- Email: [email protected]
Studying the interaction between trinuclear ruthenium complexes and human serum albumin by means of fluorescence quenching
Journal of Luminescence 169 (2016) 115–120
Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Studying the interaction between trinuclear ruthenium complexes and human serum albumin by means of fluorescence quenching Natacha Cacita, Sofia Nikolaou n Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. dos Bandeirantes 3900, 14040-901 Ribeirão Preto, SP, Brazil
art ic l e i nf o
a b s t r a c t
Article history: Received 15 January 2015 Received in revised form 30 July 2015 Accepted 21 August 2015 Available online 7 September 2015
This work reports for the first time a systematic investigation of the interaction between HSA and trinuclear ruthenium complexes, by using fluorescence spectroscopy and the Stern–Volmer model. The compounds investigated have general formula [Ru3O(CH3COO)6(3-pic)2(L)]PF6, where L¼ NO or H2O and 3-pic ¼3-methylpyridine. For both complexes it was observed that the increase of Ksv values (in the order of 104 M 1) with increasing temperature, signaling a dynamic quenching of HSA fluorescence. However, analysis of the quenching rate constants and Kb values shows that the contribution of the static quenching is significant. Particularly in the case of the nitrosyl complex, the relatively high value of Kb observed (178.44 103 M 1, 308 K) suggests that this compound can be efficiently stored and transported in the body by HSA. The interaction of the complexes with HSA is spontaneous (ΔG o 0). Complex [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 displays interaction with HSA by hydrophobic forces (ΔH ¼215 kJ mol 1 and ΔS ¼796 J mol 1 K 1), likely because of the nitrosyl lipophilicity, while complex [Ru3O (CH3COO)6(3-pic)2(H2O)]PF6 is involved in the formation of hydrogen bonds with HSA (ΔH ¼ 75.5 kJ mol 1 and ΔS ¼ 231 J mol 1 K 1), through its aquo ligand. & 2015 Elsevier B.V. All rights reserved.
Keywords: Triruthenium μ-oxo nitrosyl complexes Human serum albumin Fluorescence quenching Stern–Volmer
1. Introduction Human serum albumin (HSA) is produced in the liver and is the most abundant protein present in blood plasma. This protein acts in several physiological processes, including the regulation of osmotic pressure, transmission, distribution and metabolism of several ligands, and it is responsible for regulating blood pH [1–6]. Binding affinity to HSA is highly related to the distribution, free concentration and metabolism of ligands or drugs, therefore it is of great importance to study these interactions [7,8]. To study the interaction between a drug and HSA protein, the fluorescence technique is widely used. It is possible to verify from which residue the observed fluorescence is derived, thus determining the number of interacting sites between the drug and the protein. It is also feasible that the determination of the binding type and the mechanism by which this binding occurs, as well as the determination of the distance that this interaction site is from the tryptophan residue [9–15]. Since the discovery of the use of ruthenium compounds as metallodrugs, many studies are being developed to synthesize new compounds that may be used in the treatment of various n
Corresponding author. E-mail address: sofi[email protected] (S. Nikolaou).
http://dx.doi.org/10.1016/j.jlumin.2015.08.066 0022-2313/& 2015 Elsevier B.V. All rights reserved.
diseases, including cancer. The development of metallodrugs allows the replacement of currently used drugs, since the coordination to a metal center may increase the activity of a given drug, as well as reduce its side effects [16]. Such as iron, ruthenium ion has low toxicity due to their various oxidation states available in physiological environment (II, III, IV) [17]. Also, their complexes can act both as NO scavengers or releasers, increasing the interest on the development of a variety of nitrosyl ruthenium complexes [17–26]. Of interest to this work, trinuclear ruthenium complexes of general formula [Ru3O(CH3COO)6(L)3]PF6, with L ¼N-heterocycles, have been widely studied in recent decades due to its rich mixed-valence chemistry and catalytic properties [27]. Also of interest is the nitric oxide molecule. NO is synthesized endogenously by the oxidation of L-arginine nitrogen, which is converted in L-citruline, catalyzed by NO synthase (NOS) [28,29]. An alternative for NO release from coordination compounds in physiological environment is Photodynamic Therapy, which involves the NO labilization mediated by light stimuli. Also, electrochemical stimuli of coordinated NO triggers its delivery, taking into account, in both cases, that NO0 has low affinity for metal centers in lower oxidation states [30]. Recently we have reported the synthesis and characterization of the novel NO releaser [Ru3O(CH3COO)6(3-pic)2(NO)]PF6, as well as its ability to relax pre-contract rat aorta [31]. In order to extend the investigation of bioinorganic aspects of this candidate to a
116
N. Cacita, S. Nikolaou / Journal of Luminescence 169 (2016) 115–120
Fig. 1. Pictorial view of the structures of compounds [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 and [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6.
metallodrug, the study presented herein describes the interaction between this trinuclear ruthenium compound and the corresponding aquo complex [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 with HSA. Investigating the aquo complex is worth because it constitutes the product of NO release from [Ru3O(CH3COO)6(3-pic)2(NO)]PF6. To our knowledge, it is the first report on this type of investigation for the trinuclear ruthenium μ-oxo bridged class of compounds (Fig. 1).
2. Material and methods 2.1. Materials and methods HSA was purchased from Sigma-Aldrich. The average molecular weight value of 66,500 g/mol was used in the preparation of protein solutions. Prior to each experiment, all solutions were freshly prepared in phosphate buffer (pH 7.4). Deionized water was used in the preparation of buffer solution. All chemicals were of analytical grade and were used without further purification. The complexes were previously prepared, purified and characterized [31]. Stock solutions (2.31 10 3 M) were prepared by dissolving an adequate mass of each complex in acetonitrile.
2.2. Kinetic experiments
2.3. Fluorescence spectroscopy Fluorescence spectra of the solution of HSA in the absence and presence of the complexes (0–18.5 10 6 M) were recorded in a Shimadzu fluorescence spectrophotometer model RF-5301PC, using a quartz cell with 1.0 cm optical path. During a typical fluorescence measurement, 3.0 mL of HSA solution (1.0 10 6 M) was firstly added to a 1.0 cm quartz cell and the fluorescence spectrum was recorded. Then, the complex solution aliquots were gradually added to the cell using a micropipette and the solution was incubated in the presence of ambient light for 5 min and for 120 min, before data acquisition. The wavelength 280 nm was used for sample excitation (tryptophan excitation) [33, 34]. The fluorescence spectrophotometer was set up with a slit width of 5 nm. The fluorescence emission spectra were measured at 298, 303, and 308 K. The intensity of the fluorescence was corrected to eliminate the inner filter effect of HSA and complexes using the following equation [15]:
Fcorr = Fobs e ( Aex + Aem ) /2
(3)
where Fcorr and Fobs are the corrected and observed fluorescence intensities, respectively. Aex and Aem are the absorbance values of the drugs at the excitation and emission wavelengths, respectively.
3. Results and discussion
The absorption spectra were recorded in an Agilent 8453 spectrophotometer in the 190–1100 nm region, using a quartz cell with 1.0 cm optical path. A solution of the complex [Ru3O (CH3COO)6(3-pic)2(NO)]PF6 (2.31 10 3 M) was prepared in acetonitrile and an aliquot of this solution was added to a buffer solution containing albumin (1 10 6 M) in order to provide a concentration of 5.37 10 6 M. The solution was incubated at 303 K in the presence of ambient light and the absorption spectra were recorded as a function of time. The kinetic constant and the half life time were calculated using the following equations [32]:
[A] = ⎡⎣ A 0 ⎤⎦ e−kT
(1)
t1/2 = ln 2/k
(2)
where A and A0 are the absorbance and the initial absorbance respectively, T is the temperature, t1/2 is the half life time and k is the kinectic constant.
3.1. Fluorescence-quenching of HSA by [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 and [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 The fluorescence of the HSA molecule is largely due to the presence of the tryptophan residue and, in a smaller extent due to tyrosine, depending on the excitation wavelength [35,36]. The fluorescence intensity decreases when certain substrates are added to a HSA solution. Therefore, fluorescence spectroscopy is widely used to investigate the interaction between the protein and other molecules [12–15]. The photochemical behavior of trinuclear ruthenium clusters with a NO ligand such as [Ru3O(CH3COO)6(3-pic)2(NO)]PF6, has been described in the literature [31,37]. Although the nitrosyls are thermally stable, in solution and in presence of light, NO substitution by a solvent molecule may occur [20,37]. Because of this known reactivity, a control experiment was performed. The complex [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 was incubated with a HSA in buffer solution in order to verify, for a given time interval and in the presence of ambient light and of albumin, whether is the
N. Cacita, S. Nikolaou / Journal of Luminescence 169 (2016) 115–120
117
Fig. 2. Changes in spectral profile (up) and absorbance at 452 nm with time (bottom) for a buffer solution of the complex [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 (5.37 10 6 M) in the presence of HSA (1 10 6 M), incubated at 303 K.
nitrosyl or the aquo complex that actually interacts with the protein (Fig. 2). As one can see, the decrease in intensity of the band at 452 nm and the appearance of a band around 680 nm indicates the partial loss of the NO ligand, with generation of the [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 species, which presents a band at 679 nm (insert of Fig. 2). From the plots depicted in Fig. 2, the pseudo-first-order rate constant for the aquation of complex [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 in the presence of HSA was calculated to be kobs ¼ 1.36 10 4 s 1 and the τ1/2 ¼85 min. From this results, it was determined that a 5 min incubation of compound [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 with HSA guarantees that virtually only the nitrosyl is present in solution. On the other hand, an incubation period of 120 min leads us predominantly to the analysis of the aquo species [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 interaction with HSA. Accordingly, Fig. 3 shows that for both incubation times, HSA fluorescence intensity decreases when the concentration of the complex in solution increases. Two types of fluorescence quenching may occur: static and/or dynamic type. When the dynamic suppression mechanism predominates, the interaction between the quencher and the excited state of the fluorophore occurs through collisions during the lifetime of the fluorophore excite state, and the process has diffusion control. On the other hand, when quenching is static, the interaction between the fluorophore and quencher occurs in the ground state, with formation of a new species which is not luminescent [14,38]. The Stern–Volmer equation (Eq. (4)), is widely used to determine the type of suppression mechanism by analyzing the relation F0/F at different temperatures:
Fig. 3. Fluorescence spectra of HSA in presence of [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 (A) for 5 min incubation; concentrations of quencher: 0; 2.57; 5.15; 7.70; 10.2; 12.8; 15.4; 17.9; 20.5; 23.1 10 6 M, and (B) for 120 min incubation; concentrations of quencher 0; 3.08; 6.16; 9.24; 12.3; 15.4; 18.5 10 6 M. Both experiments were carried out at 303 K, λexc ¼ 280 nm.
F0/F = KSV [Q ] + 1 = τ 0 k q [Q ] + 1
(4)
where F0 and F are the fluorescence intensities in the absence and presence of the quencher, respectively, KSV is the Stern–Volmer quenching constant, kq is the bimolecular quenching constant, τ0 is the average life time of the fluorophore without the quencher ( 10 8 s) [39], and [Q] is the concentration of the quencher. Fig. 4 presents the Stern–Volmer plots for the HSA interaction with both compounds under investigation in this work. The KSV values were determined from the slopes of the F0/F versus [Q] plots. Then, the kq values were calculated using Eq. (4) (Table 1). The first conclusion drawn from the data depicted in Fig. 4 and Table 1 is that, presumably, only one-type of quenching process occurs in the concentrations range investigated. The analysis of KSV dependence on temperature allows one to estimate the quenching mechanism for the system under investigation. Based exclusively on the analysis of the Stern–Volmer plot, for the nitrosyl species, the value of KSV increases with increasing temperature in the presence of the complexes, suggesting that the mechanism involved is dynamic. For the aquo compound [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6, the Ksv values follow the same trend, although it increases less than for the nitrosyl species. Nevertheless, the values of the quenching rate constant (Table 1) appeared to be larger than the maximum diffusion constant
118
N. Cacita, S. Nikolaou / Journal of Luminescence 169 (2016) 115–120
(2 1010 M 1 s 1), which is characteristic of the static quenching mechanism [40]. Overall, despite the linearity observed for the Stern–Volmer plots, the analysis of Ksv and kq suggests the occurrence of both types of quenching mechanisms. Especially in the case of the aquo complex, for which the Ksv increases less with temperature, and taking into account
that the quenching of fluorescence might be initiated by the formation of a complex between HSA and [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 [15,40–43]. This observation is quite consistent if one considers that the aquo species [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 contains in its structure one labile solvent molecule, providing a free coordination site to interact with amino acid residues available in the protein. Moreover, considering the incubation time used for the two species (5 min for the nitrosyl and 120 min for the aquo species), it is more likely that the formation of a protein-quencher complex in the ground state occurs to a greater extent for [Ru3O(CH3COO)6(3-pic)2H2O)]PF6 than for [Ru3O(CH3COO)6(3-pic)2(NO)]PF6. 3.2. Binding parameters Since we observed that the static mechanism contributes to the quenching of HSA, possibly to different extents for the nitrosyl and aquo species, it was performed an analysis of the binding constants of compounds to HSA. The binding constant (Kb) and number of binding sites (n) are calculated by using the modified Stern–Volmer equation:
log ( F0 − F )/F = log Kb + n log [Q ]
Fig. 4. Stern–Volmer plots for (A)HSA-[Ru3O(CH3COO)6(3-pic)2(NO)]PF6 and (B) HSA-[Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 system at different temperatures, λexc ¼ 280 nm.
(5)
where F0 and F are the fluorescence intensities in the absence and presence of the quencher, respectively, Kb is the binding constant and n is the average number of biding site per protein molecule. The dependence of log(F0 F)/F on the value of log[Q] is linear with a slope equal to n, and Kb is fixed on the ordinate. The results are given in Table 2. The analysis of Kb values is rather useful to infer how a molecular species, particularly a drug, will be distributed in blood plasma. In general, lower values of Kb mean that the interaction between drug and a given transport protein is weak, leading to poor distribution in plasma. On the other hand, high values of Kb lead to a decrease in the concentration of free drug in plasma, since the interaction with the protein is stronger [43]. The relatively high Kb values ( 103 M) obtained for the compounds under investigation suggest that there is a strong interaction between the complexes and the HSA, showing that the static component of quenching must be significant. Also, it is observed that the binding constant for the HSA-[Ru3O(CH3COO)6 (3-pic)2(NO)]PF6 system increases with increasing temperature, which further supports the contribution of the static quenching
Table 1 Stern–Volmer quenching constants for HSA-[Ru3O(CH3COO)6(3-pic)2(NO)]PF6 system and HSA-[Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 system at different temperatures. HSA-[Ru3O(CH3COO)6(3-pic)2(NO)]PF6 4
1
T (K)
Ksv (10 M
)
298 303 308
3.677 0.0538 4.29 7 0.1094 6.30 7 0.0895
HSA-[Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 kq (10
12
M
1
s
1
)
3.6770.0538 4.29 70.1094 6.30 70.0895
R
Ksv (104 M 1)
kq (1012 M 1 s 1)
R
0.999 0.996 0.998
4.6770.3381 5.18 70.2335 5.31 70.2272
4.6770.3381 5.18 70.2335 5.31 70.2272
0.996 0.998 0.998
Table 2 The binding constant Kb, and the number of binding sites n of the HSA-[Ru3O(CH3COO)6(3-pic)2(NO)]PF6 and HSA-[Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 system at different temperatures. HSA-[Ru3O(CH3COO)6(3-pic)2(NO)]PF6
HSA-[Ru3O(CH3COO)6(3-pic)2(H2O)]PF6
T (K)
Kb (103 M 1)
n
R
Kb (103 M 1)
n
R
298 303 308
10.487 0.085 15.55 7 0.135 178.447 0.137
0.95 0.91 1.08
0.999 0.997 0.997
12.88 7 0.0327 8.717 0.0721 4.78 7 0.0443
0.86 0.82 0.77
0.995 0.996 0.991
N. Cacita, S. Nikolaou / Journal of Luminescence 169 (2016) 115–120
mechanism, since in this case the stability of the association protein-complex increase with increasing temperatures. It is worth noting that there is a large increase in the Kb value for the nitrosyl at 308 K, implying that this complex can be stored and transported in the body by HSA. However, for the system HSA/[Ru3O(CH3COO)6(3-pic)2(H2O)]PF6, the Kb values decrease with increasing temperatures, which impairs the ability of HSA to store and transport the aquo complex in a temperature closer to the body temperatures. It is also observed in Table 2 that the number of binding sites n is next to 1 in all cases, suggesting the presence of a single binding site for the complex in the HSA molecule. 3.3. Thermodynamic parameters and binding modes In general, the interaction between small molecules and biological macromolecules can be described by four types of interaction: hydrophobic, hydrogen bonding, van der Waals and electrostatic interactions [44,45].
119
The enthalpy (ΔH) and entropy (ΔS) values can be determined from van’t Hoff equation:
ln Kb = − ΔH /RT + ΔS/R
(6)
where Kb is the binding constant at a temperature T and R is the gas constant. ΔH and ΔS values are calculated from the slope and intercept values of a ln Kb versus 1/T plot, respectively (Fig. 5). Then, ΔG values are determined from the following equation:
(7)
ΔG = − RT ln Kb
The thermodynamic parameters are dependent on temperature. Changes in free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) are used to characterize the interaction forces or types of bond. ΔG is related to the spontaneity of reaction, while ΔH and ΔS are the main quantities to assess the strength of a bond. Negative ΔH and ΔS values indicate the presence of hydrogen bonds and/or van der Waals forces; negative ΔH and positive ΔS values suggests the presence of electrostatic interactions and positive ΔH and ΔS values indicate the presence of hydrophobic interactions [38,46]. Van’t Hoff plots for the systems under investigation are depicted in Fig. 5 and the calculated thermodynamics parameters are given in Table 3. It can be seen in Table 3 that all ΔG values are negative. This result indicates the spontaneity of the complexes binding to HSA. In the case of the nitrosyl [Ru3O(CH3COO)6(3-pic)2(NO)]PF6, the positive values of ΔH and ΔS suggest that hydrophobic forces play a major role in its binding to HSA, while the negative values of ΔH and ΔS indicates the presence of hydrogen bonds and/or van der Waals forces in the binding reactions for the aquo compound [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6. In both cases, the presence of a methyl group on the 3-picoline ligand confers some hydrophobicity to the complexes that, otherwise, would be more prone to present electrostatic interactions with the protein, since they are cationic molecules. However, the NO ligand constitutes a lipophilic site [28] which could assist the hydrophobic interaction suggested by the calculated values of ΔH and ΔS. The aquo complex in its turn, features the water molecule in its structure that, together with the 3-picoline methyl group, should be considered as sites for formation of hydrogen bonds.
4. Conclusions
Fig. 5. Van't Hoff plot for the binding of (A) [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 to HSA and (B) [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 to HSA.
In this study we investigated the interaction between HSA and the [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 and [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 complexes, by using fluorescence spectroscopy and the Stern–Volmer model. For both species studied, the fluorescence quenching was observed with increasing concentration of complexes, the reaction ratios with HSA are suggested to be 1:1 HSA: complex and the processes are spontaneous (ΔGo0). It was observed that both dynamic and static quenching mechanisms are present and, despite the Ksv values increase with increasing temperature, our data suggests an important contribution of static quenching. Complex [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 displays interaction with HSA by
Table 3 Thermodynamic parameters for the HSA-[Ru3O(CH3COO)6(3-pic)2(NO)]PF6 and the HSA-[Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 system. HSA-[Ru3O(CH3COO)6(3-pic)2(NO)]PF6
HSA-[Ru3O(CH3COO)6(3-pic)2(H2O)]PF6
T (K)
ΔH (kJ mol 1)
ΔS (J mol 1 K 1)
ΔG (kJ mol 1)
ΔH (kJ mol 1)
ΔS (J mol 1 K 1)
ΔG (kJ mol 1)
298 303 308
215
796
22.94 24.32 30.96
75.5
231
6.34 5.44 3.99
120
N. Cacita, S. Nikolaou / Journal of Luminescence 169 (2016) 115–120
hydrophobic forces, likely because of the lipophilicity of the nitrosyl ligand, while complex [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 is involved in the formation of hydrogen bonds with HSA, through its aquo ligand. It is worth mentioning that the results described in this study will be important to help define the distribution and transport of these candidates to metallodrugs in blood plasma. The high Kb value observed for the nitrosyl complex at 308 K suggests that this compound can be efficiently stored and transported in the body by HSA.
Acknowledgments We thank Dr. Juliana Cristina Moraes Biazzotto, Clovis Junior and Prof. Dr. Roberto Santana da Silva for their collaboration in the acquisition of part of the data. This work was supported by a scholarship and grants 2012/ 23245-6 and 141892/2014-55 from Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), respectively.
References [1] U. Kragh-Hansen, V.T.G. Chuang, M. Otagiri, Biol. Pharm. Bull. 25 (2002) 695. [2] P.B. Kandagal, S. Ashoka, J. Seetharamappa, S.M.T. Shaikh, Y. Jadegoud, O.B. Ijare, J. Pharm. Biomed. Anal. 41 (2006) 393. [3] S.M.T. Shaikh, J. Seetharamappa, P.B. Kandagal, S. Ashoka, J. Mol. Struct. 786 (2006) 46. [4] A. Samanta, B.K. Paul, N. Guchhait, Biophys. Chem. 156 (2011) 128. [5] Z. Cheng, Mol. Biol. Rep. 39 (2012) 9493. [6] J. Jayabharathi, V. Thanikachalam, M.V. Perumal, J. Lumin. 132 (2012) 707. [7] M. Xu, F.J. Chen, L. Huang, P. Xi, Z. Zeng, J. Lumin. 131 (2011) 1557. [8] B.H.M. Hussein, J. Lumin. 131 (2011) 900. [9] A. Gong, X. Zhu, Y. Hu, S. Yu, Talanta 73 (2007) 668. [10] G. Suji, S.A. Khedkar, S.K. Singh, N. Kishore, E.C. Coutinho, V.M. Bhor, S. Sivakami, Protein J. 27 (2008) 205. [11] F. Mohammadi, A.K. Bordbar, A. Divsalar, K. Mohammadi, A.A. Saboury, Protein J. 28 (2009) 189. [12] S. Tayyab, S.K. Haq, M.A. Aziz Sabeeha, M.M. Khan, S. Muzammil, Int. J. Biol. Macromol. 26 (1999) 173. [13] M.A. Khan, S. Muzammil, J. Musarrat, Int. J. Biol. Macromol. 30 (2002) 243.
[14] J. Min, X. Meng-Xia, Z. Dong, L. Yuan, L. Xiao-Yu, C. Xing, J. Mol. Struct. 692 (2004) 71. [15] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed, Kluwer Academic Publisher, New York, Boston, Dordrecht, London, Moscow, 2006. [16] D.O. Silva, Anticancer Agents Med. Chem. 10 (2010) 312. [17] C.S. Allardyce, P.J. Dyson, Platin. Met. Rev. 45 (2001) 62. [18] Z.N. da Rocha, R.G. de Lima, F.G. Doro, E. Tfouni, R.S. da Silva, Inorg. Chem. Commun. 11 (2008) 737. [19] H.E. Toma, A.D.P. Alexiou, S. Dovidauskas, Eur. J. Inorg. Chem. 11 (2002) 3010. [20] H.E. Toma, A.D.P. Alexiou, A.L.B. Formiga, M. Nakamura, S. Dovidauskas, M. N. Eberlin, D.M. Tomazela, Inorg. Chim. Acta 358 (2005) 2891–2899. [21] R.C.L. Zampieri, G.V. Poelhsitz, A.A. Batista, O.R. Nascimento, J. Ellena, E.E. Castellano, J. Inorg. Biochem. 92 (2002) 82. [22] S.A. Cicillini, A.C.L. Prazias, A.C. Tedesco, O.A. Serra, R.S. da Silva, Polyhedron 28 (2009) 2766. [23] F. Marquele-Oliveira, D.C.A. Santana, S.F. Taveira, D.M. Vermeulen, A.R.M. de Oliveira, R.S. da Silva, R.F.V. Lopez, J. Pharm. Biomed. Anal. 53 (2010) 843. [24] E. Tfouni, F.G. Doro, A.J. Gomes, R.S. da Silva, G. Metzker, P.G.Z. Benini, D.W. Franco, Coord. Chem. Rev. 254 (2010) 355. [25] M.G. Sauaia, F.S. Oliveira, R.G. de Lima, A.L. Cacciari, E. Tfouni, R.S. da Silva, Inorg. Chem. Commun. 8 (2005) 347. [26] R.G. de Lima, M.G. Sauaia, C. Ferezin, I.M. Pepe, N.M. José, L.M. Bendhack, Z.N. Rocha, R.S. da Silva, Polyhedron 26 (2007) 4620. [27] H.E. Toma, K. Araki, A.D.P. Alexiou, S. Nikolaou, S. Dovidauskas, Coord. Chem. Rev. 221 (2001) 187. [28] S. Moncada, R.M.J. Palmer, E.A. Higgs, Pharmacol. Rev. 43 (1991) 109. [29] M.A. Marletta, J. Biol. Chem. 268 (1993) 12231. [30] G. Stochel, A. Wanat, E. Kulis, Z. Stasicka, Coord. Chem. Rev. 171 (1998) 203. [31] N. Cacita, B. Possato, C.F.N. da Silva, M. Paulo, A.L.B. Formiga, L.M. Bendhack, S. Nikolaou, Inorg. Chim. Acta 429 (2015) 114. [32] P. Atkins, L. Jones, L. Laverman, Chemical Principles, (2012) 1024. [33] O. Duman, S. Tunç, B. Kancı. Bozoğlan, J. Fluoresc. 23 (2013) 659. [34] S. Tunç, A. Cetinkaya, O. Duman, J. Photochem. Photobiol. B. 120 (2013) 59. [35] T.J. Peters, All about Albumin: Biochemistry, Genetics, and Medical Applications, first ed., Academic Press, San Diego, 1996. [36] M.X. Xie, X.Y. Xu, Y.D. Wang, Biochim. Biophys. Acta 1724 (2005) 215. [37] Z.A. Carneiro, J.C. Biazzotto, A.D.P. Alexiou, S. Nikolaou, J. Inorg. Biochem. 134 (2014) 36. [38] R. Liu, X. Yu, W. Gao, D. Ji, F. Yang, X. Li, J. Chen, H. Tao, H. Huang, P. Yi, Spectrochim. Acta A 78 (2011) 1535. [39] T.G. Dewey, Biophysical and Biochemical Aspects of Fluorescence Spectroscopy, first ed., Springer, US, New York, 1991. [40] W.R. Ware, J. Phys. Chem. 66 (1962) 455. [41] R.E. Maurice, A.G. Camillo, Anal. Biochem. 114 (1981) 199. [42] H.N. Hou, Z.D. Qi, Y.W. OuYang, F.L. Liao, Y. Zhang, Y. Liu, J. Pharm. Biomed. Anal. 47 (2008) 134. [43] N. Bijari, Y. Shokoohinia, M.R. Ashrafi-Kooshk, S. Ranjbar, S. Parvaneh, M. Moieni-Arya, R. Khodarahmi, J. Lumin. 143 (2013) 328. [44] Y. Hu, S. Xu, X. Zhu, A. Gong, Spectrochim. Acta A 74 (2009) 526. [45] H. Xu, Q. Liu, Y. Zuo, Y. Bi, S. Gao, J. Solut. Chem. 38 (2009) 15. [46] P.D. Ross, S. Subramanian, Biochemistry 20 (1981) 3096.
Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Studying the interaction between trinuclear ruthenium complexes and human serum albumin by means of fluorescence quenching Natacha Cacita, Sofia Nikolaou n Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. dos Bandeirantes 3900, 14040-901 Ribeirão Preto, SP, Brazil
art ic l e i nf o
a b s t r a c t
Article history: Received 15 January 2015 Received in revised form 30 July 2015 Accepted 21 August 2015 Available online 7 September 2015
This work reports for the first time a systematic investigation of the interaction between HSA and trinuclear ruthenium complexes, by using fluorescence spectroscopy and the Stern–Volmer model. The compounds investigated have general formula [Ru3O(CH3COO)6(3-pic)2(L)]PF6, where L¼ NO or H2O and 3-pic ¼3-methylpyridine. For both complexes it was observed that the increase of Ksv values (in the order of 104 M 1) with increasing temperature, signaling a dynamic quenching of HSA fluorescence. However, analysis of the quenching rate constants and Kb values shows that the contribution of the static quenching is significant. Particularly in the case of the nitrosyl complex, the relatively high value of Kb observed (178.44 103 M 1, 308 K) suggests that this compound can be efficiently stored and transported in the body by HSA. The interaction of the complexes with HSA is spontaneous (ΔG o 0). Complex [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 displays interaction with HSA by hydrophobic forces (ΔH ¼215 kJ mol 1 and ΔS ¼796 J mol 1 K 1), likely because of the nitrosyl lipophilicity, while complex [Ru3O (CH3COO)6(3-pic)2(H2O)]PF6 is involved in the formation of hydrogen bonds with HSA (ΔH ¼ 75.5 kJ mol 1 and ΔS ¼ 231 J mol 1 K 1), through its aquo ligand. & 2015 Elsevier B.V. All rights reserved.
Keywords: Triruthenium μ-oxo nitrosyl complexes Human serum albumin Fluorescence quenching Stern–Volmer
1. Introduction Human serum albumin (HSA) is produced in the liver and is the most abundant protein present in blood plasma. This protein acts in several physiological processes, including the regulation of osmotic pressure, transmission, distribution and metabolism of several ligands, and it is responsible for regulating blood pH [1–6]. Binding affinity to HSA is highly related to the distribution, free concentration and metabolism of ligands or drugs, therefore it is of great importance to study these interactions [7,8]. To study the interaction between a drug and HSA protein, the fluorescence technique is widely used. It is possible to verify from which residue the observed fluorescence is derived, thus determining the number of interacting sites between the drug and the protein. It is also feasible that the determination of the binding type and the mechanism by which this binding occurs, as well as the determination of the distance that this interaction site is from the tryptophan residue [9–15]. Since the discovery of the use of ruthenium compounds as metallodrugs, many studies are being developed to synthesize new compounds that may be used in the treatment of various n
Corresponding author. E-mail address: sofi[email protected] (S. Nikolaou).
http://dx.doi.org/10.1016/j.jlumin.2015.08.066 0022-2313/& 2015 Elsevier B.V. All rights reserved.
diseases, including cancer. The development of metallodrugs allows the replacement of currently used drugs, since the coordination to a metal center may increase the activity of a given drug, as well as reduce its side effects [16]. Such as iron, ruthenium ion has low toxicity due to their various oxidation states available in physiological environment (II, III, IV) [17]. Also, their complexes can act both as NO scavengers or releasers, increasing the interest on the development of a variety of nitrosyl ruthenium complexes [17–26]. Of interest to this work, trinuclear ruthenium complexes of general formula [Ru3O(CH3COO)6(L)3]PF6, with L ¼N-heterocycles, have been widely studied in recent decades due to its rich mixed-valence chemistry and catalytic properties [27]. Also of interest is the nitric oxide molecule. NO is synthesized endogenously by the oxidation of L-arginine nitrogen, which is converted in L-citruline, catalyzed by NO synthase (NOS) [28,29]. An alternative for NO release from coordination compounds in physiological environment is Photodynamic Therapy, which involves the NO labilization mediated by light stimuli. Also, electrochemical stimuli of coordinated NO triggers its delivery, taking into account, in both cases, that NO0 has low affinity for metal centers in lower oxidation states [30]. Recently we have reported the synthesis and characterization of the novel NO releaser [Ru3O(CH3COO)6(3-pic)2(NO)]PF6, as well as its ability to relax pre-contract rat aorta [31]. In order to extend the investigation of bioinorganic aspects of this candidate to a
116
N. Cacita, S. Nikolaou / Journal of Luminescence 169 (2016) 115–120
Fig. 1. Pictorial view of the structures of compounds [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 and [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6.
metallodrug, the study presented herein describes the interaction between this trinuclear ruthenium compound and the corresponding aquo complex [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 with HSA. Investigating the aquo complex is worth because it constitutes the product of NO release from [Ru3O(CH3COO)6(3-pic)2(NO)]PF6. To our knowledge, it is the first report on this type of investigation for the trinuclear ruthenium μ-oxo bridged class of compounds (Fig. 1).
2. Material and methods 2.1. Materials and methods HSA was purchased from Sigma-Aldrich. The average molecular weight value of 66,500 g/mol was used in the preparation of protein solutions. Prior to each experiment, all solutions were freshly prepared in phosphate buffer (pH 7.4). Deionized water was used in the preparation of buffer solution. All chemicals were of analytical grade and were used without further purification. The complexes were previously prepared, purified and characterized [31]. Stock solutions (2.31 10 3 M) were prepared by dissolving an adequate mass of each complex in acetonitrile.
2.2. Kinetic experiments
2.3. Fluorescence spectroscopy Fluorescence spectra of the solution of HSA in the absence and presence of the complexes (0–18.5 10 6 M) were recorded in a Shimadzu fluorescence spectrophotometer model RF-5301PC, using a quartz cell with 1.0 cm optical path. During a typical fluorescence measurement, 3.0 mL of HSA solution (1.0 10 6 M) was firstly added to a 1.0 cm quartz cell and the fluorescence spectrum was recorded. Then, the complex solution aliquots were gradually added to the cell using a micropipette and the solution was incubated in the presence of ambient light for 5 min and for 120 min, before data acquisition. The wavelength 280 nm was used for sample excitation (tryptophan excitation) [33, 34]. The fluorescence spectrophotometer was set up with a slit width of 5 nm. The fluorescence emission spectra were measured at 298, 303, and 308 K. The intensity of the fluorescence was corrected to eliminate the inner filter effect of HSA and complexes using the following equation [15]:
Fcorr = Fobs e ( Aex + Aem ) /2
(3)
where Fcorr and Fobs are the corrected and observed fluorescence intensities, respectively. Aex and Aem are the absorbance values of the drugs at the excitation and emission wavelengths, respectively.
3. Results and discussion
The absorption spectra were recorded in an Agilent 8453 spectrophotometer in the 190–1100 nm region, using a quartz cell with 1.0 cm optical path. A solution of the complex [Ru3O (CH3COO)6(3-pic)2(NO)]PF6 (2.31 10 3 M) was prepared in acetonitrile and an aliquot of this solution was added to a buffer solution containing albumin (1 10 6 M) in order to provide a concentration of 5.37 10 6 M. The solution was incubated at 303 K in the presence of ambient light and the absorption spectra were recorded as a function of time. The kinetic constant and the half life time were calculated using the following equations [32]:
[A] = ⎡⎣ A 0 ⎤⎦ e−kT
(1)
t1/2 = ln 2/k
(2)
where A and A0 are the absorbance and the initial absorbance respectively, T is the temperature, t1/2 is the half life time and k is the kinectic constant.
3.1. Fluorescence-quenching of HSA by [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 and [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 The fluorescence of the HSA molecule is largely due to the presence of the tryptophan residue and, in a smaller extent due to tyrosine, depending on the excitation wavelength [35,36]. The fluorescence intensity decreases when certain substrates are added to a HSA solution. Therefore, fluorescence spectroscopy is widely used to investigate the interaction between the protein and other molecules [12–15]. The photochemical behavior of trinuclear ruthenium clusters with a NO ligand such as [Ru3O(CH3COO)6(3-pic)2(NO)]PF6, has been described in the literature [31,37]. Although the nitrosyls are thermally stable, in solution and in presence of light, NO substitution by a solvent molecule may occur [20,37]. Because of this known reactivity, a control experiment was performed. The complex [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 was incubated with a HSA in buffer solution in order to verify, for a given time interval and in the presence of ambient light and of albumin, whether is the
N. Cacita, S. Nikolaou / Journal of Luminescence 169 (2016) 115–120
117
Fig. 2. Changes in spectral profile (up) and absorbance at 452 nm with time (bottom) for a buffer solution of the complex [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 (5.37 10 6 M) in the presence of HSA (1 10 6 M), incubated at 303 K.
nitrosyl or the aquo complex that actually interacts with the protein (Fig. 2). As one can see, the decrease in intensity of the band at 452 nm and the appearance of a band around 680 nm indicates the partial loss of the NO ligand, with generation of the [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 species, which presents a band at 679 nm (insert of Fig. 2). From the plots depicted in Fig. 2, the pseudo-first-order rate constant for the aquation of complex [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 in the presence of HSA was calculated to be kobs ¼ 1.36 10 4 s 1 and the τ1/2 ¼85 min. From this results, it was determined that a 5 min incubation of compound [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 with HSA guarantees that virtually only the nitrosyl is present in solution. On the other hand, an incubation period of 120 min leads us predominantly to the analysis of the aquo species [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 interaction with HSA. Accordingly, Fig. 3 shows that for both incubation times, HSA fluorescence intensity decreases when the concentration of the complex in solution increases. Two types of fluorescence quenching may occur: static and/or dynamic type. When the dynamic suppression mechanism predominates, the interaction between the quencher and the excited state of the fluorophore occurs through collisions during the lifetime of the fluorophore excite state, and the process has diffusion control. On the other hand, when quenching is static, the interaction between the fluorophore and quencher occurs in the ground state, with formation of a new species which is not luminescent [14,38]. The Stern–Volmer equation (Eq. (4)), is widely used to determine the type of suppression mechanism by analyzing the relation F0/F at different temperatures:
Fig. 3. Fluorescence spectra of HSA in presence of [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 (A) for 5 min incubation; concentrations of quencher: 0; 2.57; 5.15; 7.70; 10.2; 12.8; 15.4; 17.9; 20.5; 23.1 10 6 M, and (B) for 120 min incubation; concentrations of quencher 0; 3.08; 6.16; 9.24; 12.3; 15.4; 18.5 10 6 M. Both experiments were carried out at 303 K, λexc ¼ 280 nm.
F0/F = KSV [Q ] + 1 = τ 0 k q [Q ] + 1
(4)
where F0 and F are the fluorescence intensities in the absence and presence of the quencher, respectively, KSV is the Stern–Volmer quenching constant, kq is the bimolecular quenching constant, τ0 is the average life time of the fluorophore without the quencher ( 10 8 s) [39], and [Q] is the concentration of the quencher. Fig. 4 presents the Stern–Volmer plots for the HSA interaction with both compounds under investigation in this work. The KSV values were determined from the slopes of the F0/F versus [Q] plots. Then, the kq values were calculated using Eq. (4) (Table 1). The first conclusion drawn from the data depicted in Fig. 4 and Table 1 is that, presumably, only one-type of quenching process occurs in the concentrations range investigated. The analysis of KSV dependence on temperature allows one to estimate the quenching mechanism for the system under investigation. Based exclusively on the analysis of the Stern–Volmer plot, for the nitrosyl species, the value of KSV increases with increasing temperature in the presence of the complexes, suggesting that the mechanism involved is dynamic. For the aquo compound [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6, the Ksv values follow the same trend, although it increases less than for the nitrosyl species. Nevertheless, the values of the quenching rate constant (Table 1) appeared to be larger than the maximum diffusion constant
118
N. Cacita, S. Nikolaou / Journal of Luminescence 169 (2016) 115–120
(2 1010 M 1 s 1), which is characteristic of the static quenching mechanism [40]. Overall, despite the linearity observed for the Stern–Volmer plots, the analysis of Ksv and kq suggests the occurrence of both types of quenching mechanisms. Especially in the case of the aquo complex, for which the Ksv increases less with temperature, and taking into account
that the quenching of fluorescence might be initiated by the formation of a complex between HSA and [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 [15,40–43]. This observation is quite consistent if one considers that the aquo species [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 contains in its structure one labile solvent molecule, providing a free coordination site to interact with amino acid residues available in the protein. Moreover, considering the incubation time used for the two species (5 min for the nitrosyl and 120 min for the aquo species), it is more likely that the formation of a protein-quencher complex in the ground state occurs to a greater extent for [Ru3O(CH3COO)6(3-pic)2H2O)]PF6 than for [Ru3O(CH3COO)6(3-pic)2(NO)]PF6. 3.2. Binding parameters Since we observed that the static mechanism contributes to the quenching of HSA, possibly to different extents for the nitrosyl and aquo species, it was performed an analysis of the binding constants of compounds to HSA. The binding constant (Kb) and number of binding sites (n) are calculated by using the modified Stern–Volmer equation:
log ( F0 − F )/F = log Kb + n log [Q ]
Fig. 4. Stern–Volmer plots for (A)HSA-[Ru3O(CH3COO)6(3-pic)2(NO)]PF6 and (B) HSA-[Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 system at different temperatures, λexc ¼ 280 nm.
(5)
where F0 and F are the fluorescence intensities in the absence and presence of the quencher, respectively, Kb is the binding constant and n is the average number of biding site per protein molecule. The dependence of log(F0 F)/F on the value of log[Q] is linear with a slope equal to n, and Kb is fixed on the ordinate. The results are given in Table 2. The analysis of Kb values is rather useful to infer how a molecular species, particularly a drug, will be distributed in blood plasma. In general, lower values of Kb mean that the interaction between drug and a given transport protein is weak, leading to poor distribution in plasma. On the other hand, high values of Kb lead to a decrease in the concentration of free drug in plasma, since the interaction with the protein is stronger [43]. The relatively high Kb values ( 103 M) obtained for the compounds under investigation suggest that there is a strong interaction between the complexes and the HSA, showing that the static component of quenching must be significant. Also, it is observed that the binding constant for the HSA-[Ru3O(CH3COO)6 (3-pic)2(NO)]PF6 system increases with increasing temperature, which further supports the contribution of the static quenching
Table 1 Stern–Volmer quenching constants for HSA-[Ru3O(CH3COO)6(3-pic)2(NO)]PF6 system and HSA-[Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 system at different temperatures. HSA-[Ru3O(CH3COO)6(3-pic)2(NO)]PF6 4
1
T (K)
Ksv (10 M
)
298 303 308
3.677 0.0538 4.29 7 0.1094 6.30 7 0.0895
HSA-[Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 kq (10
12
M
1
s
1
)
3.6770.0538 4.29 70.1094 6.30 70.0895
R
Ksv (104 M 1)
kq (1012 M 1 s 1)
R
0.999 0.996 0.998
4.6770.3381 5.18 70.2335 5.31 70.2272
4.6770.3381 5.18 70.2335 5.31 70.2272
0.996 0.998 0.998
Table 2 The binding constant Kb, and the number of binding sites n of the HSA-[Ru3O(CH3COO)6(3-pic)2(NO)]PF6 and HSA-[Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 system at different temperatures. HSA-[Ru3O(CH3COO)6(3-pic)2(NO)]PF6
HSA-[Ru3O(CH3COO)6(3-pic)2(H2O)]PF6
T (K)
Kb (103 M 1)
n
R
Kb (103 M 1)
n
R
298 303 308
10.487 0.085 15.55 7 0.135 178.447 0.137
0.95 0.91 1.08
0.999 0.997 0.997
12.88 7 0.0327 8.717 0.0721 4.78 7 0.0443
0.86 0.82 0.77
0.995 0.996 0.991
N. Cacita, S. Nikolaou / Journal of Luminescence 169 (2016) 115–120
mechanism, since in this case the stability of the association protein-complex increase with increasing temperatures. It is worth noting that there is a large increase in the Kb value for the nitrosyl at 308 K, implying that this complex can be stored and transported in the body by HSA. However, for the system HSA/[Ru3O(CH3COO)6(3-pic)2(H2O)]PF6, the Kb values decrease with increasing temperatures, which impairs the ability of HSA to store and transport the aquo complex in a temperature closer to the body temperatures. It is also observed in Table 2 that the number of binding sites n is next to 1 in all cases, suggesting the presence of a single binding site for the complex in the HSA molecule. 3.3. Thermodynamic parameters and binding modes In general, the interaction between small molecules and biological macromolecules can be described by four types of interaction: hydrophobic, hydrogen bonding, van der Waals and electrostatic interactions [44,45].
119
The enthalpy (ΔH) and entropy (ΔS) values can be determined from van’t Hoff equation:
ln Kb = − ΔH /RT + ΔS/R
(6)
where Kb is the binding constant at a temperature T and R is the gas constant. ΔH and ΔS values are calculated from the slope and intercept values of a ln Kb versus 1/T plot, respectively (Fig. 5). Then, ΔG values are determined from the following equation:
(7)
ΔG = − RT ln Kb
The thermodynamic parameters are dependent on temperature. Changes in free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) are used to characterize the interaction forces or types of bond. ΔG is related to the spontaneity of reaction, while ΔH and ΔS are the main quantities to assess the strength of a bond. Negative ΔH and ΔS values indicate the presence of hydrogen bonds and/or van der Waals forces; negative ΔH and positive ΔS values suggests the presence of electrostatic interactions and positive ΔH and ΔS values indicate the presence of hydrophobic interactions [38,46]. Van’t Hoff plots for the systems under investigation are depicted in Fig. 5 and the calculated thermodynamics parameters are given in Table 3. It can be seen in Table 3 that all ΔG values are negative. This result indicates the spontaneity of the complexes binding to HSA. In the case of the nitrosyl [Ru3O(CH3COO)6(3-pic)2(NO)]PF6, the positive values of ΔH and ΔS suggest that hydrophobic forces play a major role in its binding to HSA, while the negative values of ΔH and ΔS indicates the presence of hydrogen bonds and/or van der Waals forces in the binding reactions for the aquo compound [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6. In both cases, the presence of a methyl group on the 3-picoline ligand confers some hydrophobicity to the complexes that, otherwise, would be more prone to present electrostatic interactions with the protein, since they are cationic molecules. However, the NO ligand constitutes a lipophilic site [28] which could assist the hydrophobic interaction suggested by the calculated values of ΔH and ΔS. The aquo complex in its turn, features the water molecule in its structure that, together with the 3-picoline methyl group, should be considered as sites for formation of hydrogen bonds.
4. Conclusions
Fig. 5. Van't Hoff plot for the binding of (A) [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 to HSA and (B) [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 to HSA.
In this study we investigated the interaction between HSA and the [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 and [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 complexes, by using fluorescence spectroscopy and the Stern–Volmer model. For both species studied, the fluorescence quenching was observed with increasing concentration of complexes, the reaction ratios with HSA are suggested to be 1:1 HSA: complex and the processes are spontaneous (ΔGo0). It was observed that both dynamic and static quenching mechanisms are present and, despite the Ksv values increase with increasing temperature, our data suggests an important contribution of static quenching. Complex [Ru3O(CH3COO)6(3-pic)2(NO)]PF6 displays interaction with HSA by
Table 3 Thermodynamic parameters for the HSA-[Ru3O(CH3COO)6(3-pic)2(NO)]PF6 and the HSA-[Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 system. HSA-[Ru3O(CH3COO)6(3-pic)2(NO)]PF6
HSA-[Ru3O(CH3COO)6(3-pic)2(H2O)]PF6
T (K)
ΔH (kJ mol 1)
ΔS (J mol 1 K 1)
ΔG (kJ mol 1)
ΔH (kJ mol 1)
ΔS (J mol 1 K 1)
ΔG (kJ mol 1)
298 303 308
215
796
22.94 24.32 30.96
75.5
231
6.34 5.44 3.99
120
N. Cacita, S. Nikolaou / Journal of Luminescence 169 (2016) 115–120
hydrophobic forces, likely because of the lipophilicity of the nitrosyl ligand, while complex [Ru3O(CH3COO)6(3-pic)2(H2O)]PF6 is involved in the formation of hydrogen bonds with HSA, through its aquo ligand. It is worth mentioning that the results described in this study will be important to help define the distribution and transport of these candidates to metallodrugs in blood plasma. The high Kb value observed for the nitrosyl complex at 308 K suggests that this compound can be efficiently stored and transported in the body by HSA.
Acknowledgments We thank Dr. Juliana Cristina Moraes Biazzotto, Clovis Junior and Prof. Dr. Roberto Santana da Silva for their collaboration in the acquisition of part of the data. This work was supported by a scholarship and grants 2012/ 23245-6 and 141892/2014-55 from Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), respectively.
References [1] U. Kragh-Hansen, V.T.G. Chuang, M. Otagiri, Biol. Pharm. Bull. 25 (2002) 695. [2] P.B. Kandagal, S. Ashoka, J. Seetharamappa, S.M.T. Shaikh, Y. Jadegoud, O.B. Ijare, J. Pharm. Biomed. Anal. 41 (2006) 393. [3] S.M.T. Shaikh, J. Seetharamappa, P.B. Kandagal, S. Ashoka, J. Mol. Struct. 786 (2006) 46. [4] A. Samanta, B.K. Paul, N. Guchhait, Biophys. Chem. 156 (2011) 128. [5] Z. Cheng, Mol. Biol. Rep. 39 (2012) 9493. [6] J. Jayabharathi, V. Thanikachalam, M.V. Perumal, J. Lumin. 132 (2012) 707. [7] M. Xu, F.J. Chen, L. Huang, P. Xi, Z. Zeng, J. Lumin. 131 (2011) 1557. [8] B.H.M. Hussein, J. Lumin. 131 (2011) 900. [9] A. Gong, X. Zhu, Y. Hu, S. Yu, Talanta 73 (2007) 668. [10] G. Suji, S.A. Khedkar, S.K. Singh, N. Kishore, E.C. Coutinho, V.M. Bhor, S. Sivakami, Protein J. 27 (2008) 205. [11] F. Mohammadi, A.K. Bordbar, A. Divsalar, K. Mohammadi, A.A. Saboury, Protein J. 28 (2009) 189. [12] S. Tayyab, S.K. Haq, M.A. Aziz Sabeeha, M.M. Khan, S. Muzammil, Int. J. Biol. Macromol. 26 (1999) 173. [13] M.A. Khan, S. Muzammil, J. Musarrat, Int. J. Biol. Macromol. 30 (2002) 243.
[14] J. Min, X. Meng-Xia, Z. Dong, L. Yuan, L. Xiao-Yu, C. Xing, J. Mol. Struct. 692 (2004) 71. [15] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed, Kluwer Academic Publisher, New York, Boston, Dordrecht, London, Moscow, 2006. [16] D.O. Silva, Anticancer Agents Med. Chem. 10 (2010) 312. [17] C.S. Allardyce, P.J. Dyson, Platin. Met. Rev. 45 (2001) 62. [18] Z.N. da Rocha, R.G. de Lima, F.G. Doro, E. Tfouni, R.S. da Silva, Inorg. Chem. Commun. 11 (2008) 737. [19] H.E. Toma, A.D.P. Alexiou, S. Dovidauskas, Eur. J. Inorg. Chem. 11 (2002) 3010. [20] H.E. Toma, A.D.P. Alexiou, A.L.B. Formiga, M. Nakamura, S. Dovidauskas, M. N. Eberlin, D.M. Tomazela, Inorg. Chim. Acta 358 (2005) 2891–2899. [21] R.C.L. Zampieri, G.V. Poelhsitz, A.A. Batista, O.R. Nascimento, J. Ellena, E.E. Castellano, J. Inorg. Biochem. 92 (2002) 82. [22] S.A. Cicillini, A.C.L. Prazias, A.C. Tedesco, O.A. Serra, R.S. da Silva, Polyhedron 28 (2009) 2766. [23] F. Marquele-Oliveira, D.C.A. Santana, S.F. Taveira, D.M. Vermeulen, A.R.M. de Oliveira, R.S. da Silva, R.F.V. Lopez, J. Pharm. Biomed. Anal. 53 (2010) 843. [24] E. Tfouni, F.G. Doro, A.J. Gomes, R.S. da Silva, G. Metzker, P.G.Z. Benini, D.W. Franco, Coord. Chem. Rev. 254 (2010) 355. [25] M.G. Sauaia, F.S. Oliveira, R.G. de Lima, A.L. Cacciari, E. Tfouni, R.S. da Silva, Inorg. Chem. Commun. 8 (2005) 347. [26] R.G. de Lima, M.G. Sauaia, C. Ferezin, I.M. Pepe, N.M. José, L.M. Bendhack, Z.N. Rocha, R.S. da Silva, Polyhedron 26 (2007) 4620. [27] H.E. Toma, K. Araki, A.D.P. Alexiou, S. Nikolaou, S. Dovidauskas, Coord. Chem. Rev. 221 (2001) 187. [28] S. Moncada, R.M.J. Palmer, E.A. Higgs, Pharmacol. Rev. 43 (1991) 109. [29] M.A. Marletta, J. Biol. Chem. 268 (1993) 12231. [30] G. Stochel, A. Wanat, E. Kulis, Z. Stasicka, Coord. Chem. Rev. 171 (1998) 203. [31] N. Cacita, B. Possato, C.F.N. da Silva, M. Paulo, A.L.B. Formiga, L.M. Bendhack, S. Nikolaou, Inorg. Chim. Acta 429 (2015) 114. [32] P. Atkins, L. Jones, L. Laverman, Chemical Principles, (2012) 1024. [33] O. Duman, S. Tunç, B. Kancı. Bozoğlan, J. Fluoresc. 23 (2013) 659. [34] S. Tunç, A. Cetinkaya, O. Duman, J. Photochem. Photobiol. B. 120 (2013) 59. [35] T.J. Peters, All about Albumin: Biochemistry, Genetics, and Medical Applications, first ed., Academic Press, San Diego, 1996. [36] M.X. Xie, X.Y. Xu, Y.D. Wang, Biochim. Biophys. Acta 1724 (2005) 215. [37] Z.A. Carneiro, J.C. Biazzotto, A.D.P. Alexiou, S. Nikolaou, J. Inorg. Biochem. 134 (2014) 36. [38] R. Liu, X. Yu, W. Gao, D. Ji, F. Yang, X. Li, J. Chen, H. Tao, H. Huang, P. Yi, Spectrochim. Acta A 78 (2011) 1535. [39] T.G. Dewey, Biophysical and Biochemical Aspects of Fluorescence Spectroscopy, first ed., Springer, US, New York, 1991. [40] W.R. Ware, J. Phys. Chem. 66 (1962) 455. [41] R.E. Maurice, A.G. Camillo, Anal. Biochem. 114 (1981) 199. [42] H.N. Hou, Z.D. Qi, Y.W. OuYang, F.L. Liao, Y. Zhang, Y. Liu, J. Pharm. Biomed. Anal. 47 (2008) 134. [43] N. Bijari, Y. Shokoohinia, M.R. Ashrafi-Kooshk, S. Ranjbar, S. Parvaneh, M. Moieni-Arya, R. Khodarahmi, J. Lumin. 143 (2013) 328. [44] Y. Hu, S. Xu, X. Zhu, A. Gong, Spectrochim. Acta A 74 (2009) 526. [45] H. Xu, Q. Liu, Y. Zuo, Y. Bi, S. Gao, J. Solut. Chem. 38 (2009) 15. [46] P.D. Ross, S. Subramanian, Biochemistry 20 (1981) 3096.