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Investigation of interaction of fluorescent marker Bengal Rose with human serum albumin at various values of pH
Journal of Molecular Structure 1016 (2012) 1–7
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Investigation of interaction of fluorescent marker Bengal Rose with human serum albumin at various values of pH Irina M. Vlasova ⇑, Anna A. Kuleshova 1, Andrey I. Panchishin 1, Alexander A. Vlasov 2 Physical Department, M.V. Lomonosov Moscow State University, Vorob’evi Gori, 119991 Moscow, Russia
a r t i c l e
i n f o
Article history: Received 18 October 2011 Received in revised form 5 February 2012 Accepted 6 February 2012 Available online 24 February 2012 Keywords: Fluorescent nanomarker Human serum albumin Bengal Rose Fluorescence analysis Raman spectroscopy
a b s t r a c t The interaction of fluorescent marker Bengal Rose with human serum albumin (HSA) at various values of pH has been studied by steady-state fluorescence, absorption spectroscopy, and Raman spectroscopy. The decrease of degree of molecular association of Bengal Rose in solutions at HSA addition is revealed. It is shown that degree of molecular association of Bengal Rose monotonously decreases with increase of values of pH. It is registered that in solutions with HSA there are quenching of fluorescence and red shift of maximum of fluorescence spectrum of Bengal Rose. Various theoretical models are used to determine the constants of quenching of fluorescence of Bengal Rose by HSA, corresponding to binding of Bengal Rose to HSA molecules, at various values of pH. It is shown that binding of Bengal Rose with HSA takes place through Binding Center I of HSA. The received results of researches of interaction of Bengal Rose with HSA show the perspectives of this marker in structural researches of HSA and in modeling of binding of some medical drugs to HSA. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction The analysis of the mechanism of binding of proteins molecules with different ligands (such as markers) is extremely interesting as from the point of view of a biomedicine and pharmaceuticals, so from the point of view of bionanotechnology. The fluorescent markers are widely applied in research of structurally-dynamic states of protein molecules [1–17]. Unique property of molecule of human serum albumin (HSA) to bind wide range of ligands determines one of fundamental functions of this protein – the transport of drugs and physiological metabolites. The structural mobility of molecule of HSA (isoelectric point pI 4.7), provided by unique loop folding of one polypeptide chain of 585 amino acidic residues, ensures the interaction of molecule of HSA with various ligands. The secondary structure of HSA consists of a-helix segments and segments of random coil. Nowadays the domain model of HSA tertiary structure is accepted, according to which the molecule of HSA consists of three practically identical domains. The mechanism of ligand binding to HSA molecule is determined by availability of specific sites on protein – binding Centers. Six main binding Centers of HSA are singled out: Center
⇑ Corresponding author. Tel./fax: +7 495 9391489. E-mail addresses: [email protected] (I.M. Vlasova), ak2210first@yandex. ru (A.A. Kuleshova), [email protected] (A.I. Panchishin), [email protected] (A.A. Vlasov). 1 Tel./fax: +7 495 9391489. 2 Tel.: +7 495 9391647. 0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2012.02.008
I and II – for binding of small organic molecules, Centers III and IV – for binding of long fatty acid chains, Center V – for binding of ligands with free SH-group, Center VI – for binding of ions of metals [1]. The molecular mechanisms of binding of HSA with different ligands still completely are not discovered [18–24], and nowadays structure and physicochemical properties of binding Centers of this protein are widely studied with the help of fluorescent markers. For research of binding Centers of HSA and structurally dynamic state of HSA molecules are widely used fluorescent anionic at physiological pH (7.4) markers such as Bengal Rose that is tetra-chloride-tetra-iodinate derivative of fluorescein. Also to fluorescent markers of fluorescein family belong the initial compound – fluorescein, iodinated derivative of fluorescein–erythrosin and brominated derivative of fluorescein–eosin. Earlier we studied processes of molecular association of this three marker (fluorescein, erythrosin, eosin) in HSA solutions [12], their fluorescent characteristics in HSA solutions [13,15], also mechanisms of their binding to HSA were investigated [14,16] and constants of binding of these markers to HSA were determined [15]. This work is dedicated to investigation of interaction of fluorescent marker Bengal Rose with HSA at various pH. The following investigations were done in this work: analysis of steady-state fluorescence and molecular association of Bengal Rose in HSA solutions, analysis of chemical bonds in HSA-marker association, estimation of functional conformational rearrangements of HSA at binding of Bengal Rose, and calculation of quenching constants of Bengal Rose fluorescence by HSA, corresponding to binding of Bengal Rose to HSA.
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Investigations of binding of fluorescent markers, such as Bengal Rose, to HSA allow one to receive the information on a structure and properties of binding Centers of HSA, including its medicinal Sites. This has the important applied medical and pharmacological aspect because many medical drugs are transferred in a blood in form of ligand bound to HSA.
Using these measured spectrograms of solutions two difference spectra were composed:
D1 = spectrograms (3) – spectrograms (1). D2 = spectrograms (4) – spectrograms (2).
The following buffer solutions were prepared: 0.1 M CH3COOH– KOH (pH 3.0–5.0) and 0.1 M KH2PO4 – 0.1 M NaOH (pH 6.0–8.0). On base of buffer solutions were prepared solutions of Bengal Rose (3–50 lM) without or with addition of 150 lM HSA (from donor plasma, ‘‘Microgen’’, Russia) at different values of pH (3.5–8.0). Measurements of absorption spectra of Bengal Rose in solutions with or without HSA were made on spectrophotometer Lambda 35 (Perkin Elmer). Prepared samples were placed at room temperature in 1 cm – cuvette. Absorption spectra of Bengal Rose in solutions were registered in the range 400 –700 nm.
Owing to the composition of D1 the contribution of low molecular weight components of buffer solutions is subtracted. The remaining Raman peaks correspond to chemical bonds in native HSA molecules. Owing to the composition of D2 the contribution of low molecular weight components of buffer solutions is subtracted, the contribution of Bengal Rose, not bound to protein, also is subtracted. The remaining Raman peaks correspond to chemical bonds in HSA molecules and also to chemical bonds, which have arisen from Bengal Rose binding to HSA. We have concentrated our attention on investigation of Raman peaks corresponding only to chemical bonds between HSA and Bengal Rose. In order to analysis the conformational rearrangements of HSA at Bengal Rose binding the spectral bands of Amide-I and Amide-III in difference spectra D1 and D2 are investigated.
2.2. Steady-state fluorescence of Bengal Rose in HSA solutions
3. Results and discussion
In order to determine fluorescent characteristics of Bengal Rose in solutions of HSA the solutions of marker Bengal Rose (3 lM) without or with HSA (150 lM) at different pH (3.5–8.0) were prepared. In order to determine constants of quenching of Bengal Rose fluorescence by HSA the solutions of marker Bengal Rose (3 lM) without or with HSA (10–150 lM) at different pH (3.5–8.0) were prepared. Investigations of steady-state fluorescence characteristics of Bengal Rose were made with the help of spectrofluorimeter LS 55 (Perkin Elmer) at room temperature. Fluorescence of Bengal Rose in solutions with or without HSA was excited with the wavelength kexcit = 540 nm.
The molecular markers Bengal Rose, eosin, erythrosin and fluorescein belong to one homologous family of derivatives of fluorescein. Eosin is the tetra-brominated derivative of fluorescein, erythrosin is the tetra-iodinated derivative of fluorescein, Bengal Rose is the tetra-chloride-tetra-iodinate derivative of fluorescein. The insert of halogen atoms (Cl, Br or I instead of H) in a molecule of fluorescein reduces values of pK of its carboxyl (COOH) and hydroxyl (OH) groups and changes characteristics of these markers. The values of pK for fluorescein, erythrosin, eosin and Bengal Rose, cited in the literature, rather strong differ from one work to another. We choose the following values: 1) fluorescein – pK (OH)1 5.0–5.5 and pK(OH)2 6.8, pK(COOH) 8.0; 2) erythrosin – pK(OH) 3.6, pK(COOH) 5.5; 3) eosin – pK(OH) 3.0, pK(COOH) 5.0; 4) Bengal Rose (Fig. 1) – pK(OH) 2.6, pK(COOH) 4.0. The binding of fluorescein, erythrosin and eosin to HSA was investigated earlier [12–16]. In this work we investigated binding of Bengal Rose to HSA. At pH < 2.6 the molecules of Bengal Rose are electrically neutral. In region pH 2.6–4.0 molecules of Bengal Rose are weakly negatively charged (monoanions). At pH > 4.0 the molecules of Bengal Rose are strongly negatively charged (dianions).
2. Materials and methods 2.1. Absorption spectroscopy for analysis of Bengal Rose molecular association in HSA solutions
2.3. Raman spectroscopy in determination of chemical bonds in HSA– Bengal Rose association and conformational rearrangements of HSA at binding of Bengal Rose The mechanism of binding of Bengal Rose to HSA was studied by Near – Infrared Raman spectroscopy method. The investigations were carried out 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 (Nd-YAG laser). The laser power was 450 mW, and 200 scans (12 min.) were used for one sample of solution. These parameters (laser power and number of scans) were chosen to preserve safety of samples, since the increase of these parameters resulted in thermal denaturation of protein. The spectral range was 400–3000 cm1, the spectral resolution was 2 cm1. The samples of solutions were placed in the quartz cell at room temperature. The obtained Raman spectra were treated by the program OPUS 4.2 (Bruker). The measurements of the following spectrograms are made: 1. Solutions (pH 3.5–8.0) without HSA and Bengal Rose. 2. Solutions (pH 3.5–8.0) with Bengal Rose (5 lM), but without HSA. 3. Solutions (pH 3.5–8.0) with HSA (150 lM), but without Bengal Rose. 4. Solutions (pH 3.5–8.0), with both Bengal Rose (5 lM) and HSA (150 lM).
3.1. Molecular association of Bengal Rose in HSA solutions In the obtained absorption spectra of Bengal Rose in solutions with and without HSA two maximums of absorption are detected. The long-wave maximum belongs to monomers of marker, the short-wave maximum – to associates (dimers) of marker. In our work the value of the degree of molecular association (DMA, 1 X) of marker Bengal Rose in solutions without and with HSA is determined at different values of pH. The DMA of solution of marker is the part (equal to 1 X) of marker molecules in associative (dimer) form, where X is the part of marker molecules in monomer form, and is determined by standard procedure [12]. In Fig. 2 the dependences of DMA (1 X) of Bengal Rose on pH of solutions without protein (a) and with HSA (b) are shown. It is seen (Fig. 2) that at increase of pH for every taken concentration of Bengal Rose the decrease of degree of association of its molecules both in solutions with HSA, and in solutions without HSA is observed. This effect one can interpret by electrostatic
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Fig. 1. Structural formulas of nanomarker Bengal Rose and values of pK of its ionized groups.
Fig. 2. Dependence of degree of molecular association (1 X) of Bengal Rose on pH of solutions without protein (a) and with 150 lM HSA (b) at various concentration of Bengal Rose: 3 lM (1), 10 lM (2), 20 lM (3), 30 lM (4), 50 lM (5).
mechanisms. With increase of pH the negative charge of Bengal Rose increases, the greater is the value of pH – the greater is the negative charge of molecules and the stronger is their repulsion from each other. At values of pH, smaller than 4.0, but larger than 2.6, molecules of Bengal Rose keep a negative charge only at hydroxyl group, and processes of their association occur more intensively than at high values of pH, larger than 4.0. Also it is seen (Fig. 2) that the degree of association of Bengal Rose, both in solutions without HSA and in solutions with HSA, increases with increase of concentration of Bengal Rose for every taken value of pH. It is seen (Fig. 2) that the degree of Bengal Rose molecular association in HSA solutions is less than degree of Bengal Rose molecular association in solutions without HSA at corresponding values of pH and corresponding concentration of Bengal Rose that speaks for binding of Bengal Rose to HSA molecules and, accordingly, for decrease of part of molecules of Bengal Rose unbound to HSA, capable to formation of associates. The decrease of degree of molecular association of Bengal Rose in HSA solutions, found out in given work, is similar to decrease of degree of molecular association of three others markers of fluorescein family – fluorescein, erythrosin and eosin – in HSA solutions [12,15]. From comparison of values of degree of Bengal Rose molecular association, obtained in the given work, and values of degree of molecular association of others three markers of fluorescein family at corresponding values of pH [15] it is seen that values of degree of molecular association decrease at increase in electronegativity of the atoms in structural formulas of markers. Electronegativity of atoms in structural formulas of markers of fluorescein family increases in a following direction: fluorescein, erythrosin, eosin, Bengal Rose. 3.2. Fluorescent characteristics of Bengal Rose in HSA solutions During this work the fluorescent characteristics of marker Bengal Rose in HSA solutions at various values of pH are investigated.
It is revealed that maxima of spectra of fluorescence of Bengal Rose in solutions with HSA are shifted in red range in comparison with maxima of spectra of fluorescence of Bengal Rose in solutions without HSA: for example, at pH 3.5 maximum of spectrum of fluorescence of Bengal Rose in solutions without HSA takes place at 563 nm, and in solutions with HSA – at 572 nm. The red shift one can interpret by effect of binding of Bengal Rose to HSA. In Fig. 3 the dependences of intensity in a maximum of spectrum of fluorescence of Bengal Rose on pH in solutions without HSA and in solutions with HSA are presented, and also a difference
Fig. 3. The dependence of intensity in maximum of fluorescence spectrum of Bengal Rose (3 lM) on pH in solutions without HSA (curve 1), with 150 lM HSA (curve 2), and difference of intensity of fluorescence of Bengal Rose in solutions without HSA and with HSA, i.e. curve 1–curve 2 (curve 3).
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of intensities of fluorescence of Bengal Rose, made by subtraction of intensity of fluorescence of Bengal Rose in HSA solutions from intensity of fluorescence of Bengal Rose in solutions without HSA, is presented. It is seen (Fig. 3) that in solutions without HSA the intensity in a maximum of spectrum of fluorescence of Bengal Rose decreases and then leaves on constant value with increase of pH. At pH, larger than 4.0 (pK of COOH-group of Bengal Rose), molecules of Bengal Rose are strongly negatively charged (dianions) that causes its screening by dipole molecules of water. At pH, smaller than 4.0, but larger than 3.5 (pK of OH-group of Bengal Rose is equal to 2.6), molecules of Bengal Rose are weakly negatively charged (monoanions), therefore screening by dipole molecules of water practically is absent. In HSA solutions (Fig. 3) the quenching of Bengal Rose fluorescence occurs at all investigated values of pH. The low value of intensity of Bengal Rose fluorescence in HSA solutions at values of pH, smaller than approximately 5.0, one can interpret by intensive binding of Bengal Rose with protein: at values of pH, smaller than approximately 5.0, HSA molecules are charged positively (pI 4.7), and Bengal Rose molecules are or in the monoanion form at 3.5 < pH < 4.0 or in the dianion form at 4.0 < pH < 5.0, as a consequence, the number of molecules of Bengal Rose unbound with HSA is small. The increase of intensity of fluorescence of Bengal Rose in HSA solutions at increase of pH more than 5.0 one can interpret by nonintensive binding of dianions of Bengal Rose to negatively charged molecules of HSA, as a consequence, the number of molecules of Bengal Rose unbound with HSA is not small. The red shift of spectra of fluorescence and the quenching of Bengal Rose fluorescence in HSA solutions, found out in the given work, are similar to red shift of spectra of fluorescence and quenching of fluorescence of three others markers of fluorescein family – fluorescein, erythrosin and eosin – in HSA solutions [13,15]. 3.3. Quenching of fluorescence of Bengal Rose in HSA solutions and different models of determination of constants of binding of Bengal Rose to HSA For various values of pH (Fig. 4) the dependences of (F0/F) – 1 on [Q] are plotted, where F0 – intensity of fluorescence Bengal Rose in absence of HSA, F – intensity of fluorescence Bengal Rose in presence of HSA, [Q] – concentration of HSA (approximately it is the general concentration of protein in a solution). It is seen that the received dependences of (F0/F) – 1 on [Q] have nonlinear character. In solutions of fluorescent marker with protein in the conditions, described in the given work, the formation of complexes plays central role in quenching of fluorescence of marker at interaction with protein, thus quenching of fluorescence is ‘‘the static’’. In this case deviations from linearity of dependences of (F0/F) – 1 on [Q] speak for several types of mechanisms (presumably, ionic and covalent) of formations of complexes of marker with protein. The constants of fluorescence quenching, determined from quenching of Bengal Rose fluorescence by addition of various concentration of HSA, correspond to binding of marker to protein. In this work it is considered three various models of determination of constants of quenching of fluorescence of Bengal Rose at its interaction with HSA. 3.3.1. The first model. Simple model of Stern–Volmer At quenching of Bengal Rose fluorescence by various concentration of quencher – HSA [25,26] it is possible to use the equation according to Stern–Volmer theory:
F0 ¼ 1 þ K ½Q ; F
Fig. 4. The plot of quenching of fluorescence of Bengal Rose (3 lM) by addition of various concentration of HSA at various values of pH: 3.5 (1), 4.0 (2), 5.0 (3), 6.0 (4), 7.0 (5), 8.0 (6).
where K is the effective constant of quenching of Bengal Rose fluorescence, corresponding to binding of Bengal Rose to protein (M1). The given Model describes linear dependence of (F0/F) – 1 on [Q], therefore for difficult nonlinear cases it is possible to speak only about a line of trend of the given dependences and, consequently, about a constant of trend. Within the frames of given Model 1 it is possible one to determine the certain effective constants of quenching of fluorescence (constant of trend, Keff) of Bengal Rose in HSA solutions at various values of pH (Table 1). Determined by line of trend of dependences of (F0/F) – 1 on [Q] the constants Keff averagely reflect all mechanisms of interactions of marker with protein. As it is seen (Table 1), for Bengal Rose the monotonously decreasing dependence of Keff with increase of pH is characteristic, what speaks for correlation of electric charges of Bengal Rose and HSA: at increase of pH a protein becomes negatively charged and binding of Bengal Rose to HSA is bad. 3.3.2. The second model. Expansion of dependences of (F0/F) – 1 on [Q] in two linear cases For more precise description of the received dependences of (F0/ F) – 1 on [Q] it is possible to expand them within the frames of Model 2 in two components – in the range of concentration of HSA up to 20 lM and in the range of concentration of HSA more than 20 lM – each of which can be approximated linearly, and received, accordingly, two constants (Table 2) of quenching of fluorescence of Bengal Rose, corresponding to various types of interactions of marker with HSA.
Table 1 Effective constants of quenching of Bengal Rose fluorescence by HSA (Model 1). pH
Keff, M1
3.5 4.0 5.0 6.0 7.0 8.0
(9.3 ± 0.6) 103 (6.7 ± 0.4) 103 (4.8 ± 0.3) 103 (2.3 ± 0.2) 103 (0.8 ± 0.1) 103 (0.3 ± 0.1) 103
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From Table 2 it is seen that both constants of quenching of fluorescence (K1 and K2) of Bengal Rose, received in the given Model 2, have monotonously decrease with increase of pH that repeats the character of dependence of an effective constant (Keff) of quenching of Bengal Rose fluorescence, received in Model 1. 3.3.3. The third model. The description of dependences of (F0/F) – 1 by sigmoid function For non-linear character of dependence of (F0/F) – 1 on [Q] for quenching of fluorescence of marker by various concentration of protein it is possible to use the following equation:
F0 ¼ 1 þ K ½Q n ; F where n is the Hill coefficient or factor of cooperativity, Ksigm is constant of quenching of fluorescence of marker pffiffiffiffi (corresponding to binding of marker to protein, M1), K sigm ¼ n K . Within the frames of the given Model 3, based on use of sigmoid (at n < 1) function of the description of quenching of fluorescence, the constants of quenching of Bengal Rose fluorescence by HSA, and factors of cooperativity are determined (Table 3). From Table 3 it is seen, that, as in the previous models, the constant of quenching of Bengal Rose fluorescence decreases with increase of pH. From Table 3 it is seen that there is a phenomenon of anticooperativity between various mechanisms of binding of Bengal Rose to HSA. From comparison of values of effective constants (Model 1) of quenching of Bengal Rose fluorescence by HSA, determined in the given work, and effective constants of quenching of fluorescence of three others markers of fluorescein family by HSA, determined in [15], it is revealed that the presence in a molecule of marker of more electronegative atom leads to decrease of values of effective constants of quenching of fluorescence of marker by HSA in range from pI CAX (4.7) to physiological pH (7.4). 3.4. Determination of chemical bonds in HSA–Bengal Rose association As it is well known, changes in Raman peaks characterize changes in chemical bonds. The Raman peaks, corresponding to binding of Bengal Rose to HSA, were registered at different pH (Table 4). The maximal binding of Bengal Rose to HSA takes place at pH 3.5–5.0 (Table 4), i.e. in that range, where Bengal Rose is weakly negatively charged (HSA at these values of pH is positively charged). At pH 5.0–8.0 the binding of Bengal Rose to HSA decreases due to increase of negative charge of Bengal Rose (HSA at these values of pH is charged negatively). It is seen that the dependence of number of types of chemical bonds between the Bengal Rose and HSA on pH has monotone decreasing behavior with increase of pH. On the data of Table 4, it is seen, that at binding of Bengal Rose to HSA and at binding of other three markers of fluorescein family
Table 2 Constants (Model 2) of quenching of fluorescence (K1 – in the range of concentration of HSA up to 20 lM, K2 – in the range of concentration of HSA more than 20 lM) of Bengal Rose in HSA solutions. pH 3.5 4.0 5.0 6.0 7.0 8.0
K1 (M1)
K2 (M1) 4
(4.8 ± 0.1) 10 (2.2 ± 0.1) 104 (1.5 ± 0.1) 104 (1.1 ± 0.1) 104 (1.5 ± 0.1) 103 (3.0 ± 0.4) 102
(3.6 ± 0.1) 103 (3.0 ± 0.1) 103 (2.2 ± 0.1) 103 (2.0 ± 0.1) 103 (5.4 ± 0.2) 102 (1.5 ± 0.1) 102
Table 3 Constants (Ksigm) of quenching of fluorescence (Model 3) of Bengal Rose in HSA solutions, corresponding to binding of marker to protein, and factors of cooperativity (n). pH
Ksigm (M1)
n
3.5 4.0 5.0 6.0 7.0 8.0
(14.3 ± 0.1) 103 (7.2 ± 0.1) 103 (44.0 ± 0.5) 102 (11.0 ± 0.1) 102 (40.0 ± 0.4) 10 (10.0 ± 0.1) 10
0.51 0.62 0.64 0.64 0.65 0.86
to HSA [14] the Raman peaks are registered in identical ranges of spectra. In Table 4 the decryption of Raman peaks, corresponding to binding of Bengal Rose to HSA, is shown. The residues of only polar amino acids of HSA participate in binding of Bengal Rose: the residues of threonine (Thr), serine (Ser), lysine (Lys), arginine (Arg), glutamine (Gln) and glutaminic acid (Glu). 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 attribute Centers I and II of HSA. 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 HSA. Tyrosine (411), leucine (413) and valine (415, 418) are in Center II. On the data of Table 4 we can conclude that Bengal Rose binds to Center I of HSA. 3.5. Determination of functional conformational rearrangements of HSA at Bengal Rose binding In the given work by the analysis of spectral bands of Amide-I and Amide-III the changes in secondary structure of HSA at binding of Bengal Rose at different values of pH are investigated. The spectral bands of Amide-I and Amide-III, used for research of secondary structure of proteins, have the following values: (1) Amide-I – 1645–1655 cm1 (a-helix), 1670–1680 cm1 (b-structure), 1660–1670 cm1 (random coil); (2) Amide-III – 1270–1280 cm1 (a-helix), 1230–1240 cm1 (b-structure), 1240–1260 cm1 (random coil). The secondary structure of native (intact, i.e. before binding of marker) HSA, stabilized by hydrogen bonds between peptide groups of amino acid chain, consists from a-helix segments and segments of random coil. In spectral bands of Amide-I and Amide-III the lines, which are responsible to a-helix and to random coil segments of HSA before and after binding of Bengal Rose, are investigated. The percentage of a-helix in secondary structure of native molecules of HSA (before binding of marker) and also percentage of a-helix in secondary structure of HSA after binding of Bengal Rose at different values of pH were determined (Table 5). It is seen (Table 5) that in the absence of marker in solutions the percentage of a-helix in secondary structure of native HSA molecules almost constantly and does not depend on pH in the range 3.5–8.0, when there is no acid-induced denaturation of protein. At binding of Bengal Rose to HSA the percentage of a-helix in secondary structure of HSA monotone increases with increase of pH. The maximal decrease of a-helix in secondary structure of HSA at binding of Bengal Rose takes place at pH 3.5–5.0, i.e. in that range, where the maximum binding of Bengal Rose to HSA takes place. These conformational rearrangements of HSA at binding of Bengal Rose at all values of pH are functional conformational rearrangements and not-denaturation conformational rearrangements.
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Table 4 Spectral lines in Raman spectra, corresponding to binding of Bengal Rose to HSA. Location of maximum of intensity of spectral line (cm1)
Presence of spectral line at various values of pH 3.5
4.0
5.0
6.0
7.0
8.0
1105
+
+
+
+
+
+
Decryption
Binding of Bengal Rose to threonine of HSA. 1151
+
+
+
+
1392
+
+
+
+
1515
+
1540
+
+
1558
+
+
1581
+
Binding of Bengal Rose to serine of HSA. +
+
Binding of Bengal Rose to lysine of HSA. Binding of Bengal Rose to glutamine of HSA. Binding of Bengal Rose to arginine of HSA.
+
+
+
+
Binding of Bengal Rose to lysine of HSA. Binding of Bengal Rose to threonine of HSA.
1608
+
+
+
1702
+
+
+
+
+
+
1740
+
+
+
+
+
+
Binding of Bengal Rose to serine of HSA.
Table 5 Percentage of a-helix in secondary structure of HSA before and after addition of Bengal Rose in solutions at different values of pH. pH % of a-helix
3.5
4.0
5.0
6.0
7.0
8.0
Before addition of Bengal Rose After addition of Bengal Rose
66.1 51.3
66.3 52.4
66.8 54.1
66.7 57.3
66.4 59.4
66.2 60.3
4. Conclusion In this work the analysis of fluorescent characteristics and molecular association of Bengal Rose in HSA solutions at various values of pH is done, the constants of quenching of Bengal Rose fluorescence by HSA, corresponding to binding of Bengal Rose to HSA, are determined, the binding Center on HSA, responsible for binding of Bengal Rose, is found out. The decrease of degree of molecular association of Bengal Rose in solutions at addition of HSA is revealed. It is shown that degree of molecular association of Bengal Rose monotonously decreases with increase of values of pH, both in solutions with HSA, and in solutions without HSA. In the given work it is revealed that on values of degree of molecular association of Bengal Rose, as well as others markers of fluorescein family, influences the value of electronegativity of the radicals in the structural formula of marker: the increase in electronegativity leads to decrease of its degree of molecular association. In the given work it is registered that in solutions with HSA there is a quenching of fluorescence and red shift of maximum of spectrum of fluorescence of Bengal Rose, characteristic for marker of fluorescein family and registered by us for eosin, erythrosin and fluorescein in [13,15]. By analysis of quenching of fluorescence of Bengal Rose by various concentration of HSA the constants of quenching fluorescence of Bengal Rose by HSA, corresponding to binding of marker to HSA, at various values of pH are determined by application of various theoretical models. It is shown that the constants of quenching of fluorescence of Bengal Rose by HSA, received in various models, monotonously decrease with increase of pH. This behavior of
Binding of Bengal Rose to glutaminic acid of HSA. Binding of Bengal Rose to glutaminic acid of HSA.
dependences of constants on pH is characteristic and for other halogens-derivatives of fluorescein, such as eosin and erythrosin [15], and is under the influence of value of electronegativity of atoms in structural formulas of markers. By the analysis of Raman spectra it is shown that binding of Bengal Rose to HSA occurs through binding Center I of HSA and at this binding the functional conformational rearrangements of protein, depending on pH, occur, what is characteristic also for other representatives of fluorescein family – fluorescein and its halogens-derivatives eosin and erythrosin [14]. As it is known, there are two types of conformational rearrangements of proteins – functional rearrangements with preservation of functional activity of protein and denaturation rearrangements with loss of functional activity of protein. At binding of Bengal Rose to HSA there are only functional rearrangements of HSA. 5. Summary The interaction of fluorescent marker Bengal Rose with human serum albumin (HSA) at various values of pH has been studied using steady-state fluorescence, absorption spectroscopy, and Raman spectroscopy. It is shown that degree of molecular association of Bengal Rose monotonously decreases with increase of values of pH. Various theoretical models are used to determine the constants of quenching of fluorescence of Bengal Rose by HSA, corresponding to binding of Bengal Rose to HSA molecules, at various values of pH. It is shown that binding of Bengal Rose with HSA takes place through Binding Center I of HSA. The received regularities of fluorescent characteristics and molecular association of Bengal Rose in HSA solutions, peculiarities of binding of Bengal Rose to HSA at various values of pH are explained by accessory of Bengal Rose to markers of fluorescein family. References [1] T. Peters, All About Albumin, Academic Press, New York, 1996. [2] S. Pelet, M. Gratzel, J.E. Moser, J. Phys. Chem. B 107 (2003) 3215–3224. [3] L. Birla, B. Prieto, T. Noguel, J. Vigo, A.-C. Ribou, Rev. Roum. Chim. 52 (2007) 639–646. [4] I.M. Vlasova, A.Yu. Zemlyansky, A.M. Saletsky, J. Appl. Spectr. 73 (2006) 743– 747.
I.M. Vlasova et al. / Journal of Molecular Structure 1016 (2012) 1–7 [5] D. Gao, Y. Tian, F. Liang, D. Jin, Y. Chen, H. Zhang, A. Yu, J. Lumin. 127 (2007) 515–522. [6] D.E. Buravtcov, I.M. Vlasova, A.M. Saletsky, Photomed. Las. Surg. 26 (2008) 181–187. [7] I.M. Vlasova, A.M. Polyansky, A.M. Saletsky, Las. Phys. Lett. 4 (2007) 390–394. [8] I.M. Vlasova, A.M. Saletsky, Las. Phys. Lett. 5 (2008) 384–389. [9] A. Sulkowska, M. Maciazek-Jurczyk, B. Bojko, J. Rownicka, I. Zubik-Skupien, E. Temba, D. Pentak, W.W. Sulkowski, J. Mol. Struct. doi: 10.1016/ j.molstruc.2007.09.001. [10] Feng Wang, Wei Huang, Zhongxiang Dai, J. Mol. Struct. doi: 10.1016/ j.molstruc.2007.05.034. [11] Zaolian Yu, Daojin Li, Baoming Ji, Jianjun Chen, J. Mol. Struct. doi: 10.1016/ j.molstruc.2008.03.13. [12] E.M. Buharova, I.M. Vlasova, A.M. Saletsky, J. Appl. Spectr. 75 (2008) 785–790. [13] I.M. Vlasova, A.M. Saletsky, Curr. Appl. Phys. 9 (2009) 1027–1031. [14] I.M. Vlasova, A.M. Saletsky, Las. Phys. Lett. 5 (2008) 834–839.
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Investigation of interaction of fluorescent marker Bengal Rose with human serum albumin at various values of pH Irina M. Vlasova ⇑, Anna A. Kuleshova 1, Andrey I. Panchishin 1, Alexander A. Vlasov 2 Physical Department, M.V. Lomonosov Moscow State University, Vorob’evi Gori, 119991 Moscow, Russia
a r t i c l e
i n f o
Article history: Received 18 October 2011 Received in revised form 5 February 2012 Accepted 6 February 2012 Available online 24 February 2012 Keywords: Fluorescent nanomarker Human serum albumin Bengal Rose Fluorescence analysis Raman spectroscopy
a b s t r a c t The interaction of fluorescent marker Bengal Rose with human serum albumin (HSA) at various values of pH has been studied by steady-state fluorescence, absorption spectroscopy, and Raman spectroscopy. The decrease of degree of molecular association of Bengal Rose in solutions at HSA addition is revealed. It is shown that degree of molecular association of Bengal Rose monotonously decreases with increase of values of pH. It is registered that in solutions with HSA there are quenching of fluorescence and red shift of maximum of fluorescence spectrum of Bengal Rose. Various theoretical models are used to determine the constants of quenching of fluorescence of Bengal Rose by HSA, corresponding to binding of Bengal Rose to HSA molecules, at various values of pH. It is shown that binding of Bengal Rose with HSA takes place through Binding Center I of HSA. The received results of researches of interaction of Bengal Rose with HSA show the perspectives of this marker in structural researches of HSA and in modeling of binding of some medical drugs to HSA. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction The analysis of the mechanism of binding of proteins molecules with different ligands (such as markers) is extremely interesting as from the point of view of a biomedicine and pharmaceuticals, so from the point of view of bionanotechnology. The fluorescent markers are widely applied in research of structurally-dynamic states of protein molecules [1–17]. Unique property of molecule of human serum albumin (HSA) to bind wide range of ligands determines one of fundamental functions of this protein – the transport of drugs and physiological metabolites. The structural mobility of molecule of HSA (isoelectric point pI 4.7), provided by unique loop folding of one polypeptide chain of 585 amino acidic residues, ensures the interaction of molecule of HSA with various ligands. The secondary structure of HSA consists of a-helix segments and segments of random coil. Nowadays the domain model of HSA tertiary structure is accepted, according to which the molecule of HSA consists of three practically identical domains. The mechanism of ligand binding to HSA molecule is determined by availability of specific sites on protein – binding Centers. Six main binding Centers of HSA are singled out: Center
⇑ Corresponding author. Tel./fax: +7 495 9391489. E-mail addresses: [email protected] (I.M. Vlasova), ak2210first@yandex. ru (A.A. Kuleshova), [email protected] (A.I. Panchishin), [email protected] (A.A. Vlasov). 1 Tel./fax: +7 495 9391489. 2 Tel.: +7 495 9391647. 0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2012.02.008
I and II – for binding of small organic molecules, Centers III and IV – for binding of long fatty acid chains, Center V – for binding of ligands with free SH-group, Center VI – for binding of ions of metals [1]. The molecular mechanisms of binding of HSA with different ligands still completely are not discovered [18–24], and nowadays structure and physicochemical properties of binding Centers of this protein are widely studied with the help of fluorescent markers. For research of binding Centers of HSA and structurally dynamic state of HSA molecules are widely used fluorescent anionic at physiological pH (7.4) markers such as Bengal Rose that is tetra-chloride-tetra-iodinate derivative of fluorescein. Also to fluorescent markers of fluorescein family belong the initial compound – fluorescein, iodinated derivative of fluorescein–erythrosin and brominated derivative of fluorescein–eosin. Earlier we studied processes of molecular association of this three marker (fluorescein, erythrosin, eosin) in HSA solutions [12], their fluorescent characteristics in HSA solutions [13,15], also mechanisms of their binding to HSA were investigated [14,16] and constants of binding of these markers to HSA were determined [15]. This work is dedicated to investigation of interaction of fluorescent marker Bengal Rose with HSA at various pH. The following investigations were done in this work: analysis of steady-state fluorescence and molecular association of Bengal Rose in HSA solutions, analysis of chemical bonds in HSA-marker association, estimation of functional conformational rearrangements of HSA at binding of Bengal Rose, and calculation of quenching constants of Bengal Rose fluorescence by HSA, corresponding to binding of Bengal Rose to HSA.
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Investigations of binding of fluorescent markers, such as Bengal Rose, to HSA allow one to receive the information on a structure and properties of binding Centers of HSA, including its medicinal Sites. This has the important applied medical and pharmacological aspect because many medical drugs are transferred in a blood in form of ligand bound to HSA.
Using these measured spectrograms of solutions two difference spectra were composed:
D1 = spectrograms (3) – spectrograms (1). D2 = spectrograms (4) – spectrograms (2).
The following buffer solutions were prepared: 0.1 M CH3COOH– KOH (pH 3.0–5.0) and 0.1 M KH2PO4 – 0.1 M NaOH (pH 6.0–8.0). On base of buffer solutions were prepared solutions of Bengal Rose (3–50 lM) without or with addition of 150 lM HSA (from donor plasma, ‘‘Microgen’’, Russia) at different values of pH (3.5–8.0). Measurements of absorption spectra of Bengal Rose in solutions with or without HSA were made on spectrophotometer Lambda 35 (Perkin Elmer). Prepared samples were placed at room temperature in 1 cm – cuvette. Absorption spectra of Bengal Rose in solutions were registered in the range 400 –700 nm.
Owing to the composition of D1 the contribution of low molecular weight components of buffer solutions is subtracted. The remaining Raman peaks correspond to chemical bonds in native HSA molecules. Owing to the composition of D2 the contribution of low molecular weight components of buffer solutions is subtracted, the contribution of Bengal Rose, not bound to protein, also is subtracted. The remaining Raman peaks correspond to chemical bonds in HSA molecules and also to chemical bonds, which have arisen from Bengal Rose binding to HSA. We have concentrated our attention on investigation of Raman peaks corresponding only to chemical bonds between HSA and Bengal Rose. In order to analysis the conformational rearrangements of HSA at Bengal Rose binding the spectral bands of Amide-I and Amide-III in difference spectra D1 and D2 are investigated.
2.2. Steady-state fluorescence of Bengal Rose in HSA solutions
3. Results and discussion
In order to determine fluorescent characteristics of Bengal Rose in solutions of HSA the solutions of marker Bengal Rose (3 lM) without or with HSA (150 lM) at different pH (3.5–8.0) were prepared. In order to determine constants of quenching of Bengal Rose fluorescence by HSA the solutions of marker Bengal Rose (3 lM) without or with HSA (10–150 lM) at different pH (3.5–8.0) were prepared. Investigations of steady-state fluorescence characteristics of Bengal Rose were made with the help of spectrofluorimeter LS 55 (Perkin Elmer) at room temperature. Fluorescence of Bengal Rose in solutions with or without HSA was excited with the wavelength kexcit = 540 nm.
The molecular markers Bengal Rose, eosin, erythrosin and fluorescein belong to one homologous family of derivatives of fluorescein. Eosin is the tetra-brominated derivative of fluorescein, erythrosin is the tetra-iodinated derivative of fluorescein, Bengal Rose is the tetra-chloride-tetra-iodinate derivative of fluorescein. The insert of halogen atoms (Cl, Br or I instead of H) in a molecule of fluorescein reduces values of pK of its carboxyl (COOH) and hydroxyl (OH) groups and changes characteristics of these markers. The values of pK for fluorescein, erythrosin, eosin and Bengal Rose, cited in the literature, rather strong differ from one work to another. We choose the following values: 1) fluorescein – pK (OH)1 5.0–5.5 and pK(OH)2 6.8, pK(COOH) 8.0; 2) erythrosin – pK(OH) 3.6, pK(COOH) 5.5; 3) eosin – pK(OH) 3.0, pK(COOH) 5.0; 4) Bengal Rose (Fig. 1) – pK(OH) 2.6, pK(COOH) 4.0. The binding of fluorescein, erythrosin and eosin to HSA was investigated earlier [12–16]. In this work we investigated binding of Bengal Rose to HSA. At pH < 2.6 the molecules of Bengal Rose are electrically neutral. In region pH 2.6–4.0 molecules of Bengal Rose are weakly negatively charged (monoanions). At pH > 4.0 the molecules of Bengal Rose are strongly negatively charged (dianions).
2. Materials and methods 2.1. Absorption spectroscopy for analysis of Bengal Rose molecular association in HSA solutions
2.3. Raman spectroscopy in determination of chemical bonds in HSA– Bengal Rose association and conformational rearrangements of HSA at binding of Bengal Rose The mechanism of binding of Bengal Rose to HSA was studied by Near – Infrared Raman spectroscopy method. The investigations were carried out 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 (Nd-YAG laser). The laser power was 450 mW, and 200 scans (12 min.) were used for one sample of solution. These parameters (laser power and number of scans) were chosen to preserve safety of samples, since the increase of these parameters resulted in thermal denaturation of protein. The spectral range was 400–3000 cm1, the spectral resolution was 2 cm1. The samples of solutions were placed in the quartz cell at room temperature. The obtained Raman spectra were treated by the program OPUS 4.2 (Bruker). The measurements of the following spectrograms are made: 1. Solutions (pH 3.5–8.0) without HSA and Bengal Rose. 2. Solutions (pH 3.5–8.0) with Bengal Rose (5 lM), but without HSA. 3. Solutions (pH 3.5–8.0) with HSA (150 lM), but without Bengal Rose. 4. Solutions (pH 3.5–8.0), with both Bengal Rose (5 lM) and HSA (150 lM).
3.1. Molecular association of Bengal Rose in HSA solutions In the obtained absorption spectra of Bengal Rose in solutions with and without HSA two maximums of absorption are detected. The long-wave maximum belongs to monomers of marker, the short-wave maximum – to associates (dimers) of marker. In our work the value of the degree of molecular association (DMA, 1 X) of marker Bengal Rose in solutions without and with HSA is determined at different values of pH. The DMA of solution of marker is the part (equal to 1 X) of marker molecules in associative (dimer) form, where X is the part of marker molecules in monomer form, and is determined by standard procedure [12]. In Fig. 2 the dependences of DMA (1 X) of Bengal Rose on pH of solutions without protein (a) and with HSA (b) are shown. It is seen (Fig. 2) that at increase of pH for every taken concentration of Bengal Rose the decrease of degree of association of its molecules both in solutions with HSA, and in solutions without HSA is observed. This effect one can interpret by electrostatic
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Fig. 1. Structural formulas of nanomarker Bengal Rose and values of pK of its ionized groups.
Fig. 2. Dependence of degree of molecular association (1 X) of Bengal Rose on pH of solutions without protein (a) and with 150 lM HSA (b) at various concentration of Bengal Rose: 3 lM (1), 10 lM (2), 20 lM (3), 30 lM (4), 50 lM (5).
mechanisms. With increase of pH the negative charge of Bengal Rose increases, the greater is the value of pH – the greater is the negative charge of molecules and the stronger is their repulsion from each other. At values of pH, smaller than 4.0, but larger than 2.6, molecules of Bengal Rose keep a negative charge only at hydroxyl group, and processes of their association occur more intensively than at high values of pH, larger than 4.0. Also it is seen (Fig. 2) that the degree of association of Bengal Rose, both in solutions without HSA and in solutions with HSA, increases with increase of concentration of Bengal Rose for every taken value of pH. It is seen (Fig. 2) that the degree of Bengal Rose molecular association in HSA solutions is less than degree of Bengal Rose molecular association in solutions without HSA at corresponding values of pH and corresponding concentration of Bengal Rose that speaks for binding of Bengal Rose to HSA molecules and, accordingly, for decrease of part of molecules of Bengal Rose unbound to HSA, capable to formation of associates. The decrease of degree of molecular association of Bengal Rose in HSA solutions, found out in given work, is similar to decrease of degree of molecular association of three others markers of fluorescein family – fluorescein, erythrosin and eosin – in HSA solutions [12,15]. From comparison of values of degree of Bengal Rose molecular association, obtained in the given work, and values of degree of molecular association of others three markers of fluorescein family at corresponding values of pH [15] it is seen that values of degree of molecular association decrease at increase in electronegativity of the atoms in structural formulas of markers. Electronegativity of atoms in structural formulas of markers of fluorescein family increases in a following direction: fluorescein, erythrosin, eosin, Bengal Rose. 3.2. Fluorescent characteristics of Bengal Rose in HSA solutions During this work the fluorescent characteristics of marker Bengal Rose in HSA solutions at various values of pH are investigated.
It is revealed that maxima of spectra of fluorescence of Bengal Rose in solutions with HSA are shifted in red range in comparison with maxima of spectra of fluorescence of Bengal Rose in solutions without HSA: for example, at pH 3.5 maximum of spectrum of fluorescence of Bengal Rose in solutions without HSA takes place at 563 nm, and in solutions with HSA – at 572 nm. The red shift one can interpret by effect of binding of Bengal Rose to HSA. In Fig. 3 the dependences of intensity in a maximum of spectrum of fluorescence of Bengal Rose on pH in solutions without HSA and in solutions with HSA are presented, and also a difference
Fig. 3. The dependence of intensity in maximum of fluorescence spectrum of Bengal Rose (3 lM) on pH in solutions without HSA (curve 1), with 150 lM HSA (curve 2), and difference of intensity of fluorescence of Bengal Rose in solutions without HSA and with HSA, i.e. curve 1–curve 2 (curve 3).
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of intensities of fluorescence of Bengal Rose, made by subtraction of intensity of fluorescence of Bengal Rose in HSA solutions from intensity of fluorescence of Bengal Rose in solutions without HSA, is presented. It is seen (Fig. 3) that in solutions without HSA the intensity in a maximum of spectrum of fluorescence of Bengal Rose decreases and then leaves on constant value with increase of pH. At pH, larger than 4.0 (pK of COOH-group of Bengal Rose), molecules of Bengal Rose are strongly negatively charged (dianions) that causes its screening by dipole molecules of water. At pH, smaller than 4.0, but larger than 3.5 (pK of OH-group of Bengal Rose is equal to 2.6), molecules of Bengal Rose are weakly negatively charged (monoanions), therefore screening by dipole molecules of water practically is absent. In HSA solutions (Fig. 3) the quenching of Bengal Rose fluorescence occurs at all investigated values of pH. The low value of intensity of Bengal Rose fluorescence in HSA solutions at values of pH, smaller than approximately 5.0, one can interpret by intensive binding of Bengal Rose with protein: at values of pH, smaller than approximately 5.0, HSA molecules are charged positively (pI 4.7), and Bengal Rose molecules are or in the monoanion form at 3.5 < pH < 4.0 or in the dianion form at 4.0 < pH < 5.0, as a consequence, the number of molecules of Bengal Rose unbound with HSA is small. The increase of intensity of fluorescence of Bengal Rose in HSA solutions at increase of pH more than 5.0 one can interpret by nonintensive binding of dianions of Bengal Rose to negatively charged molecules of HSA, as a consequence, the number of molecules of Bengal Rose unbound with HSA is not small. The red shift of spectra of fluorescence and the quenching of Bengal Rose fluorescence in HSA solutions, found out in the given work, are similar to red shift of spectra of fluorescence and quenching of fluorescence of three others markers of fluorescein family – fluorescein, erythrosin and eosin – in HSA solutions [13,15]. 3.3. Quenching of fluorescence of Bengal Rose in HSA solutions and different models of determination of constants of binding of Bengal Rose to HSA For various values of pH (Fig. 4) the dependences of (F0/F) – 1 on [Q] are plotted, where F0 – intensity of fluorescence Bengal Rose in absence of HSA, F – intensity of fluorescence Bengal Rose in presence of HSA, [Q] – concentration of HSA (approximately it is the general concentration of protein in a solution). It is seen that the received dependences of (F0/F) – 1 on [Q] have nonlinear character. In solutions of fluorescent marker with protein in the conditions, described in the given work, the formation of complexes plays central role in quenching of fluorescence of marker at interaction with protein, thus quenching of fluorescence is ‘‘the static’’. In this case deviations from linearity of dependences of (F0/F) – 1 on [Q] speak for several types of mechanisms (presumably, ionic and covalent) of formations of complexes of marker with protein. The constants of fluorescence quenching, determined from quenching of Bengal Rose fluorescence by addition of various concentration of HSA, correspond to binding of marker to protein. In this work it is considered three various models of determination of constants of quenching of fluorescence of Bengal Rose at its interaction with HSA. 3.3.1. The first model. Simple model of Stern–Volmer At quenching of Bengal Rose fluorescence by various concentration of quencher – HSA [25,26] it is possible to use the equation according to Stern–Volmer theory:
F0 ¼ 1 þ K ½Q ; F
Fig. 4. The plot of quenching of fluorescence of Bengal Rose (3 lM) by addition of various concentration of HSA at various values of pH: 3.5 (1), 4.0 (2), 5.0 (3), 6.0 (4), 7.0 (5), 8.0 (6).
where K is the effective constant of quenching of Bengal Rose fluorescence, corresponding to binding of Bengal Rose to protein (M1). The given Model describes linear dependence of (F0/F) – 1 on [Q], therefore for difficult nonlinear cases it is possible to speak only about a line of trend of the given dependences and, consequently, about a constant of trend. Within the frames of given Model 1 it is possible one to determine the certain effective constants of quenching of fluorescence (constant of trend, Keff) of Bengal Rose in HSA solutions at various values of pH (Table 1). Determined by line of trend of dependences of (F0/F) – 1 on [Q] the constants Keff averagely reflect all mechanisms of interactions of marker with protein. As it is seen (Table 1), for Bengal Rose the monotonously decreasing dependence of Keff with increase of pH is characteristic, what speaks for correlation of electric charges of Bengal Rose and HSA: at increase of pH a protein becomes negatively charged and binding of Bengal Rose to HSA is bad. 3.3.2. The second model. Expansion of dependences of (F0/F) – 1 on [Q] in two linear cases For more precise description of the received dependences of (F0/ F) – 1 on [Q] it is possible to expand them within the frames of Model 2 in two components – in the range of concentration of HSA up to 20 lM and in the range of concentration of HSA more than 20 lM – each of which can be approximated linearly, and received, accordingly, two constants (Table 2) of quenching of fluorescence of Bengal Rose, corresponding to various types of interactions of marker with HSA.
Table 1 Effective constants of quenching of Bengal Rose fluorescence by HSA (Model 1). pH
Keff, M1
3.5 4.0 5.0 6.0 7.0 8.0
(9.3 ± 0.6) 103 (6.7 ± 0.4) 103 (4.8 ± 0.3) 103 (2.3 ± 0.2) 103 (0.8 ± 0.1) 103 (0.3 ± 0.1) 103
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From Table 2 it is seen that both constants of quenching of fluorescence (K1 and K2) of Bengal Rose, received in the given Model 2, have monotonously decrease with increase of pH that repeats the character of dependence of an effective constant (Keff) of quenching of Bengal Rose fluorescence, received in Model 1. 3.3.3. The third model. The description of dependences of (F0/F) – 1 by sigmoid function For non-linear character of dependence of (F0/F) – 1 on [Q] for quenching of fluorescence of marker by various concentration of protein it is possible to use the following equation:
F0 ¼ 1 þ K ½Q n ; F where n is the Hill coefficient or factor of cooperativity, Ksigm is constant of quenching of fluorescence of marker pffiffiffiffi (corresponding to binding of marker to protein, M1), K sigm ¼ n K . Within the frames of the given Model 3, based on use of sigmoid (at n < 1) function of the description of quenching of fluorescence, the constants of quenching of Bengal Rose fluorescence by HSA, and factors of cooperativity are determined (Table 3). From Table 3 it is seen, that, as in the previous models, the constant of quenching of Bengal Rose fluorescence decreases with increase of pH. From Table 3 it is seen that there is a phenomenon of anticooperativity between various mechanisms of binding of Bengal Rose to HSA. From comparison of values of effective constants (Model 1) of quenching of Bengal Rose fluorescence by HSA, determined in the given work, and effective constants of quenching of fluorescence of three others markers of fluorescein family by HSA, determined in [15], it is revealed that the presence in a molecule of marker of more electronegative atom leads to decrease of values of effective constants of quenching of fluorescence of marker by HSA in range from pI CAX (4.7) to physiological pH (7.4). 3.4. Determination of chemical bonds in HSA–Bengal Rose association As it is well known, changes in Raman peaks characterize changes in chemical bonds. The Raman peaks, corresponding to binding of Bengal Rose to HSA, were registered at different pH (Table 4). The maximal binding of Bengal Rose to HSA takes place at pH 3.5–5.0 (Table 4), i.e. in that range, where Bengal Rose is weakly negatively charged (HSA at these values of pH is positively charged). At pH 5.0–8.0 the binding of Bengal Rose to HSA decreases due to increase of negative charge of Bengal Rose (HSA at these values of pH is charged negatively). It is seen that the dependence of number of types of chemical bonds between the Bengal Rose and HSA on pH has monotone decreasing behavior with increase of pH. On the data of Table 4, it is seen, that at binding of Bengal Rose to HSA and at binding of other three markers of fluorescein family
Table 2 Constants (Model 2) of quenching of fluorescence (K1 – in the range of concentration of HSA up to 20 lM, K2 – in the range of concentration of HSA more than 20 lM) of Bengal Rose in HSA solutions. pH 3.5 4.0 5.0 6.0 7.0 8.0
K1 (M1)
K2 (M1) 4
(4.8 ± 0.1) 10 (2.2 ± 0.1) 104 (1.5 ± 0.1) 104 (1.1 ± 0.1) 104 (1.5 ± 0.1) 103 (3.0 ± 0.4) 102
(3.6 ± 0.1) 103 (3.0 ± 0.1) 103 (2.2 ± 0.1) 103 (2.0 ± 0.1) 103 (5.4 ± 0.2) 102 (1.5 ± 0.1) 102
Table 3 Constants (Ksigm) of quenching of fluorescence (Model 3) of Bengal Rose in HSA solutions, corresponding to binding of marker to protein, and factors of cooperativity (n). pH
Ksigm (M1)
n
3.5 4.0 5.0 6.0 7.0 8.0
(14.3 ± 0.1) 103 (7.2 ± 0.1) 103 (44.0 ± 0.5) 102 (11.0 ± 0.1) 102 (40.0 ± 0.4) 10 (10.0 ± 0.1) 10
0.51 0.62 0.64 0.64 0.65 0.86
to HSA [14] the Raman peaks are registered in identical ranges of spectra. In Table 4 the decryption of Raman peaks, corresponding to binding of Bengal Rose to HSA, is shown. The residues of only polar amino acids of HSA participate in binding of Bengal Rose: the residues of threonine (Thr), serine (Ser), lysine (Lys), arginine (Arg), glutamine (Gln) and glutaminic acid (Glu). 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 attribute Centers I and II of HSA. 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 HSA. Tyrosine (411), leucine (413) and valine (415, 418) are in Center II. On the data of Table 4 we can conclude that Bengal Rose binds to Center I of HSA. 3.5. Determination of functional conformational rearrangements of HSA at Bengal Rose binding In the given work by the analysis of spectral bands of Amide-I and Amide-III the changes in secondary structure of HSA at binding of Bengal Rose at different values of pH are investigated. The spectral bands of Amide-I and Amide-III, used for research of secondary structure of proteins, have the following values: (1) Amide-I – 1645–1655 cm1 (a-helix), 1670–1680 cm1 (b-structure), 1660–1670 cm1 (random coil); (2) Amide-III – 1270–1280 cm1 (a-helix), 1230–1240 cm1 (b-structure), 1240–1260 cm1 (random coil). The secondary structure of native (intact, i.e. before binding of marker) HSA, stabilized by hydrogen bonds between peptide groups of amino acid chain, consists from a-helix segments and segments of random coil. In spectral bands of Amide-I and Amide-III the lines, which are responsible to a-helix and to random coil segments of HSA before and after binding of Bengal Rose, are investigated. The percentage of a-helix in secondary structure of native molecules of HSA (before binding of marker) and also percentage of a-helix in secondary structure of HSA after binding of Bengal Rose at different values of pH were determined (Table 5). It is seen (Table 5) that in the absence of marker in solutions the percentage of a-helix in secondary structure of native HSA molecules almost constantly and does not depend on pH in the range 3.5–8.0, when there is no acid-induced denaturation of protein. At binding of Bengal Rose to HSA the percentage of a-helix in secondary structure of HSA monotone increases with increase of pH. The maximal decrease of a-helix in secondary structure of HSA at binding of Bengal Rose takes place at pH 3.5–5.0, i.e. in that range, where the maximum binding of Bengal Rose to HSA takes place. These conformational rearrangements of HSA at binding of Bengal Rose at all values of pH are functional conformational rearrangements and not-denaturation conformational rearrangements.
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Table 4 Spectral lines in Raman spectra, corresponding to binding of Bengal Rose to HSA. Location of maximum of intensity of spectral line (cm1)
Presence of spectral line at various values of pH 3.5
4.0
5.0
6.0
7.0
8.0
1105
+
+
+
+
+
+
Decryption
Binding of Bengal Rose to threonine of HSA. 1151
+
+
+
+
1392
+
+
+
+
1515
+
1540
+
+
1558
+
+
1581
+
Binding of Bengal Rose to serine of HSA. +
+
Binding of Bengal Rose to lysine of HSA. Binding of Bengal Rose to glutamine of HSA. Binding of Bengal Rose to arginine of HSA.
+
+
+
+
Binding of Bengal Rose to lysine of HSA. Binding of Bengal Rose to threonine of HSA.
1608
+
+
+
1702
+
+
+
+
+
+
1740
+
+
+
+
+
+
Binding of Bengal Rose to serine of HSA.
Table 5 Percentage of a-helix in secondary structure of HSA before and after addition of Bengal Rose in solutions at different values of pH. pH % of a-helix
3.5
4.0
5.0
6.0
7.0
8.0
Before addition of Bengal Rose After addition of Bengal Rose
66.1 51.3
66.3 52.4
66.8 54.1
66.7 57.3
66.4 59.4
66.2 60.3
4. Conclusion In this work the analysis of fluorescent characteristics and molecular association of Bengal Rose in HSA solutions at various values of pH is done, the constants of quenching of Bengal Rose fluorescence by HSA, corresponding to binding of Bengal Rose to HSA, are determined, the binding Center on HSA, responsible for binding of Bengal Rose, is found out. The decrease of degree of molecular association of Bengal Rose in solutions at addition of HSA is revealed. It is shown that degree of molecular association of Bengal Rose monotonously decreases with increase of values of pH, both in solutions with HSA, and in solutions without HSA. In the given work it is revealed that on values of degree of molecular association of Bengal Rose, as well as others markers of fluorescein family, influences the value of electronegativity of the radicals in the structural formula of marker: the increase in electronegativity leads to decrease of its degree of molecular association. In the given work it is registered that in solutions with HSA there is a quenching of fluorescence and red shift of maximum of spectrum of fluorescence of Bengal Rose, characteristic for marker of fluorescein family and registered by us for eosin, erythrosin and fluorescein in [13,15]. By analysis of quenching of fluorescence of Bengal Rose by various concentration of HSA the constants of quenching fluorescence of Bengal Rose by HSA, corresponding to binding of marker to HSA, at various values of pH are determined by application of various theoretical models. It is shown that the constants of quenching of fluorescence of Bengal Rose by HSA, received in various models, monotonously decrease with increase of pH. This behavior of
Binding of Bengal Rose to glutaminic acid of HSA. Binding of Bengal Rose to glutaminic acid of HSA.
dependences of constants on pH is characteristic and for other halogens-derivatives of fluorescein, such as eosin and erythrosin [15], and is under the influence of value of electronegativity of atoms in structural formulas of markers. By the analysis of Raman spectra it is shown that binding of Bengal Rose to HSA occurs through binding Center I of HSA and at this binding the functional conformational rearrangements of protein, depending on pH, occur, what is characteristic also for other representatives of fluorescein family – fluorescein and its halogens-derivatives eosin and erythrosin [14]. As it is known, there are two types of conformational rearrangements of proteins – functional rearrangements with preservation of functional activity of protein and denaturation rearrangements with loss of functional activity of protein. At binding of Bengal Rose to HSA there are only functional rearrangements of HSA. 5. Summary The interaction of fluorescent marker Bengal Rose with human serum albumin (HSA) at various values of pH has been studied using steady-state fluorescence, absorption spectroscopy, and Raman spectroscopy. It is shown that degree of molecular association of Bengal Rose monotonously decreases with increase of values of pH. Various theoretical models are used to determine the constants of quenching of fluorescence of Bengal Rose by HSA, corresponding to binding of Bengal Rose to HSA molecules, at various values of pH. It is shown that binding of Bengal Rose with HSA takes place through Binding Center I of HSA. The received regularities of fluorescent characteristics and molecular association of Bengal Rose in HSA solutions, peculiarities of binding of Bengal Rose to HSA at various values of pH are explained by accessory of Bengal Rose to markers of fluorescein family. References [1] T. Peters, All About Albumin, Academic Press, New York, 1996. [2] S. Pelet, M. Gratzel, J.E. Moser, J. Phys. Chem. B 107 (2003) 3215–3224. [3] L. Birla, B. Prieto, T. Noguel, J. Vigo, A.-C. Ribou, Rev. Roum. Chim. 52 (2007) 639–646. [4] I.M. Vlasova, A.Yu. Zemlyansky, A.M. Saletsky, J. Appl. Spectr. 73 (2006) 743– 747.
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