, P0 e limiting degree of polarization of fluorescence. Varying the viscosity of solutions, and plotting 1/P as ordinate and T/h as abscissa, we get a straight line which cuts on axis of ordinates an interval equal to 1/P0. Tangent of angle between this straight line and axis of abscissa is equal to:
tg4 ¼
1 1 P0 3
ks $ 0; V
this tangent at known value s0 allows one to determine the value of molecular volume
V ¼
ð3 P0 Þ$k$s0 ; 3$P0 $tg 4
and, hence, Einstein’s effective hydrodynamic radius a. Using this data, it is possible to determine the time of rotational relaxation (or disorder as a consequence of thermal diffusion) x of fluorophor:
x ¼ Fig. 1. Degree of polarization of fluorescence of 30 mM fluorescein (1, 2), 30 mM erythrosin (3, 4), 30 mM eosin (5, 6) in solutions without HSA (1, 3, 5) and in solutions of 150 mM HSA (2, 4, 6) at different values of pH.
values of Ik and It in maximum of emission spectrum of nanomarker fluorescence. It is shown (Fig. 1) that the value of P of all three nanomarkers of fluorescein family in solutions with HSA is greater than in solutions without HSA. As the solutions of nanomarkers of the given concentration are diluted solutions, in absence of non-radiation transfer of energy between molecules of nanomarkers the main reason of depolarization of fluorescence is the rotational diffusion of molecules of nanomarkers. In solutions with HSA the rotational diffusion of nanomarkers molecules decreases owing to the binding to protein, what leads to increase of P. Both in solutions without protein, and in HSA solutions, P of fluorescein (Fig. 1) non-linearly depends on pH with maximum at pH 6.0. Dependence of P of fluorescein on pH is caused by changes of rotational diffusion of molecules of fluorescein at variation of pH. Both in solutions without protein, and in HSA solutions, P of erythrosin (Fig. 1) and eosin (Fig. 1) does not depend on pH (in range 3.5 pH 8.0), that is explained by independence of rotational diffusion of molecules of erythrosin and eosin on variation of pH, related with electronegativity of the atoms in structural formulas of nanomarkers. Qualitatively the behavior P of erythrosin repeats the behavior P of eosin, and quantitatively the value of P of erythrosin takes intermediate position between P of fluorescein and eosin. 3.2. Rotational diffusion of nanomarkers of fluorescein family in HSA solutions at different pH The estimation of parameters of rotational diffusion of fluorophors is large application of method of polarized fluorescence. In the theory of rotational depolarization there is a following formula [14]:
1 1 1 1 kT s0 $ þ ; ¼ Vh P P0 P0 3 where T e absolute temperature, h e viscosity of solution, V e volume, k e constant of Boltzmann, s0 e average time of life of
Vh ; Tk
where V e molecular volume. For fluorophors it is possible to determine the coefficient of rotational diffusion Drot [14]:
Drot ¼
kT : 6hV
In the given work by a variation of viscosity of solutions by addition of various concentration of sucrose the time of rotational relaxation, the coefficient of rotational diffusion and the Einstein’s effective radius of nanomarkers of fluorescein family were determined, both in HSA solutions, and in solutions without protein, at different values of pH. At all values of pH in solutions with HSA the coefficient of rotational diffusion Drot of nanomarkers of fluorescein family is smaller than in solutions without HSA (Fig. 2), that reflects a binding of nanomarkers to protein. Both in HSA solutions, and in solutions without HSA (Fig. 2), Drot of fluorescein non-linear depends on pH with a minimum at pH 6.0, and Drot of eosin and erythrosin does not depend on pH (at 3.5 pH 8.0). At presence of rather weak electronegative atoms of Hydrogen in structural formula of fluorescein and, hence, at small interaction with polar environment of solutions the strong dependences on pH of processes of molecular association of fluorescein and of efficiency of its binding to protein [6,7,10] influence greatly on rotational diffusion of nanomarker that results in dependence of rotational diffusion of nanomarker on pH and in dependence of polarization of fluorescence of fluorescein on pH. Therefore at pH 6.0, at which the maximum binding of nanomarker to HSA and the maximum association of nanomarker molecules occur, the value of Drot of fluorescein has the lowest value, both in HSA solutions, and in solutions without HSA. Despite of processes of molecular association and efficiency of binding to protein, depending on pH and having maximum at pH, smaller 5.0 [6,7,10], the value of Drot of erythrosin and eosin does not depend on pH. At presence of strongly electronegative atoms (Bromide and Iodine) in structural formulas of eosin and erythrosin the strong interaction of nanomarkers with polar environment takes place, that results in independence of rotational diffusion of nanomarker on its molecular association and efficiency of its binding to protein, depending on pH, and, therefore, to independence of rotational diffusion of nanomarkers on pH and to
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Fig. 3. Einstein’s effective radius a of fluorescein (1, 2), erythrosin (3, 4), eosin (5, 6) in solutions without HSA (1, 3, 5) and in solutions with HSA (2, 4, 6) at different values of pH.
Fig. 2. Dependences of coefficient of rotational diffusion and time of rotational relaxation (inserted figure) of fluorescein (1, 2), erythrosin (3, 4), eosin (5, 6) in the solutions without HSA (1, 3, 5) and in solutions with HSA (2, 4, 6) on pH.
in solutions with HSA, radius a of eosin and erythrosin practically does not depend on pH in a range pH 3.5e8.0. 3.3. Binding of nanomarkers of fluorescein family to HSA at different pH
independence of polarization of fluorescence of these nanomarkers on pH. In solutions without HSA the time of rotational relaxation x (Fig. 2) of nanomarkers of fluorescein family, describing rotational mobility and corresponding to time of disorder of fluorophor owing to thermal diffusion, is essentially smaller, than in HSA solutions at all investigated values of pH (3.5e8.0). Nanomarkers of fluorescein family, not bound to protein, have small x, smaller then times of life of their fluorescence (s0 of fluorescein e 3.5 ns, s0 of erythrosin e 3.2 ns, s0 of eosin e 2.9 ns) that leads to weak polarization of fluorescence of nanomarkers in solutions without protein. Nanomarkers of fluorescein family, bound to HSA, have x, greater then times of life of their fluorescence that leads to increase in polarization of fluorescence of nanomarkers in solutions with HSA in comparison with solutions without HSA. Also in this work the values of Einstein’s effective radius a of nanomarkers of fluorescein family (Fig. 3), both in HSA solutions, and in solutions without HSA, are determinated at various values of pH. In solutions without HSA the value of a of nanomarker represents the real size of molecule of nanomarker, and in solutions with HSA the value of a is not the real size and represents the effective size which determinates percent of nanomarker bound to HSA from its total concentration in solution. The increase of Einstein’s effective radius a of nanomarkers of fluorescein family in HSA solutions in comparison with solutions without HSA (Fig. 3) speaks about binding of nanomarkers to protein molecules, but there is also some amount of nanomarkers, not bound to protein (radius of HSA is equal to 4.0e4.5 nm). It is seen (Fig. 3) that, both in solutions without HSA, and in solutions with HSA, radius a of fluorescein non-linear depends on pH with a maximum at pH 6.0. Both in solutions without HSA, and
In Table 1 those ranges of spectra are shown where the Raman peaks, corresponding to chemical bonds of nanomarkers of fluorescein family to HSA at different pH are registered. From the data of Table 1, it is seen, that at binding of these three nanomarkers to HSA the Raman peaks in identical ranges of spectra are registered. In Table 1 the decryption of Raman peaks, corresponding to binding of nanomarkers of fluorescein family with HSA, is shown. As it follows from the decryption of obtained Raman spectrograms, the residues of only polar amino acids of HSA participate in binding of these nanomarkers: the residues of threonine (Thr), serine (Ser), lysine (Lys), arginine (Arg), glutamine (Gln) and glutaminic acid (Glu). The fact that the identical amino acidic residues of HSA participate in binding of each of three nanomarkers is possible to explain by the similar chemical structure of these nanomarkers and their belonging to one homologous family. The mechanism of binding of ligands to HSA molecule is determined by availability of binding Centers on protein. To binding Centers of small organic molecules, such as fluorescein, eosin and erythrosin, attribute Center I and II. Center I is in the domain II of HSA. Lysine (195, 199), threonine (125, 243), glutaminic acid (167, 244), glutamine (196), serine (202), arginine (197) are in Center I. Center II is in the domain III of albumin molecule. Tyrosine (411) and lying near leucine (413) and valine (415, 418) are in Center II. On obtained Raman spectra one can conclude, that nanomarkers of fluorescein family bind to Center I of HSA. Though the same amino acidic residues, which are included in a structure of binding Center I of HSA, participate in binding of these three nanomarkers, there are differences in binding of these nanomarkers with protein in dependence on pH of solutions (Table 1). The dependence of number of types of chemical bonds between the nanomarker and HSA on pH for fluorescein has a nonlinear behavior with a maximum at pH 5.0e6.0: at these values of
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Table 1 The decryption of Raman peaks, corresponding to binding of nanomarkers of fluorescein family (1 e fluorescein, 2 e erythrosin, 3 e eosin) to HSA at different values of pH. Range, cm1
Decryption
Mechanism of binding
Range of pH of action of type of binding 1
2
3
1105e1115
Binding of nanomarker through its hydroxyl group with threonine (Thr).
3.5e8.0
3.5e8.0
3.5e8.0
1150e1160
Binding of nanomarker through its hydroxyl group with serine (Ser).
5.0e7.0
3.5e7.0
3.5e7.0
1380e1405
Binding of nanomarker through its carboxyl group with lysine (Lys).
3.5e8.0
3.5e8.0
3.5e8.0
1510e1520
Binding of nanomarker through its hydroxyl group with glutamine (Gln).
5.0e7.0
3.5e5.0
3.5e5.0
1530e1545
Binding of nanomarker through its hydroxyl group with arginine (Arg).
6.0
3.5e6.0
3.5e6.0
1550e1565
Binding of nanomarker through its hydroxyl group with lysine (Lys).
5.0e8.0
3.5e8.0
3.5e8.0
1580e1590
Binding of nanomarker through its carboxyl group with threonine (Thr).
4.0e8.0
3.5
3.5e4.0
1600e1610
Binding of nanomarker through its carboxyl group with serine (Ser).
5.0e8.0
3.5e5.0
3.5e4.0
1695e1720
Binding of nanomarker through its hydroxyl group with glutaminic acid (Glu).
5.0e6.0
3.5e8.0
3.5e8.0
1730e1755
Binding of nanomarker through its carboxyl group with glutaminic acid (Glu).
3.5e8.0
3.5e8.0
3.5e8.0
pH there is a greatest number of types of chemical bonds. For halogen-derivatives of fluorescein the dependence of number of types of chemical bonds between the nanomarker and HSA on pH has a linearly decreasing behavior with an increase of pH. The greatest number of types of chemical bonds between eosin and HSA takes place at pH 3.5e4.0. The greatest number of types of chemical bonds between erythrosin and HSA takes place at pH 3.5e5.0. The differences in dependences of binding of nanomarkers to protein on pH are explained by difference in lateral radicals of structural formulas of these nanomarkers: the fluorescein has atoms of Hydrogen (H) as lateral radicals, and in its halogenderivatives these atoms of hydrogen are changed to halogens (Bromide Br e for eosin, Iodine I e for erythrosin). The increase of electronegativity goes in the following direction: Hydrogen (for fluorescein) e Iodine (for erythrosin) e Bromide (for eosin). The availability of more electronegative atom in a molecule of the nanomarker results in strong decreasing of values of pK(COOH) and pK(OH) of these nanomarkers. The found differences in binding of these three nanomarkers to HSA are determined by value of electronegativity of atoms of lateral radicals in structural formulas of nanomarkers and, therefore, by value of pK of their ionized groups. Let’s consider the ionized conditions of fluorescein depending on pH. At values of pH, smaller 5.5, the molecules of fluorescein are in gently positively charged form. At values of pH 5.5e6.8 molecules of fluorescein are electrically neutral. Value of pK(OH) of fluorescein e 6.8. At pH 6.8e8.0 molecules of fluorescein are gently
negatively charged and are in the form of monoanions. Value of pK(COOH) of fluorescein e 8.0. At pH, larger 8.0, the molecules of fluorescein are strongly negatively charged and are in the form of dianions. The maximum binding of fluorescein to HSA takes place at pH 5.0e6.0, i.e. when fluorescein either is gently positively charged, or is neutral. Let’s consider the ionized conditions of erythrosin depending on pH. The erythrosin has the following values of pK of ionized groups: pK(OH) e 3.6 and pK(COOH) e 5.5. In range of values of pH, smaller 3.6, the molecules of erythrosin are electrically neutral. In range of pH 3.6e5.5 molecules of erythrosin are gently negatively charged (monoanions). In range of values of pH, larger 5.5, the molecules of erythrosin are strongly negatively charged (dianions). The maximum binding of erythrosin to HSA takes place at pH 3.5e5.0, i.e. in that range, where the erythrosin is gently negatively charged (pH 3.6e5.0) or is electrically neutral (3.5e3.6), and HSA at these pH is positively charged as a whole. Let’s consider the ionized conditions of eosin depending on pH. At values of pH, smaller 3.0, the molecules of eosin are electrically neutral. Value of pK(OH) of eosin is approximately 3.0. At values of pH, larger 3.0, but smaller 5.0, molecule of eosin is gently negatively charged (monoanion). Value of pK(COOH) of eosin is approximately 5.0. At values of pH, larger 5.0, the eosin is strongly negatively charged (dianion). The maximum binding of eosin to HSA takes place at pH 3.5e4.0, i.e. in that range, where the eosin is gently negatively charged and is in the form of monoanion (HSA at these values of pH is positively charged as a whole).
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3.4. Secondary structure of HSA at interaction of protein with nanomarkers of fluorescein family at different pH The Raman spectral bands of Amide-I and Amide-III, used for investigation of secondary structure of proteins, have the following values: 1) Amide-I e 1645e1655 cm1 (a-helix), 1670e1680 cm1 (b-structure), 1660e1670 cm1 (random clew); 2) Amide-III e 1270e1280 cm1 (a-helix), 1230e1240 cm1 (b-structure), 1240e1260 cm1 (random clew). The secondary structure of native (intact, i.e. before binding of nanomarkers) HSA consists from a-helix segments and segments of random clew. In spectral bands of Amide-I and Amide-III the Raman peaks, which are responsible to a-helix and random clew segments of HSA before and after binding of one of three nanomarkers, are investigated. On base of the calculated areas under peaks in spectral bands of Amide-I and Amide-III the percentage of a-helix in secondary structure of native molecules of HSA (before binding of nanomarkers) and also percentage of a-helix in secondary structure of HSA after binding of nanomarkers at different values of pH were determinated (Fig. 4). In the absence of nanomarkers in solutions the percentage of ahelix in secondary structure of native HSA molecules almost constantly and does not depend on pH in the range 3.5e8.0, when there is no acid-induced denaturation of protein. For HSA with bound to it fluorescein the non-linear dependence of percentage of a-helix in secondary structure of protein on pH with minimum at pH 6.0 is vividly seen. At binding of halogensderivatives of fluorescein (eosin and erythrosin) by HSA the percentage of a-helix in secondary structure of protein linearly increases with increasing of pH. The found laws in percentage of a-helix in secondary structure of HSA at binding of nanomarkers of fluorescein family are explained by dependences of this binding on pH, determinated by electronegativity of atoms in structural formulas of nanomarkers and values of pK of its ionized groups. The greatest changes in secondary structure of HSA, consisting in decreasing of a-helix sites, and occurring at binding of nanomarkers of fluorescein family, take place at those values of pH when a maximal binding of these nanomarkers to HSA occurs.
Fig. 4. The percentage of a-helix (%) in secondary structure of HSA at different values of pH before and after binding of nanomarkers of fluorescein family: 1 e before binding, 2 e after binding of fluorescein, 3 e after binding of erythrosin, 4 e after binding of eosin.
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Table 2 Effective SterneVolmer constants of fluorescence quenching (K . M1) of nanomarkers of fluorescein family in HSA solutions at different values of pH. pH
Fluorescein-HSA
Erythrosin-HSA
Eosin-HSA
3.5 4.0 5.0 6.0 7.0 8.0
850 3610 24390 24790 6170 4970
24950 20010 16140 6510 5210 2520
9580 7850 5180 2570 840 340
3.5. Effective SterneVolmer constants of fluorescence quenching of nanomarkers of fluorescein family in HSA solutions The effective constants of quenching of fluorescence of nanomarkers of fluorescein family in HSA solutions are determinated by SterneVolmer equation [14,15]:
F0 ¼ 1 þ K$½Q ; F where F0 e intensity of fluorescence of nanomarker in absence of quencher (HSA), F e intensity of fluorescence of nanomarker in presence of quencher (HSA), [Q] e concentration of quencher (HSA), K e effective SterneVolmer constants of fluorescence quenching of nanomarkers (M1). The slope of plot of dependence (F0/F) 1 on [Q] is determinated by type of interaction and by amount of binding sites. From our Raman investigation we concluded that nanomarkers of fluorescein family are bound to Center I of HSA, consequently there is one binding site on HSA for these nanomarkers. The plot of dependence (F0/F) 1 on [Q] represents a straight line with tangent of angle of slope K in case of one mechanism of binding of nanomarker to protein, but if there is several mechanisms of binding of nanomarker to protein some deviations from linearity are seen. On the obtained spectra of fluorescence of each nanomarker in solutions with different concentration of HSA at different values of pH the dependences (F0/F) 1 on [Q] are constructed. It is shown, that for each nanomarker the practically linear dependence (F0/F) 1 on [Q] is observed at pH 6.0, that speaks about one (probably through chemical bonds) covalent mechanism of binding of nanomarker to HSA. While at pH <6.0 the deviations from linearity of dependence (F0/F) 1 on [Q] are observed, that indicates two mechanisms of binding (covalent and ionic) of each nanomarker to HSA. For the account of both mechanisms (covalent and ionic) of interactions of nanomarkers with HSA and for comparison of nanomarkers among themselves the effective SterneVolmer constants of fluorescence quenching of nanomarkers in HSA solutions at different pH are determinated by linear approximation (Table 2). For fluorescein the non-linear dependence of effective SterneVolmer constant on pH with maximum approximately at pH 6.0 is characteristic. Halogen-derivatives of fluorescein are characterized by almost linear decrease of effective SterneVolmer constant with increasing of pH. It is accepted that the maximum SterneVolmer constant allowed for the dynamic quenching of biomolecules is 100 M1 [16]. From obtained SterneVolmer constants (Table 2) it is concluded that the quenching is static process occurring in the HSA e nanomarker complex. From the comparison of values of effective SterneVolmer constants in the range of pH from pI of HSA (4.7) and up to physiological value of pH (7.4) it is seen (Table 2), that effective constant has its greatest value in the case of fluorescein, the lowest value e in the case of eosin, and the intermediate value e in the case of erythrosin. The found differences in the effective SterneVolmer constants of fluorescence quenching of nanomarkers are determined by value of an electronegativity of atoms in structural
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formulas of nanomarkers. The presence of more electronegative atoms in nanomarker results in decrease of effective SterneVolmer constant in range from pI of HSA (4.7) and up to pH 7.4. 4. Conclusion The comparative analysis of interaction of nanomarkers of fluorescein family with HSA at different values of pH was carried out by spectroscopic methods. The conclusion is made that the found differences in polarization of fluorescence of nanomarkers, in parameters of rotational diffusion of nanomarkers, in binding mechanisms of nanomarkers to HSA, in changes of secondary structure of HSA at binding of nanomarkers, in effective SterneVolmer constants of fluorescence quenching of nanomarkers in HSA solutions are determined by value of an electronegativity of atoms of lateral radicals in structural formulas of nanomarkers and values of pK of their ionized groups. The increase of electronegativity is observed in a following direction: Hydrogen (for fluorescein) e Iodine (for erythrosin) e Bromide (for eosin). The presence of more electronegative atom in molecule of nanomarker results in strong decrease of values of pK(COOH) and pK(OH) of these nanomarkers. The increase in electronegativity of atoms of lateral radicals in structural formulas of nanomarkers leads to increase of degree of polarization of their fluorescence, to decrease of their coefficient of rotational diffusion, to increase of their time of rotational relaxation, to increase of Einstein’s effective radius, to decrease of effective SterneVolmer constants of fluorescence quenching of nanomarkers in HSA solutions in range from pI of HSA (4.7) and up to pH 7.4. It is shown that depending on pH the mechanism of binding of fluorescein with HSA differs from character of binding of fluorescein halogens-derivatives with HSA. It is registered that the greatest changes in secondary structure of HSA, occurring at binding of nanomarkers of fluorescein family, take place at those values of pH when a maximal binding of these nanomarkers to HSA occurs. The insertion of atoms with different electronegativity in structural formulas of nanomarkers of fluorescein family allows
one to receive the nanomarkers with different optical and molecular characteristics and different character of binding to HSA. Chemical and electrostatic interactions play the leading role in interaction between nanomarkers and HSA. The data about binding of fluorescent nanomarkers to HSA and about properties of these nanomarkers in HSA solutions allow one to receive the information about mechanisms of interaction of fluorescent nanomarkers with HSA, what can be useful at research of structure and properties of binding Centers (drug-binding Centers) of HSA, and this is of great importance in medical investigations of binding of drugs to HSA. Acknowledgements The work is supported by RFBR (N 07-02-00459). References [1] T. Peters, All About Albumin. Academic Press, New York, 1996. [2] J.R. Lakowicz, Principles of Fluorescence Spectroscopy. Plenum, New York, 1999. [3] S. Pelet, M. Gratzel, J.-E. Moser, Journal of Physical Chemistry B 107 (2003) 3215e3224. [4] L. Birla, B. Prieto, T. Noguel, et al., Revue Roumaine de Chimie 52 (2007) 639e646. [5] A.A. Waheed, K. Sridhar, P.D. Gupta, Analytical Biochemistry 287 (2000) 73e79. [6] I.M. Vlasova, A.M. Saletsky, Current Applied Physics 9 (2009) 1027e1031. [7] E.M. Buharova, I.M. Vlasova, A.M. Saletsky, Journal of Applied Spectroscopy 75 (2008) 785e790. [8] I.M. Vlasova, D.V. Polyansky, A.M. Saletsky, Laser Physics Letters 4 (2007) 390e394. [9] I.M. Vlasova, A.M. Saletsky, Laser Physics Letters 5 (2008) 384e389. [10] I.M. Vlasova, A.M. Saletsky, Journal Molecular Structure 936 (2009) 220e227. [11] S. Pilotto, M.T.T. Pacheco, L. Silveira, A. Balbin Villaverde, R.A. Zangaro, Lasers in Medical Science 16 (2001) 2e9. [12] H. Sato, H. Chiba, H. Tashiro, Y. Ozaki, Journal of Biomedical Optics 6 (2001) 366e370. [13] A.J. Berger, T.-W. Koo, I. Itzkan, G. Horowitz, M.S. Feld, Applied Optics 38 (1999) 2916e2926. [14] W. Schmidt, Optical Spectroscopy in Chemistry and Life Sciences. WILEY-VCN Verlag GmbH & Co. KgaA, 2005. [15] A.G. Marshall, Biophysical Chemistry. John Wiley & Sons, New York, 1978. [16] W. An, Y. Jiao, C. Dong, C. Yang, Y. Inoue, S. Shuang, Dyes and Pigments 81 (2009). doi:10.1016/j.dyepig.2008.08.004.
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Current Applied Physics journal homepage: www.elsevier.com/locate/cap
Spectroscopic investigations of interaction of fluorescent nanomarkers of fluorescein family with human serum albumin at different values of pH I.M. Vlasova*, E.M. Bukharova, A.A. Kuleshova, A.M. Saletsky Physical Department, M.V. Lomonosov Moscow State University, Leninskie Gori, 119991 Moscow, Russia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 May 2010 Received in revised form 6 February 2011 Accepted 8 February 2011 Available online 24 February 2011
This work is dedicated to investigation of interaction of nanomarkers of fluorescein family (initial composition e fluorescein and its halogens-derivatives e erythrosin and eosin) with human serum albumin (HSA) at different values of pH. Were detected differences in polarization of fluorescence of nanomarkers, in parameters of rotational diffusion of nanomarkers, in binding mechanisms of nanomarkers to HSA, in change of secondary structure of HSA at binding of nanomarkers, in effective Stern eVolmer constants of quenching of fluorescence of nanomarkers, which are determinated by value of electronegativity of atoms of nanomarkers. The increase of electronegativity of atoms in nanomarkers leads to increase of polarization of its fluorescence, to decrease of its coefficient of rotational diffusion, to increase of its time of rotational relaxation, to increase of Einstein’s effective radius of nanomarkers, to decrease of effective SterneVolmer constants of quenching of fluorescence of nanomarkers in range from pI of HSA (4.7) to physiological pH (7.4). Ó 2011 Published by Elsevier B.V.
Keywords: Nanomarker Fluorescein Erythrosin Eosin Fluorescent analysis Raman spectroscopy Human serum albumin Binding centers of protein
1. Introduction The analysis of the mechanism of binding of biomolecules with different nanoligands is extremely interesting as from the point of view of a biomedicine and pharmaceuticals, so from the point of view of bionanotechnology: for example, at researches of influence of different pathogenic factors on organisms, at creation of new drugs and their tests etc. The human serum albumin (HSA) is the globular protein of human blood plasma (66.4 kDa, isoelectric point pI 4.7). Unique property of molecule of HSA to bind wide range of organic and inorganic ligands determines one of fundamental functions of this protein e the transport of physiological metabolites. The structural mobility of HSA molecule, provided by unique loop folding of one polypeptide chain of 585 amino acidic residues, ensures the interaction of HSA molecule with ligands [1]. Secondary structure of HSA consists from a-helix segments and segments of random clew: for example, at physiological value of pH about 50e67% of amino acidic residues of HSA are folded in the ahelix. Nowadays the domain model of HSA tertiary structure is
* Corresponding author. Fax: þ7 495 9391489. E-mail address: [email protected] (I.M. Vlasova). 1567-1739/$ e see front matter Ó 2011 Published by Elsevier B.V. doi:10.1016/j.cap.2011.02.004
accepted, according to which the molecule of HSA consists of 3 practically identical domains, each of them in turn consists of 3 loops or subdomains (2 large loops and 1 small loop). The internal zone of each domain consists of hydrophobic amino acid residues, and the external zone of each domain consists of hydrophilic amino acid residues. The domains are placed at some angles to each other, and their mutual relationship is described by model of “heart”. The mechanism of ligand binding to HSA molecule is determined by availability of binding Centers on protein. Six main binding Centers of HSA are singled out [1]: Center I and II e for binding of small organic molecules, Centers III and IV e for binding of long fatty acid chains, Center V e for binding of ligands with free SH-group, Center VI e for binding of ions of metals. Some binding reactions are provided by electrostatic interactions, other e by covalent interactions. The method of fluorescent nanomarkers plays the large role in studying of physical and chemical properties of binding centers of HSA (in particular, in blood plasma) [2e5]. Now mechanisms of interaction of fluorescent nanomarkers with HSA are widely investigated [6e10]. For research of HSA in vitro in blood plasma the anionic at physiological pH nanomarkers are used, such as nanomarkers of fluorescein family e initial composition fluorescein and its halogen-derivatives (brominated derivative eosin and iodinated derivative erythrosin).
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For practical use of nanomarkers of fluorescein family in HSA solutions it is interest to investigate the interactions of nanomarkers with HSA e to define the polarization of fluorescence of nanomarkers in HSA solutions, the parameters of rotational diffusion of nanomarkers in HSA solutions, the effective SterneVolmer constants of quenching of fluorescence of nanomarkers, the binding mechanisms of nanomarkers to HSA and sites on the protein, responsible for this binding, the changes in HSA structure at binding of nanomarkers at various values of pH. In the given work these researches are carried out by spectroscopic methods e Raman spectroscopy and fluorescent analysis. The fluorescent analysis has high sensitivity to changes of environment of nanomarkers at binding with protein [6,10]. The Near-Infrared Raman spectroscopy allows one to investigate the mechanism of binding of these nanomarkers of fluorescein family with proteins, avoiding a fluorescence of nanomarkers [11e13]. 2. Materials and methods 2.1. Fluorescent spectroscopy in investigations of polarization of fluorescence and rotational diffusion of nanomarkers of fluorescein family in HSA solutions at different pH The following buffer solutions were prepared: 0.1 M CH3COOH e KOH (pH 3.0e5.0) and 0.1 M KH2PO4 e 0.1 M NaOH (pH 6.0e8.0). For investigation of polarization of fluorescence of nanomarkers on base of buffer solutions were prepared solutions of nanomarkers (3 mM fluorescein; 30 mM erythrosin; 30 mM eosin) without or with addition of HSA (150 mM) at different values of pH (3.5e8.0). For research of rotational diffusion of nanomarkers by polarized fluorescence method on the base of buffer solutions the following solutions were prepared: 30 mM nanomarker (fluorescein, erythrosin, eosin) without or with HSA (150 mM) and with addition of various concentration of sucrose (10e200 mM) at different values of pH (3.5e8.0). Studies of polarized fluorescence of nanomarkers were made on spectrofluorimeter LS 55 (Perkin Elmer) at room temperature. Fluorescence of nanomarkers in solutions was excited with the following wavelengths: 1) fluorescein e lexcfl ¼ 440 nm; 2) erythrosin e lexcer ¼ 530 nm; 3) eosin e lexceo ¼ 520 nm. During measurements of polarized fluorescence were registered Ik and It e intensity of fluorescence, measured through a polarizer, the electrical axis of which one is directed parallel or perpendicular to polarization of exciting light. The investigation of polarization of fluorescence of nanomarkers in HSA solutions allows one to judge about local environment of nanomarkers at binding to HSA in dependence on value of pH and on electronegativity of atoms of structural formulas of nanomarkers. 2.2. Raman spectroscopy in investigation of interaction of nanomarkers of fluorescein family with HSA and in determination of changes of secondary structure of HSA at this interaction at different pH
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The measurements of the following Raman spectrograms were made: 1. Buffer solutions (pH 3.5e8.0) without protein and without nanomarker. 2. Buffer solutions (pH 3.5e8.0) with addition of nanomarker (5 mN), but without protein. 3. Buffer solutions (pH 3.5e8.0) with addition of HSA (150 mN), but without nanomarker. 4. Buffer solutions (pH 3.5e8.0), containing both nanomarker (5 mN), and HSA (150 mN). Using these measured Raman spectrograms of solutions for each nanomarker the two difference spectra were composed:
D1 ¼ spectrogramsð3Þ spectrogramsð1Þ; D2 ¼ spectrogramsð4Þ spectrogramsð2Þ: Owing to the composition of D1 the contribution of low molecular weight components of buffer solutions and contribution of water are subtracted. The remaining Raman peaks correspond to chemical bonds in native molecules of HSA. Owing to the composition of D2 the contribution of low molecular weight components of buffer solutions and contribution of water are subtracted, the contribution of the nanomarker, not bound to protein, also is subtracted. The remaining Raman peaks correspond to chemical bonds in HSA molecules after binding of nanomarker and also chemical bonds, which have arisen from nanomarker binding to protein. For research of interaction of nanomarkers of fluorescein family with HSA the Raman peaks, corresponding to chemical bonds between nanomarker (fluorescein, eosin, erythrosin) and HSA, were chosen for investigation at various values of pH (3.5e8.0). For studying of conformational rearrangements in secondary structure of HSA at binding of nanomarkers of fluorescein family at different pH (3.5e8.0) the spectral bands of Amide-I and Amide-III are investigated. 2.3. Fluorescent spectroscopy in researches of effective SterneVolmer constants of quenching of fluorescence of nanomarkers in HSA solution at different pH On base of mention above buffer solutions were prepared the solutions of nanomarkers (3 mM fluorescein, 30 mM eosin, 30 mM erythrosin) with addition of different concentrations of HSA (0e150 mM) at different pH (3.5e8.0). Studies of fluorescence of nanomarkers in solutions with different concentration of HSA were made on spectrofluorimeter LS 55 (Perkin Elmer) at room temperature. Fluorescence of nanomarkers in solutions was excited with the following wavelengths: 1) fluorescein e lexcfl ¼ 440 nm; 2) erythrosin e lexcer ¼ 530 nm; 3) eosin e lexceo ¼ 520 nm. 3. Results and discussion
The HSA solutions were obtained by dilution of protein up to concentration 150 mN in mention above buffer solutions. The 5 mN nanomarker (fluorescein, eosin, erythrosin) was added in HSA solutions at different pH (3.5e8.0). The investigations were carried with the help of Raman spectrometer with Fourier-Transformation EQUINOX 55 (Bruker) with FRA-106 with InGaAs detector, wavelength of excitation light was 1064 nm (NdYAG laser). The laser power was 450 mW. The spectral range was 400e3000 cm1, the spectral resolution was 2 cm1. The samples of solutions were placed in the quartz cell at room temperature.
3.1. The polarized fluorescence of nanomarkers of fluorescein family in HSA solutions at different pH For an estimation of influence of HSA on polarization of fluorescence of three nanomarkers of fluorescein family the comparative analysis of degree of polarization of fluorescence (P) of nanomarkers is carried out, both in solutions without protein, and in solutions with HSA, at different values of pH (Fig. 1). The degree of polarization of fluorescence of nanomarkers was calculated by
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excited molecules, P0 e limiting degree of polarization of fluorescence. Varying the viscosity of solutions, and plotting 1/P as ordinate and T/h as abscissa, we get a straight line which cuts on axis of ordinates an interval equal to 1/P0. Tangent of angle between this straight line and axis of abscissa is equal to:
tg4 ¼
1 1 P0 3
ks $ 0; V
this tangent at known value s0 allows one to determine the value of molecular volume
V ¼
ð3 P0 Þ$k$s0 ; 3$P0 $tg 4
and, hence, Einstein’s effective hydrodynamic radius a. Using this data, it is possible to determine the time of rotational relaxation (or disorder as a consequence of thermal diffusion) x of fluorophor:
x ¼ Fig. 1. Degree of polarization of fluorescence of 30 mM fluorescein (1, 2), 30 mM erythrosin (3, 4), 30 mM eosin (5, 6) in solutions without HSA (1, 3, 5) and in solutions of 150 mM HSA (2, 4, 6) at different values of pH.
values of Ik and It in maximum of emission spectrum of nanomarker fluorescence. It is shown (Fig. 1) that the value of P of all three nanomarkers of fluorescein family in solutions with HSA is greater than in solutions without HSA. As the solutions of nanomarkers of the given concentration are diluted solutions, in absence of non-radiation transfer of energy between molecules of nanomarkers the main reason of depolarization of fluorescence is the rotational diffusion of molecules of nanomarkers. In solutions with HSA the rotational diffusion of nanomarkers molecules decreases owing to the binding to protein, what leads to increase of P. Both in solutions without protein, and in HSA solutions, P of fluorescein (Fig. 1) non-linearly depends on pH with maximum at pH 6.0. Dependence of P of fluorescein on pH is caused by changes of rotational diffusion of molecules of fluorescein at variation of pH. Both in solutions without protein, and in HSA solutions, P of erythrosin (Fig. 1) and eosin (Fig. 1) does not depend on pH (in range 3.5 pH 8.0), that is explained by independence of rotational diffusion of molecules of erythrosin and eosin on variation of pH, related with electronegativity of the atoms in structural formulas of nanomarkers. Qualitatively the behavior P of erythrosin repeats the behavior P of eosin, and quantitatively the value of P of erythrosin takes intermediate position between P of fluorescein and eosin. 3.2. Rotational diffusion of nanomarkers of fluorescein family in HSA solutions at different pH The estimation of parameters of rotational diffusion of fluorophors is large application of method of polarized fluorescence. In the theory of rotational depolarization there is a following formula [14]:
1 1 1 1 kT s0 $ þ ; ¼ Vh P P0 P0 3 where T e absolute temperature, h e viscosity of solution, V e volume, k e constant of Boltzmann, s0 e average time of life of
Vh ; Tk
where V e molecular volume. For fluorophors it is possible to determine the coefficient of rotational diffusion Drot [14]:
Drot ¼
kT : 6hV
In the given work by a variation of viscosity of solutions by addition of various concentration of sucrose the time of rotational relaxation, the coefficient of rotational diffusion and the Einstein’s effective radius of nanomarkers of fluorescein family were determined, both in HSA solutions, and in solutions without protein, at different values of pH. At all values of pH in solutions with HSA the coefficient of rotational diffusion Drot of nanomarkers of fluorescein family is smaller than in solutions without HSA (Fig. 2), that reflects a binding of nanomarkers to protein. Both in HSA solutions, and in solutions without HSA (Fig. 2), Drot of fluorescein non-linear depends on pH with a minimum at pH 6.0, and Drot of eosin and erythrosin does not depend on pH (at 3.5 pH 8.0). At presence of rather weak electronegative atoms of Hydrogen in structural formula of fluorescein and, hence, at small interaction with polar environment of solutions the strong dependences on pH of processes of molecular association of fluorescein and of efficiency of its binding to protein [6,7,10] influence greatly on rotational diffusion of nanomarker that results in dependence of rotational diffusion of nanomarker on pH and in dependence of polarization of fluorescence of fluorescein on pH. Therefore at pH 6.0, at which the maximum binding of nanomarker to HSA and the maximum association of nanomarker molecules occur, the value of Drot of fluorescein has the lowest value, both in HSA solutions, and in solutions without HSA. Despite of processes of molecular association and efficiency of binding to protein, depending on pH and having maximum at pH, smaller 5.0 [6,7,10], the value of Drot of erythrosin and eosin does not depend on pH. At presence of strongly electronegative atoms (Bromide and Iodine) in structural formulas of eosin and erythrosin the strong interaction of nanomarkers with polar environment takes place, that results in independence of rotational diffusion of nanomarker on its molecular association and efficiency of its binding to protein, depending on pH, and, therefore, to independence of rotational diffusion of nanomarkers on pH and to
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Fig. 3. Einstein’s effective radius a of fluorescein (1, 2), erythrosin (3, 4), eosin (5, 6) in solutions without HSA (1, 3, 5) and in solutions with HSA (2, 4, 6) at different values of pH.
Fig. 2. Dependences of coefficient of rotational diffusion and time of rotational relaxation (inserted figure) of fluorescein (1, 2), erythrosin (3, 4), eosin (5, 6) in the solutions without HSA (1, 3, 5) and in solutions with HSA (2, 4, 6) on pH.
in solutions with HSA, radius a of eosin and erythrosin practically does not depend on pH in a range pH 3.5e8.0. 3.3. Binding of nanomarkers of fluorescein family to HSA at different pH
independence of polarization of fluorescence of these nanomarkers on pH. In solutions without HSA the time of rotational relaxation x (Fig. 2) of nanomarkers of fluorescein family, describing rotational mobility and corresponding to time of disorder of fluorophor owing to thermal diffusion, is essentially smaller, than in HSA solutions at all investigated values of pH (3.5e8.0). Nanomarkers of fluorescein family, not bound to protein, have small x, smaller then times of life of their fluorescence (s0 of fluorescein e 3.5 ns, s0 of erythrosin e 3.2 ns, s0 of eosin e 2.9 ns) that leads to weak polarization of fluorescence of nanomarkers in solutions without protein. Nanomarkers of fluorescein family, bound to HSA, have x, greater then times of life of their fluorescence that leads to increase in polarization of fluorescence of nanomarkers in solutions with HSA in comparison with solutions without HSA. Also in this work the values of Einstein’s effective radius a of nanomarkers of fluorescein family (Fig. 3), both in HSA solutions, and in solutions without HSA, are determinated at various values of pH. In solutions without HSA the value of a of nanomarker represents the real size of molecule of nanomarker, and in solutions with HSA the value of a is not the real size and represents the effective size which determinates percent of nanomarker bound to HSA from its total concentration in solution. The increase of Einstein’s effective radius a of nanomarkers of fluorescein family in HSA solutions in comparison with solutions without HSA (Fig. 3) speaks about binding of nanomarkers to protein molecules, but there is also some amount of nanomarkers, not bound to protein (radius of HSA is equal to 4.0e4.5 nm). It is seen (Fig. 3) that, both in solutions without HSA, and in solutions with HSA, radius a of fluorescein non-linear depends on pH with a maximum at pH 6.0. Both in solutions without HSA, and
In Table 1 those ranges of spectra are shown where the Raman peaks, corresponding to chemical bonds of nanomarkers of fluorescein family to HSA at different pH are registered. From the data of Table 1, it is seen, that at binding of these three nanomarkers to HSA the Raman peaks in identical ranges of spectra are registered. In Table 1 the decryption of Raman peaks, corresponding to binding of nanomarkers of fluorescein family with HSA, is shown. As it follows from the decryption of obtained Raman spectrograms, the residues of only polar amino acids of HSA participate in binding of these nanomarkers: the residues of threonine (Thr), serine (Ser), lysine (Lys), arginine (Arg), glutamine (Gln) and glutaminic acid (Glu). The fact that the identical amino acidic residues of HSA participate in binding of each of three nanomarkers is possible to explain by the similar chemical structure of these nanomarkers and their belonging to one homologous family. The mechanism of binding of ligands to HSA molecule is determined by availability of binding Centers on protein. To binding Centers of small organic molecules, such as fluorescein, eosin and erythrosin, attribute Center I and II. Center I is in the domain II of HSA. Lysine (195, 199), threonine (125, 243), glutaminic acid (167, 244), glutamine (196), serine (202), arginine (197) are in Center I. Center II is in the domain III of albumin molecule. Tyrosine (411) and lying near leucine (413) and valine (415, 418) are in Center II. On obtained Raman spectra one can conclude, that nanomarkers of fluorescein family bind to Center I of HSA. Though the same amino acidic residues, which are included in a structure of binding Center I of HSA, participate in binding of these three nanomarkers, there are differences in binding of these nanomarkers with protein in dependence on pH of solutions (Table 1). The dependence of number of types of chemical bonds between the nanomarker and HSA on pH for fluorescein has a nonlinear behavior with a maximum at pH 5.0e6.0: at these values of
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Table 1 The decryption of Raman peaks, corresponding to binding of nanomarkers of fluorescein family (1 e fluorescein, 2 e erythrosin, 3 e eosin) to HSA at different values of pH. Range, cm1
Decryption
Mechanism of binding
Range of pH of action of type of binding 1
2
3
1105e1115
Binding of nanomarker through its hydroxyl group with threonine (Thr).
3.5e8.0
3.5e8.0
3.5e8.0
1150e1160
Binding of nanomarker through its hydroxyl group with serine (Ser).
5.0e7.0
3.5e7.0
3.5e7.0
1380e1405
Binding of nanomarker through its carboxyl group with lysine (Lys).
3.5e8.0
3.5e8.0
3.5e8.0
1510e1520
Binding of nanomarker through its hydroxyl group with glutamine (Gln).
5.0e7.0
3.5e5.0
3.5e5.0
1530e1545
Binding of nanomarker through its hydroxyl group with arginine (Arg).
6.0
3.5e6.0
3.5e6.0
1550e1565
Binding of nanomarker through its hydroxyl group with lysine (Lys).
5.0e8.0
3.5e8.0
3.5e8.0
1580e1590
Binding of nanomarker through its carboxyl group with threonine (Thr).
4.0e8.0
3.5
3.5e4.0
1600e1610
Binding of nanomarker through its carboxyl group with serine (Ser).
5.0e8.0
3.5e5.0
3.5e4.0
1695e1720
Binding of nanomarker through its hydroxyl group with glutaminic acid (Glu).
5.0e6.0
3.5e8.0
3.5e8.0
1730e1755
Binding of nanomarker through its carboxyl group with glutaminic acid (Glu).
3.5e8.0
3.5e8.0
3.5e8.0
pH there is a greatest number of types of chemical bonds. For halogen-derivatives of fluorescein the dependence of number of types of chemical bonds between the nanomarker and HSA on pH has a linearly decreasing behavior with an increase of pH. The greatest number of types of chemical bonds between eosin and HSA takes place at pH 3.5e4.0. The greatest number of types of chemical bonds between erythrosin and HSA takes place at pH 3.5e5.0. The differences in dependences of binding of nanomarkers to protein on pH are explained by difference in lateral radicals of structural formulas of these nanomarkers: the fluorescein has atoms of Hydrogen (H) as lateral radicals, and in its halogenderivatives these atoms of hydrogen are changed to halogens (Bromide Br e for eosin, Iodine I e for erythrosin). The increase of electronegativity goes in the following direction: Hydrogen (for fluorescein) e Iodine (for erythrosin) e Bromide (for eosin). The availability of more electronegative atom in a molecule of the nanomarker results in strong decreasing of values of pK(COOH) and pK(OH) of these nanomarkers. The found differences in binding of these three nanomarkers to HSA are determined by value of electronegativity of atoms of lateral radicals in structural formulas of nanomarkers and, therefore, by value of pK of their ionized groups. Let’s consider the ionized conditions of fluorescein depending on pH. At values of pH, smaller 5.5, the molecules of fluorescein are in gently positively charged form. At values of pH 5.5e6.8 molecules of fluorescein are electrically neutral. Value of pK(OH) of fluorescein e 6.8. At pH 6.8e8.0 molecules of fluorescein are gently
negatively charged and are in the form of monoanions. Value of pK(COOH) of fluorescein e 8.0. At pH, larger 8.0, the molecules of fluorescein are strongly negatively charged and are in the form of dianions. The maximum binding of fluorescein to HSA takes place at pH 5.0e6.0, i.e. when fluorescein either is gently positively charged, or is neutral. Let’s consider the ionized conditions of erythrosin depending on pH. The erythrosin has the following values of pK of ionized groups: pK(OH) e 3.6 and pK(COOH) e 5.5. In range of values of pH, smaller 3.6, the molecules of erythrosin are electrically neutral. In range of pH 3.6e5.5 molecules of erythrosin are gently negatively charged (monoanions). In range of values of pH, larger 5.5, the molecules of erythrosin are strongly negatively charged (dianions). The maximum binding of erythrosin to HSA takes place at pH 3.5e5.0, i.e. in that range, where the erythrosin is gently negatively charged (pH 3.6e5.0) or is electrically neutral (3.5e3.6), and HSA at these pH is positively charged as a whole. Let’s consider the ionized conditions of eosin depending on pH. At values of pH, smaller 3.0, the molecules of eosin are electrically neutral. Value of pK(OH) of eosin is approximately 3.0. At values of pH, larger 3.0, but smaller 5.0, molecule of eosin is gently negatively charged (monoanion). Value of pK(COOH) of eosin is approximately 5.0. At values of pH, larger 5.0, the eosin is strongly negatively charged (dianion). The maximum binding of eosin to HSA takes place at pH 3.5e4.0, i.e. in that range, where the eosin is gently negatively charged and is in the form of monoanion (HSA at these values of pH is positively charged as a whole).
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3.4. Secondary structure of HSA at interaction of protein with nanomarkers of fluorescein family at different pH The Raman spectral bands of Amide-I and Amide-III, used for investigation of secondary structure of proteins, have the following values: 1) Amide-I e 1645e1655 cm1 (a-helix), 1670e1680 cm1 (b-structure), 1660e1670 cm1 (random clew); 2) Amide-III e 1270e1280 cm1 (a-helix), 1230e1240 cm1 (b-structure), 1240e1260 cm1 (random clew). The secondary structure of native (intact, i.e. before binding of nanomarkers) HSA consists from a-helix segments and segments of random clew. In spectral bands of Amide-I and Amide-III the Raman peaks, which are responsible to a-helix and random clew segments of HSA before and after binding of one of three nanomarkers, are investigated. On base of the calculated areas under peaks in spectral bands of Amide-I and Amide-III the percentage of a-helix in secondary structure of native molecules of HSA (before binding of nanomarkers) and also percentage of a-helix in secondary structure of HSA after binding of nanomarkers at different values of pH were determinated (Fig. 4). In the absence of nanomarkers in solutions the percentage of ahelix in secondary structure of native HSA molecules almost constantly and does not depend on pH in the range 3.5e8.0, when there is no acid-induced denaturation of protein. For HSA with bound to it fluorescein the non-linear dependence of percentage of a-helix in secondary structure of protein on pH with minimum at pH 6.0 is vividly seen. At binding of halogensderivatives of fluorescein (eosin and erythrosin) by HSA the percentage of a-helix in secondary structure of protein linearly increases with increasing of pH. The found laws in percentage of a-helix in secondary structure of HSA at binding of nanomarkers of fluorescein family are explained by dependences of this binding on pH, determinated by electronegativity of atoms in structural formulas of nanomarkers and values of pK of its ionized groups. The greatest changes in secondary structure of HSA, consisting in decreasing of a-helix sites, and occurring at binding of nanomarkers of fluorescein family, take place at those values of pH when a maximal binding of these nanomarkers to HSA occurs.
Fig. 4. The percentage of a-helix (%) in secondary structure of HSA at different values of pH before and after binding of nanomarkers of fluorescein family: 1 e before binding, 2 e after binding of fluorescein, 3 e after binding of erythrosin, 4 e after binding of eosin.
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Table 2 Effective SterneVolmer constants of fluorescence quenching (K . M1) of nanomarkers of fluorescein family in HSA solutions at different values of pH. pH
Fluorescein-HSA
Erythrosin-HSA
Eosin-HSA
3.5 4.0 5.0 6.0 7.0 8.0
850 3610 24390 24790 6170 4970
24950 20010 16140 6510 5210 2520
9580 7850 5180 2570 840 340
3.5. Effective SterneVolmer constants of fluorescence quenching of nanomarkers of fluorescein family in HSA solutions The effective constants of quenching of fluorescence of nanomarkers of fluorescein family in HSA solutions are determinated by SterneVolmer equation [14,15]:
F0 ¼ 1 þ K$½Q ; F where F0 e intensity of fluorescence of nanomarker in absence of quencher (HSA), F e intensity of fluorescence of nanomarker in presence of quencher (HSA), [Q] e concentration of quencher (HSA), K e effective SterneVolmer constants of fluorescence quenching of nanomarkers (M1). The slope of plot of dependence (F0/F) 1 on [Q] is determinated by type of interaction and by amount of binding sites. From our Raman investigation we concluded that nanomarkers of fluorescein family are bound to Center I of HSA, consequently there is one binding site on HSA for these nanomarkers. The plot of dependence (F0/F) 1 on [Q] represents a straight line with tangent of angle of slope K in case of one mechanism of binding of nanomarker to protein, but if there is several mechanisms of binding of nanomarker to protein some deviations from linearity are seen. On the obtained spectra of fluorescence of each nanomarker in solutions with different concentration of HSA at different values of pH the dependences (F0/F) 1 on [Q] are constructed. It is shown, that for each nanomarker the practically linear dependence (F0/F) 1 on [Q] is observed at pH 6.0, that speaks about one (probably through chemical bonds) covalent mechanism of binding of nanomarker to HSA. While at pH <6.0 the deviations from linearity of dependence (F0/F) 1 on [Q] are observed, that indicates two mechanisms of binding (covalent and ionic) of each nanomarker to HSA. For the account of both mechanisms (covalent and ionic) of interactions of nanomarkers with HSA and for comparison of nanomarkers among themselves the effective SterneVolmer constants of fluorescence quenching of nanomarkers in HSA solutions at different pH are determinated by linear approximation (Table 2). For fluorescein the non-linear dependence of effective SterneVolmer constant on pH with maximum approximately at pH 6.0 is characteristic. Halogen-derivatives of fluorescein are characterized by almost linear decrease of effective SterneVolmer constant with increasing of pH. It is accepted that the maximum SterneVolmer constant allowed for the dynamic quenching of biomolecules is 100 M1 [16]. From obtained SterneVolmer constants (Table 2) it is concluded that the quenching is static process occurring in the HSA e nanomarker complex. From the comparison of values of effective SterneVolmer constants in the range of pH from pI of HSA (4.7) and up to physiological value of pH (7.4) it is seen (Table 2), that effective constant has its greatest value in the case of fluorescein, the lowest value e in the case of eosin, and the intermediate value e in the case of erythrosin. The found differences in the effective SterneVolmer constants of fluorescence quenching of nanomarkers are determined by value of an electronegativity of atoms in structural
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formulas of nanomarkers. The presence of more electronegative atoms in nanomarker results in decrease of effective SterneVolmer constant in range from pI of HSA (4.7) and up to pH 7.4. 4. Conclusion The comparative analysis of interaction of nanomarkers of fluorescein family with HSA at different values of pH was carried out by spectroscopic methods. The conclusion is made that the found differences in polarization of fluorescence of nanomarkers, in parameters of rotational diffusion of nanomarkers, in binding mechanisms of nanomarkers to HSA, in changes of secondary structure of HSA at binding of nanomarkers, in effective SterneVolmer constants of fluorescence quenching of nanomarkers in HSA solutions are determined by value of an electronegativity of atoms of lateral radicals in structural formulas of nanomarkers and values of pK of their ionized groups. The increase of electronegativity is observed in a following direction: Hydrogen (for fluorescein) e Iodine (for erythrosin) e Bromide (for eosin). The presence of more electronegative atom in molecule of nanomarker results in strong decrease of values of pK(COOH) and pK(OH) of these nanomarkers. The increase in electronegativity of atoms of lateral radicals in structural formulas of nanomarkers leads to increase of degree of polarization of their fluorescence, to decrease of their coefficient of rotational diffusion, to increase of their time of rotational relaxation, to increase of Einstein’s effective radius, to decrease of effective SterneVolmer constants of fluorescence quenching of nanomarkers in HSA solutions in range from pI of HSA (4.7) and up to pH 7.4. It is shown that depending on pH the mechanism of binding of fluorescein with HSA differs from character of binding of fluorescein halogens-derivatives with HSA. It is registered that the greatest changes in secondary structure of HSA, occurring at binding of nanomarkers of fluorescein family, take place at those values of pH when a maximal binding of these nanomarkers to HSA occurs. The insertion of atoms with different electronegativity in structural formulas of nanomarkers of fluorescein family allows
one to receive the nanomarkers with different optical and molecular characteristics and different character of binding to HSA. Chemical and electrostatic interactions play the leading role in interaction between nanomarkers and HSA. The data about binding of fluorescent nanomarkers to HSA and about properties of these nanomarkers in HSA solutions allow one to receive the information about mechanisms of interaction of fluorescent nanomarkers with HSA, what can be useful at research of structure and properties of binding Centers (drug-binding Centers) of HSA, and this is of great importance in medical investigations of binding of drugs to HSA. Acknowledgements The work is supported by RFBR (N 07-02-00459). References [1] T. Peters, All About Albumin. Academic Press, New York, 1996. [2] J.R. Lakowicz, Principles of Fluorescence Spectroscopy. Plenum, New York, 1999. [3] S. Pelet, M. Gratzel, J.-E. Moser, Journal of Physical Chemistry B 107 (2003) 3215e3224. [4] L. Birla, B. Prieto, T. Noguel, et al., Revue Roumaine de Chimie 52 (2007) 639e646. [5] A.A. Waheed, K. Sridhar, P.D. Gupta, Analytical Biochemistry 287 (2000) 73e79. [6] I.M. Vlasova, A.M. Saletsky, Current Applied Physics 9 (2009) 1027e1031. [7] E.M. Buharova, I.M. Vlasova, A.M. Saletsky, Journal of Applied Spectroscopy 75 (2008) 785e790. [8] I.M. Vlasova, D.V. Polyansky, A.M. Saletsky, Laser Physics Letters 4 (2007) 390e394. [9] I.M. Vlasova, A.M. Saletsky, Laser Physics Letters 5 (2008) 384e389. [10] I.M. Vlasova, A.M. Saletsky, Journal Molecular Structure 936 (2009) 220e227. [11] S. Pilotto, M.T.T. Pacheco, L. Silveira, A. Balbin Villaverde, R.A. Zangaro, Lasers in Medical Science 16 (2001) 2e9. [12] H. Sato, H. Chiba, H. Tashiro, Y. Ozaki, Journal of Biomedical Optics 6 (2001) 366e370. [13] A.J. Berger, T.-W. Koo, I. Itzkan, G. Horowitz, M.S. Feld, Applied Optics 38 (1999) 2916e2926. [14] W. Schmidt, Optical Spectroscopy in Chemistry and Life Sciences. WILEY-VCN Verlag GmbH & Co. KgaA, 2005. [15] A.G. Marshall, Biophysical Chemistry. John Wiley & Sons, New York, 1978. [16] W. An, Y. Jiao, C. Dong, C. Yang, Y. Inoue, S. Shuang, Dyes and Pigments 81 (2009). doi:10.1016/j.dyepig.2008.08.004.