Accepted Manuscript Interaction of prodigiosin with HSA and β-Lg: Spectroscopic and molecular docking studies Banafsheh Rastegari, Hamid Reza Karbalaei-Heidari, Reza Yousefi, Sedigheh Zeinali, Masoud Nabavizadeh PII: DOI: Reference:
S0968-0896(16)30100-6 http://dx.doi.org/10.1016/j.bmc.2016.02.020 BMC 12822
To appear in:
Bioorganic & Medicinal Chemistry
Received Date: Revised Date: Accepted Date:
28 November 2015 5 February 2016 12 February 2016
Please cite this article as: Rastegari, B., Karbalaei-Heidari, H.R., Yousefi, R., Zeinali, S., Nabavizadeh, M., Interaction of prodigiosin with HSA and β-Lg: Spectroscopic and molecular docking studies, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.02.020
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Interaction
of prodigiosin
with HSA and β-Lg:
spectroscopic and molecular docking studies Authors: Banafsheh Rastegaria, Hamid Reza Karbalaei-Heidaria,*, Reza Yousefib, Sedigheh Zeinalic, Masoud Nabavizadehd
a
Molecular Biotechnolgy Laboratory,
,
Department of Biology, Faculty of Science, Shiraz
University, Shiraz, 71454, Iran b
Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz
University, Shiraz, Iran c
Department of Nanochemical Engineering, Faculty of Advanced Technologies, Shiraz University,
Shiraz, Iran d
Department of Chemistry, Faculty of Science, Shiraz University, Shiraz, Iran
* Corresponding author. Address: Molecular Biotechnology Lab., Department of Biology, Faculty of Sciences, Shiraz University, Shiraz 71454, Iran; P. O. Box: 71467-13565; Tel: +98 71 32280916; Fax: + 98 71 32280926; E-mail: [email protected]
1
Abstract Human serum albumin (HSA) and Bovine β-lactoglobulin (β-Lg) are both introduced as blood and oral carrier scaffolds with high affinity for a wide range of pharmaceutical compounds.
Prodigiosin, a natural three pyrrolic compound produced by Serratia
marcescens, exhibits many pharmaceutical properties associated with health benefits. In the present study, the interaction of prodigiosin with HSA and β-Lg was investigated using fluorescence spectroscopy, circular dichroism (CD) and computational docking. Prodigiosin interacts with the Sudlow's site I of HSA and the calyx of β-Lg with association constant of 4.41x104 and 1.99x104 M-1 to form 1:1 and 2:3 complexes at 300 K, respectively. The results indicated that binding of prodigiosin to HSA and β-Lg caused strong fluorescence quenching of both proteins through static quenching mechanism. Electrostatic and hydrophobic interactions are the major forces in the stability of PG-HSA complex with enthalpy-and entropy-driving mode, although the formation of prodigiosinβ-Lg complex is entropy-driven hydrophobic associations. CD spectra showed slight conformational changes in both proteins due to the binding of prodigiosin. Moreover, the ligand displacement assay, pH-dependent interaction and protein–ligand docking study confirmed that the prodigiosin binds to residues located in the subdomain IIA and IIIA of HSA and central calyx of β-Lg.
Key words: Prodigiosin; HSA-PG interaction; β-Lg-PG binding study.
Abbreviations: PG: prodigiosin; β-Lg: β-lactoglobulin; HSA: Human Serum Albumin; CD: circular dichroism.
2
1. Introduction Prodigiosin, a member of prodiginine family (Fig. 1), is a red three pyrrole compound which mostly produced by bacterial resources like Serratia marcescens [1]. Antimicrobial, antimalarial, immunosuppressive and remarkable anticancer properties suggest prodigiosin as an appropriate drug candidate for various pharmaceutical purposes [2-4]. The selective cytotoxicity to malignant cell-lines motivated researchers to introduce prodigiosin as a potential anticancer drug and currently placed it in phase II clinical trials [5]. Prodigiosin, in a similar manner to the other poor water soluble drugs, exhibits limited bioavailability and must be bound to available polymers such as proteins to remain in its effective concentration in blood and other water-based mixtures. In addition, biodegradable protein like β-lactoglobulin (β-Lg) has been used in preparing encapsulated drugs. This protein is stable in stomach and suggested as a suitable vehicle for protein-based drug delivery to cancerous cells of digestive tract [6, 7].
Fig. 1. Optimized structure of the prodigiosin (PG).
Human serum albumin (HSA) is the most abundant plasma protein with an exceptional binding capacity for a wide variety of hydrophobic ligands and transport of hormones, fatty acids, amino acids, steroids, metal ions, metabolites such as bilirubin and pharmaceutical drug compounds. Low toxicity and non-immunogenic nature of this 3
protein is ideal for its application as intravenous drug carrier. The protein consisted of 585 residues with three structurally similar repeating domains-I (residues 1-195), II (196-383) and III (384-585)- each of which are divided into two sub-domains, A and B. The protein has two high affinity binding sites which are Sudlow's sites I and II. Site I as the major binding site, interacts with the most neutral heterocyclic ligands, while site II is mainly binds to many aromatic compounds [8, 9]. β-lactoglobulin (β-Lg), the major whey protein of bovine milk, is a small protein that consists of 162 amino acids (~18.3 kDa). It is normally exists as a mixture of monomers and dimers, depending on the protein concentration and its environmental pH. An important functional feature of this lipocalin family protein is its ability to bind several physiologically important non-water soluble ligands such as steroids, retinoids, fatty acids, vitamin D and cholesterol [10-12]. β-Lg consists of a large β-barrel with nine β strands (called A to I) and two ligand-binding sites; including a central calyx domain of the βbarrel and a surface hydrophobic pocket in the groove between the β-barrel and R-helix [13, 14]. Good solubilization, cost effectiveness and structural resistance to acidic pH of stomach, make it an ideal vehicle for encapsulation and a controlled release of hydrophobic compounds like prodigiosin [15, 16]. Study on the binding of prodigiosin to blood and oral matrixes represent a significant importance in applied pharmacology and biotechnology. Natural biomolecules such as proteins are an attractive group of scaffold compounds compared to synthetic ones, due to their low toxicity and biodegradability. Nanoparticle technology utilized several benefits of HSA and β-Lg, two successful scaffolds carrying hydrophobic ligands to the target sites. An essential step to design protein-based nanoparticle systems is to understand the binding sites and binding affinity of the protein-drug complex [17]. Although previous studies have been shown the interaction potential of prodigiosin with some proteins such as bovine hemoglobin and bovine serum albumin, to the best of our knowledge, there is no report on detailed interaction study of HSA and β-Lg with prodigiosin in literature [18, 19]. So, the potential of these proteins (HSA and β-Lg) as the intravenous and oral carrier molecules, respectively for transportation of the prodigiosin was investigated by determining the binding properties (including binding mechanism, binding constant, and the number of binding sites) of PG to the both proteins at three temperatures under simulative physiological conditions using fluorescence technique. We have also examined 4
the effect of prodigiosin on the secondary and tertiary structures of HSA and β–Lg using CD spectra. Finally, the binding site of the ligand was predicted by molecular modeling study.
2. Materials and methods 2.1. Materials HSA, Bovine β-Lg (B variant, purity 90% ) and warfarin (98% purity), were purchased from Sigma-Aldrich Inc., St. Louis, MO and used without further purification. 1 mM stock solutions of HSA and β-Lg in Tris-HCl buffer solution at pH 7.4 was prepared and the concentrations were adjusted spectrophotometrically by using extinction coefficient of 35219 M-1 cm-1 at 280 nm for HSA [20] and 17600 M -1 cm-1 at 278 nm for β-Lg [21]. Prodigiosin (PG) was isolated from pigment producing bacterium, Serratia marcescens strain S2B and purified by acetone-HCl extraction followed by passing through petroleum ether balanced silica gel column (28 × 200 mm) [22]. The purity of the ligand was assessed by HPLC using a reversed-phase column (C18, 5 × 150 mm) (Fig. S1). Prodigiosin concentration was calculated using extinction coefficient of 112000 M-1 cm-1 at 535 nm and 10 mM stock solution were prepared in methanol.
2.2. Fluorescence Measurements Fluorescence spectra were recorded on a Perkin-Elmer LS45 fluorimeter and the temperature was controlled by a water bath circulator. Intrinsic fluorescence of HSA and β-Lg was measured upon exciting at 295 nm and recording at 310–600 nm. Both the excitation and the emission slit widths were set at 10.0 nm. Steady state fluorescence of HSA was measured by fixing the protein concentration at 0.4 μM and adding aliquots of PG from the stock solution, in the concentration range of 03.2 μM. Similar experiments were carried out using β-Lg at concentration of 1 µM. The samples were allowed to equilibrate for 10 min after each addition. The scan speed was 500 nm/min, and the sensitivity was adjusted to 700 V. The data which recorded at maximum emission wavelength were used to analyze the binding parameters. The Trp emission quenching was used to calculate the binding constant (Kb) at three different
5
temperatures (290, 300 and 310 K) using the Stern–Volmer equation [23, 24]:
where F0 and F are the fluorescence intensities in the absence and presence of quencher, [Q] is the quencher concentration, KSV Stern–Volmer quenching constant, kq is the bimolecular quenching rate constant and τ0 is the average lifetime of the fluorophore (Trp) in the absence of quencher and its value is 10 -8 s for HSA and 1.28 ×10 -9 s for β-Lg, respectively [21, 25]. Linear regression of a Fo/F versus [Q] plots represents Ksv constant. The binding constant (called Kb) and number of bound prodigiosin to HSA and β-Lg (called n) were determined by plotting the double log graph of the fluorescence intensities.
For the static quenching, the binding data must be analyzed according to the modified Stern–Volmer based on fallowing Eq.
In this case, fa is the fraction of accessible fluorescence and Ka is the effective quenching constant for the accessible fluorophore.
2.3. Thermodynamic parameters Thermodynamic parameters of ligand-protein driving forces were determined by van′t Hoff equations:
K is the Lineweaver–Burk static quenching constant at three different temperatures 290, 300 and 310 K and R is the gas constant. The values of ∆H and ∆S were determined from the linear relationship between ln K and the free energy change (∆G) and calculated by the following equation:
6
2.4. Circular Dichroism (CD) Spectroscopy Far-UV (190-260 nm) region CD spectra of HSA-PG and β-Lg-PG complexes was recorded on a Jasco J-715 spectropolarimeter (Tokyo, Japan) with path length of 1 mm. Concentration ratios of HSA and β-Lg solutions with PG were selected from 0.5 to 3 with incubation time of 15 minutes at room temperature for equilibrium attainment. Ellipticity was recorded at a speed of 100 nm/min, 1.0 nm resolution, and 1.0 nm bandwidth. Buffer background was subtracted from the raw spectra. The results were expressed in terms of MRE (Mean Residue Ellipticity) in the following equation:
Cp is the molar concentration of the protein, n the number of amino acid residues (585 and 162) and l is the path length (0.1 cm). The secondary structure contents of the proteins were calculated from DichroWeb (http://dichroweb.cryst.bbk.ac.uk), an on-line CD spectra analyzer [26]
2.5. Site-specific binding of prodigiosin Warfarin was used as site I competitive binding displacement ligand to identify the prodigiosin binding site on HSA (Zhang et al., 2010). The fluorescence intensity changes of equal concentrations (1:1) of 30 minutes incubated HSA-Warfarin complex was monitored in the wavelength ranging from 310-600 nm upon excitation at 295 nm, both in the absence and presence of different concentrations of PG (0.4-3 µM). To investigate the β-Lg-PG site-specific region, three different pH solutions of β-Lg including 3.0, 5.5 & 7.4 were prepared and the protein mixture was incubated at 25˚C for 30 minutes before prodigiosin additions. The fluorescence intensity changes were monitored as mentioned above.
7
2.6. Molecular Modeling measurements Molecular 3D conformer of prodigiosin, warfarin and palmitic acid were obtained from Pubchem with CID code 5351169, 54678486 and 985 (http://Pubchem.ncbi.nih.gov). After optimization of the ligands by Gaussian 09 software, force constants and the resulting vibrational frequencies were computed by the Freq command and docking were performed to the binding sites of HSA and β-Lg. The crystal structure of HSA and β-Lg were chosen from Protein Data Bank (entry PDB code: 4L8U & 3PNO) as protein receptor and molecular dynamic simulation was performed according to previous publication [27]. To construct the structures for docking, factors like Gasteiger partial charges addition, non-polar hydrogen merging and rotatable bonds assignment were done with AutoDock 4.2.2.1 [28]. For HSA-PG and β-Lg-PG complexes, the Grid box were defined using grid of 110 × 82 × 98 point for HSA and 16 × 28 × 12 for β-Lg, each with a grid space of 1.000 Å centered at coordinates xh = 71.903, xb=5.352, yh =53.183, yb =41.092 and zh = 53.587, zb=43.794 and Docking were performed using AutoDock Vina [29]. Cluster analysis was performed on docked results using a root-mean-square deviation (RMSD) tolerance of 2.0 Å. Then, the lowest energy docked conformation in each docking cases results was selected as the binding mode.
3. Results and discussion Encapsulation of bioactive drugs in hydrophobic binding sites of HSA and central calyx of the β-Lg as intravenous and oral drug scaffolds not only improve the solubility and bioavailability of both systems (intravenous and oral), but also reduce side effects and prevent drug from extreme pH shocks and gastric digestion. At first, the interaction of prodigiosin with HSA was surveyed by size exclusion chromatography technique. Size exclusion chromatography is commonly used to separate small molecules (like ligands) from large ones, including proteins. After loading the equilibrated protein-ligand solution on column, small molecules like salts and free ligands are retained longer within the column; while proteins and protein-bound ligand populations are passed faster. By quantifying the amount of ligand concentration in protein containing fractions, bound ligand to total ligand concentrations can be determined [30]. According to this theory, size exclusion chromatography of PG-HSA complex was conducted and results confirmed the 8
significant interaction of PG to HSA protein (Fig. S2).
3.1. Fluorescence study of prodigiosin binding Formation of ligand-protein complex sometimes induces slight changes in protein conformation and consequently its fluorescence spectra. Generally, the fluorescence spectrum is mostly due to the presence of Trp and Tyr residues. It is well characterized that most of fluorescence emission of proteins naturally comes from Trp residues specially when excited at 295 nm. The Trp214 of HSA is located in the hydrophobic cavity of the subdomain IIA that is called Sudlow’s drug-binding site I region [31]. Addition of PG to the protein solution revealed a concentration-dependent quenching of HSA emission spectra with a 4 nm blue shift in its maximum emission peak. At low temperature (290 K), the decrease in the fluorescence emission intensity of the protein upon PG addition was more pronounced for lower PG/HSA ratios and sloped off at higher ones compared to elevated temperatures. At the lower temperature, around 80% quenching was observed for HSA/PG molar ratios of 5.0, although at 300 K and 310 K around 85% quenching was observed at the molar ratio of 7.0. As illustrated in Fig. 2a, blue shift may occur when the hydrophobic microenvironment of fluorophore is increased by ligand binding while quenching is occurred by the movement of charged groups and hydrophobic changes around the fluorophore [32]. This phenomenon reflects the fact that there is a conformational change in 3D structure of the protein upon binding to prodigiosin. For fluorescence quenching mechanism characterization, quenching profile of HSA-PG complex was performed at three different temperatures (290 K, 300 K and 310 K). Results revealed the existence of an inverse correlation between K sv and temperature (Fig. 2b). In the static binding process, contrary to the dynamic one, raising the temperature attenuates formation of HSA-PG complex [24].
9
a
b
1
11
c
1
d
11
Fig. 2. Fluorescence quenching spectra of HSA and β-Lg (0.4 µm) by different concentrations of PG corresponding to 0, 0.4, 0.8, 1.0, 1.2, 1.6, 2.0, 2.4, 2.8, 3 & 3.2 µM (1-11) with the excitation wavelength at 295 nm in 20 mM Tris, pH 7.4, at room temperature (a & c). Arrow shows the intensity changes upon increasing concentration of the quencher. (b & d ) Stern–Volmer plots showing HSA and β-Lg tryptophan quenching caused by PG at three different temperatures (▲17, ■ 27, ●37 ˚C).
According to Table 1, Kq values at different temperatures (8.14×1013, 6.89×1013 and 4.04×1013) are much higher than diffusion-controlled process (2×1010 M-1 s-1) of a dynamic quenching in the binding process. This finding suggests that the quenching was not initiated by dynamic collision but via formation of protein-ligand complexes. Thus, the overall quenching of HSA fluorescence by PG can be described by the static quenching mechanism. Different parameters like binding constant (Kb) and number of binding sites (n) are obtained from logarithmic ratios of (F0-F)/F versus Log [Q]. The value of Kb indicates the moderate affinity of compound to the protein that is critical for its transport in 10
blood circulation and its release to the target site. It can also find that binding affinity between PG and HSA decreases with respect to increasing temperature. Moreover, the number of binding sites on HSA obtained from different temperatures (n=0.905, 1.004 and 0.968) revealed the presence of only a single binding site for the PG per HSA molecule [33]. The same data have been reported during the interaction of nobiletin, caffeine and theophylline and theobromine with HSA [34, 35].
Table 1. Stern–Volmer quenching constants (Ksv), binding constants (Kb) and thermodynamic parameters of HSA–PG and β-Lg-PG complexes at different temperatures. T Protein
HSA
β-Lg
Ksv 4
Kq -1
12
-1 -1
∆S
Kb R
4
-1
n
R
-1
∆H -1
∆G -1
-1
(K)
(10 M )
(10 M S )
290
8.140
8.140
0.989
4.845
0.905
0.991
300
6.891
6.891
0.984
4.408
1.004
0.987
310
4.043
4.043
0.981
3.396
0.968
0.982
-26.97
290
5.02
3.92
0.983
1.25
1.32
0.980
-22.48
300
7.28
5.69
0.985
1.99
1.45
0.984
310
10.6
8.28
0.994
5.37
1.68
0.983
(Jmol K )
(10 mol L )
(kJmol )
-26.08 +44.64
+264.06
-13.14
+54.07
In the case of β–Lg, although this protein has two tryptophan residues (Trp19 and Trp61) for monitoring intrinsic fluorescence emission, Trp61 is mostly quenched by two cysteine residues (Cys66-Cys160) nearby. Tryptophan19 is located at the central hydrophobic calyx of the protein [36]. The fluorescence spectra of β-Lg in the presence of different PG concentrations showed the quenching effect of the ligand to intrinsic fluorescence of the protein without changing the maximum emission wavelength and shape of the peaks. This behavior has been reported during interaction of curcumin (as an anti-inflammatory, anticancer drug) with β-Lg [37]. Quenching profile of β-Lg-PG complex in various temperatures (290 K, 300 K and 310 K) revealed the straight 11
(kJmol )
-26.53
-25.12 -27.76
correlation between Ksv and temperature, which demonstrates that the quenching speed and β-Lg-PG complex formation is elevated by increase of temperature. In contrast to the βLg-curcumin complex formation, in β-Lg-PG interaction at high temperature (310 K), decrease of fluorescence emission intensity of the protein was more visible in lower PG/HSA ratios and sloped off at higher ones in comparison to fluorescence intensity alterations at 290 and 300 K. As shown in Fig. 2c-d, at 310 K, around 90% of the quenching was observed at HSA/PG molar ratios of 4.0, although at 290 and 300 K around 78% and 82% of quenching were observed at the molar ratio of 7.0. As illustrated in Table 1, the estimated Kq values in different temperatures (3.92×1012, 5.69×1012 and 8.28×1012) are much higher than diffusion-controlled process (2×1010 M-1s-1) of a dynamic quenching in the binding process. Subsequently, the main mechanism of quenching should be static. Estimated Kb values represent the moderate binding affinity of the PG to β-Lg as protein carrier (see Table 1). Moreover, the estimated number of binding sites of PG on β-Lg (1.32, 1.45 and 1.68) at the three different temperatures showed binding of about 1.5 PG molecules per each β-Lg which suggests its dimer form dominancy in the mixture. As reported in the literature, β-Lg exists at the equilibrium ratio of monomer and dimer at neutral pH and the proportion of dimer is 29% when the protein concentration is 10 µM [38]. Although the estimated number of binding molecules per protein is calculated to be 3:2, at low concentrations the dominant form of the β-Lg is monomer and the oligomer formation can be ignored [39].
3.2. Thermodynamic parameters and binding forces Thermodynamic parameters at different temperatures represent the driving forces of complex formation of molecules. Van′t Hoff plots showed the values of enthalpy (ΔH˚), entropy (ΔS˚) and free energy (ΔG˚) of binding processes. According to the data from both enthalpy (ΔH) and entropy (ΔS) changes, the model of drug-protein interaction can be explained as follows: 1) ΔH>0 and ΔS>0, hydrophobic forces are significant, 2) ΔH
S0968-0896(16)30100-6 http://dx.doi.org/10.1016/j.bmc.2016.02.020 BMC 12822
To appear in:
Bioorganic & Medicinal Chemistry
Received Date: Revised Date: Accepted Date:
28 November 2015 5 February 2016 12 February 2016
Please cite this article as: Rastegari, B., Karbalaei-Heidari, H.R., Yousefi, R., Zeinali, S., Nabavizadeh, M., Interaction of prodigiosin with HSA and β-Lg: Spectroscopic and molecular docking studies, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.02.020
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Interaction
of prodigiosin
with HSA and β-Lg:
spectroscopic and molecular docking studies Authors: Banafsheh Rastegaria, Hamid Reza Karbalaei-Heidaria,*, Reza Yousefib, Sedigheh Zeinalic, Masoud Nabavizadehd
a
Molecular Biotechnolgy Laboratory,
,
Department of Biology, Faculty of Science, Shiraz
University, Shiraz, 71454, Iran b
Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz
University, Shiraz, Iran c
Department of Nanochemical Engineering, Faculty of Advanced Technologies, Shiraz University,
Shiraz, Iran d
Department of Chemistry, Faculty of Science, Shiraz University, Shiraz, Iran
* Corresponding author. Address: Molecular Biotechnology Lab., Department of Biology, Faculty of Sciences, Shiraz University, Shiraz 71454, Iran; P. O. Box: 71467-13565; Tel: +98 71 32280916; Fax: + 98 71 32280926; E-mail: [email protected]
1
Abstract Human serum albumin (HSA) and Bovine β-lactoglobulin (β-Lg) are both introduced as blood and oral carrier scaffolds with high affinity for a wide range of pharmaceutical compounds.
Prodigiosin, a natural three pyrrolic compound produced by Serratia
marcescens, exhibits many pharmaceutical properties associated with health benefits. In the present study, the interaction of prodigiosin with HSA and β-Lg was investigated using fluorescence spectroscopy, circular dichroism (CD) and computational docking. Prodigiosin interacts with the Sudlow's site I of HSA and the calyx of β-Lg with association constant of 4.41x104 and 1.99x104 M-1 to form 1:1 and 2:3 complexes at 300 K, respectively. The results indicated that binding of prodigiosin to HSA and β-Lg caused strong fluorescence quenching of both proteins through static quenching mechanism. Electrostatic and hydrophobic interactions are the major forces in the stability of PG-HSA complex with enthalpy-and entropy-driving mode, although the formation of prodigiosinβ-Lg complex is entropy-driven hydrophobic associations. CD spectra showed slight conformational changes in both proteins due to the binding of prodigiosin. Moreover, the ligand displacement assay, pH-dependent interaction and protein–ligand docking study confirmed that the prodigiosin binds to residues located in the subdomain IIA and IIIA of HSA and central calyx of β-Lg.
Key words: Prodigiosin; HSA-PG interaction; β-Lg-PG binding study.
Abbreviations: PG: prodigiosin; β-Lg: β-lactoglobulin; HSA: Human Serum Albumin; CD: circular dichroism.
2
1. Introduction Prodigiosin, a member of prodiginine family (Fig. 1), is a red three pyrrole compound which mostly produced by bacterial resources like Serratia marcescens [1]. Antimicrobial, antimalarial, immunosuppressive and remarkable anticancer properties suggest prodigiosin as an appropriate drug candidate for various pharmaceutical purposes [2-4]. The selective cytotoxicity to malignant cell-lines motivated researchers to introduce prodigiosin as a potential anticancer drug and currently placed it in phase II clinical trials [5]. Prodigiosin, in a similar manner to the other poor water soluble drugs, exhibits limited bioavailability and must be bound to available polymers such as proteins to remain in its effective concentration in blood and other water-based mixtures. In addition, biodegradable protein like β-lactoglobulin (β-Lg) has been used in preparing encapsulated drugs. This protein is stable in stomach and suggested as a suitable vehicle for protein-based drug delivery to cancerous cells of digestive tract [6, 7].
Fig. 1. Optimized structure of the prodigiosin (PG).
Human serum albumin (HSA) is the most abundant plasma protein with an exceptional binding capacity for a wide variety of hydrophobic ligands and transport of hormones, fatty acids, amino acids, steroids, metal ions, metabolites such as bilirubin and pharmaceutical drug compounds. Low toxicity and non-immunogenic nature of this 3
protein is ideal for its application as intravenous drug carrier. The protein consisted of 585 residues with three structurally similar repeating domains-I (residues 1-195), II (196-383) and III (384-585)- each of which are divided into two sub-domains, A and B. The protein has two high affinity binding sites which are Sudlow's sites I and II. Site I as the major binding site, interacts with the most neutral heterocyclic ligands, while site II is mainly binds to many aromatic compounds [8, 9]. β-lactoglobulin (β-Lg), the major whey protein of bovine milk, is a small protein that consists of 162 amino acids (~18.3 kDa). It is normally exists as a mixture of monomers and dimers, depending on the protein concentration and its environmental pH. An important functional feature of this lipocalin family protein is its ability to bind several physiologically important non-water soluble ligands such as steroids, retinoids, fatty acids, vitamin D and cholesterol [10-12]. β-Lg consists of a large β-barrel with nine β strands (called A to I) and two ligand-binding sites; including a central calyx domain of the βbarrel and a surface hydrophobic pocket in the groove between the β-barrel and R-helix [13, 14]. Good solubilization, cost effectiveness and structural resistance to acidic pH of stomach, make it an ideal vehicle for encapsulation and a controlled release of hydrophobic compounds like prodigiosin [15, 16]. Study on the binding of prodigiosin to blood and oral matrixes represent a significant importance in applied pharmacology and biotechnology. Natural biomolecules such as proteins are an attractive group of scaffold compounds compared to synthetic ones, due to their low toxicity and biodegradability. Nanoparticle technology utilized several benefits of HSA and β-Lg, two successful scaffolds carrying hydrophobic ligands to the target sites. An essential step to design protein-based nanoparticle systems is to understand the binding sites and binding affinity of the protein-drug complex [17]. Although previous studies have been shown the interaction potential of prodigiosin with some proteins such as bovine hemoglobin and bovine serum albumin, to the best of our knowledge, there is no report on detailed interaction study of HSA and β-Lg with prodigiosin in literature [18, 19]. So, the potential of these proteins (HSA and β-Lg) as the intravenous and oral carrier molecules, respectively for transportation of the prodigiosin was investigated by determining the binding properties (including binding mechanism, binding constant, and the number of binding sites) of PG to the both proteins at three temperatures under simulative physiological conditions using fluorescence technique. We have also examined 4
the effect of prodigiosin on the secondary and tertiary structures of HSA and β–Lg using CD spectra. Finally, the binding site of the ligand was predicted by molecular modeling study.
2. Materials and methods 2.1. Materials HSA, Bovine β-Lg (B variant, purity 90% ) and warfarin (98% purity), were purchased from Sigma-Aldrich Inc., St. Louis, MO and used without further purification. 1 mM stock solutions of HSA and β-Lg in Tris-HCl buffer solution at pH 7.4 was prepared and the concentrations were adjusted spectrophotometrically by using extinction coefficient of 35219 M-1 cm-1 at 280 nm for HSA [20] and 17600 M -1 cm-1 at 278 nm for β-Lg [21]. Prodigiosin (PG) was isolated from pigment producing bacterium, Serratia marcescens strain S2B and purified by acetone-HCl extraction followed by passing through petroleum ether balanced silica gel column (28 × 200 mm) [22]. The purity of the ligand was assessed by HPLC using a reversed-phase column (C18, 5 × 150 mm) (Fig. S1). Prodigiosin concentration was calculated using extinction coefficient of 112000 M-1 cm-1 at 535 nm and 10 mM stock solution were prepared in methanol.
2.2. Fluorescence Measurements Fluorescence spectra were recorded on a Perkin-Elmer LS45 fluorimeter and the temperature was controlled by a water bath circulator. Intrinsic fluorescence of HSA and β-Lg was measured upon exciting at 295 nm and recording at 310–600 nm. Both the excitation and the emission slit widths were set at 10.0 nm. Steady state fluorescence of HSA was measured by fixing the protein concentration at 0.4 μM and adding aliquots of PG from the stock solution, in the concentration range of 03.2 μM. Similar experiments were carried out using β-Lg at concentration of 1 µM. The samples were allowed to equilibrate for 10 min after each addition. The scan speed was 500 nm/min, and the sensitivity was adjusted to 700 V. The data which recorded at maximum emission wavelength were used to analyze the binding parameters. The Trp emission quenching was used to calculate the binding constant (Kb) at three different
5
temperatures (290, 300 and 310 K) using the Stern–Volmer equation [23, 24]:
where F0 and F are the fluorescence intensities in the absence and presence of quencher, [Q] is the quencher concentration, KSV Stern–Volmer quenching constant, kq is the bimolecular quenching rate constant and τ0 is the average lifetime of the fluorophore (Trp) in the absence of quencher and its value is 10 -8 s for HSA and 1.28 ×10 -9 s for β-Lg, respectively [21, 25]. Linear regression of a Fo/F versus [Q] plots represents Ksv constant. The binding constant (called Kb) and number of bound prodigiosin to HSA and β-Lg (called n) were determined by plotting the double log graph of the fluorescence intensities.
For the static quenching, the binding data must be analyzed according to the modified Stern–Volmer based on fallowing Eq.
In this case, fa is the fraction of accessible fluorescence and Ka is the effective quenching constant for the accessible fluorophore.
2.3. Thermodynamic parameters Thermodynamic parameters of ligand-protein driving forces were determined by van′t Hoff equations:
K is the Lineweaver–Burk static quenching constant at three different temperatures 290, 300 and 310 K and R is the gas constant. The values of ∆H and ∆S were determined from the linear relationship between ln K and the free energy change (∆G) and calculated by the following equation:
6
2.4. Circular Dichroism (CD) Spectroscopy Far-UV (190-260 nm) region CD spectra of HSA-PG and β-Lg-PG complexes was recorded on a Jasco J-715 spectropolarimeter (Tokyo, Japan) with path length of 1 mm. Concentration ratios of HSA and β-Lg solutions with PG were selected from 0.5 to 3 with incubation time of 15 minutes at room temperature for equilibrium attainment. Ellipticity was recorded at a speed of 100 nm/min, 1.0 nm resolution, and 1.0 nm bandwidth. Buffer background was subtracted from the raw spectra. The results were expressed in terms of MRE (Mean Residue Ellipticity) in the following equation:
Cp is the molar concentration of the protein, n the number of amino acid residues (585 and 162) and l is the path length (0.1 cm). The secondary structure contents of the proteins were calculated from DichroWeb (http://dichroweb.cryst.bbk.ac.uk), an on-line CD spectra analyzer [26]
2.5. Site-specific binding of prodigiosin Warfarin was used as site I competitive binding displacement ligand to identify the prodigiosin binding site on HSA (Zhang et al., 2010). The fluorescence intensity changes of equal concentrations (1:1) of 30 minutes incubated HSA-Warfarin complex was monitored in the wavelength ranging from 310-600 nm upon excitation at 295 nm, both in the absence and presence of different concentrations of PG (0.4-3 µM). To investigate the β-Lg-PG site-specific region, three different pH solutions of β-Lg including 3.0, 5.5 & 7.4 were prepared and the protein mixture was incubated at 25˚C for 30 minutes before prodigiosin additions. The fluorescence intensity changes were monitored as mentioned above.
7
2.6. Molecular Modeling measurements Molecular 3D conformer of prodigiosin, warfarin and palmitic acid were obtained from Pubchem with CID code 5351169, 54678486 and 985 (http://Pubchem.ncbi.nih.gov). After optimization of the ligands by Gaussian 09 software, force constants and the resulting vibrational frequencies were computed by the Freq command and docking were performed to the binding sites of HSA and β-Lg. The crystal structure of HSA and β-Lg were chosen from Protein Data Bank (entry PDB code: 4L8U & 3PNO) as protein receptor and molecular dynamic simulation was performed according to previous publication [27]. To construct the structures for docking, factors like Gasteiger partial charges addition, non-polar hydrogen merging and rotatable bonds assignment were done with AutoDock 4.2.2.1 [28]. For HSA-PG and β-Lg-PG complexes, the Grid box were defined using grid of 110 × 82 × 98 point for HSA and 16 × 28 × 12 for β-Lg, each with a grid space of 1.000 Å centered at coordinates xh = 71.903, xb=5.352, yh =53.183, yb =41.092 and zh = 53.587, zb=43.794 and Docking were performed using AutoDock Vina [29]. Cluster analysis was performed on docked results using a root-mean-square deviation (RMSD) tolerance of 2.0 Å. Then, the lowest energy docked conformation in each docking cases results was selected as the binding mode.
3. Results and discussion Encapsulation of bioactive drugs in hydrophobic binding sites of HSA and central calyx of the β-Lg as intravenous and oral drug scaffolds not only improve the solubility and bioavailability of both systems (intravenous and oral), but also reduce side effects and prevent drug from extreme pH shocks and gastric digestion. At first, the interaction of prodigiosin with HSA was surveyed by size exclusion chromatography technique. Size exclusion chromatography is commonly used to separate small molecules (like ligands) from large ones, including proteins. After loading the equilibrated protein-ligand solution on column, small molecules like salts and free ligands are retained longer within the column; while proteins and protein-bound ligand populations are passed faster. By quantifying the amount of ligand concentration in protein containing fractions, bound ligand to total ligand concentrations can be determined [30]. According to this theory, size exclusion chromatography of PG-HSA complex was conducted and results confirmed the 8
significant interaction of PG to HSA protein (Fig. S2).
3.1. Fluorescence study of prodigiosin binding Formation of ligand-protein complex sometimes induces slight changes in protein conformation and consequently its fluorescence spectra. Generally, the fluorescence spectrum is mostly due to the presence of Trp and Tyr residues. It is well characterized that most of fluorescence emission of proteins naturally comes from Trp residues specially when excited at 295 nm. The Trp214 of HSA is located in the hydrophobic cavity of the subdomain IIA that is called Sudlow’s drug-binding site I region [31]. Addition of PG to the protein solution revealed a concentration-dependent quenching of HSA emission spectra with a 4 nm blue shift in its maximum emission peak. At low temperature (290 K), the decrease in the fluorescence emission intensity of the protein upon PG addition was more pronounced for lower PG/HSA ratios and sloped off at higher ones compared to elevated temperatures. At the lower temperature, around 80% quenching was observed for HSA/PG molar ratios of 5.0, although at 300 K and 310 K around 85% quenching was observed at the molar ratio of 7.0. As illustrated in Fig. 2a, blue shift may occur when the hydrophobic microenvironment of fluorophore is increased by ligand binding while quenching is occurred by the movement of charged groups and hydrophobic changes around the fluorophore [32]. This phenomenon reflects the fact that there is a conformational change in 3D structure of the protein upon binding to prodigiosin. For fluorescence quenching mechanism characterization, quenching profile of HSA-PG complex was performed at three different temperatures (290 K, 300 K and 310 K). Results revealed the existence of an inverse correlation between K sv and temperature (Fig. 2b). In the static binding process, contrary to the dynamic one, raising the temperature attenuates formation of HSA-PG complex [24].
9
a
b
1
11
c
1
d
11
Fig. 2. Fluorescence quenching spectra of HSA and β-Lg (0.4 µm) by different concentrations of PG corresponding to 0, 0.4, 0.8, 1.0, 1.2, 1.6, 2.0, 2.4, 2.8, 3 & 3.2 µM (1-11) with the excitation wavelength at 295 nm in 20 mM Tris, pH 7.4, at room temperature (a & c). Arrow shows the intensity changes upon increasing concentration of the quencher. (b & d ) Stern–Volmer plots showing HSA and β-Lg tryptophan quenching caused by PG at three different temperatures (▲17, ■ 27, ●37 ˚C).
According to Table 1, Kq values at different temperatures (8.14×1013, 6.89×1013 and 4.04×1013) are much higher than diffusion-controlled process (2×1010 M-1 s-1) of a dynamic quenching in the binding process. This finding suggests that the quenching was not initiated by dynamic collision but via formation of protein-ligand complexes. Thus, the overall quenching of HSA fluorescence by PG can be described by the static quenching mechanism. Different parameters like binding constant (Kb) and number of binding sites (n) are obtained from logarithmic ratios of (F0-F)/F versus Log [Q]. The value of Kb indicates the moderate affinity of compound to the protein that is critical for its transport in 10
blood circulation and its release to the target site. It can also find that binding affinity between PG and HSA decreases with respect to increasing temperature. Moreover, the number of binding sites on HSA obtained from different temperatures (n=0.905, 1.004 and 0.968) revealed the presence of only a single binding site for the PG per HSA molecule [33]. The same data have been reported during the interaction of nobiletin, caffeine and theophylline and theobromine with HSA [34, 35].
Table 1. Stern–Volmer quenching constants (Ksv), binding constants (Kb) and thermodynamic parameters of HSA–PG and β-Lg-PG complexes at different temperatures. T Protein
HSA
β-Lg
Ksv 4
Kq -1
12
-1 -1
∆S
Kb R
4
-1
n
R
-1
∆H -1
∆G -1
-1
(K)
(10 M )
(10 M S )
290
8.140
8.140
0.989
4.845
0.905
0.991
300
6.891
6.891
0.984
4.408
1.004
0.987
310
4.043
4.043
0.981
3.396
0.968
0.982
-26.97
290
5.02
3.92
0.983
1.25
1.32
0.980
-22.48
300
7.28
5.69
0.985
1.99
1.45
0.984
310
10.6
8.28
0.994
5.37
1.68
0.983
(Jmol K )
(10 mol L )
(kJmol )
-26.08 +44.64
+264.06
-13.14
+54.07
In the case of β–Lg, although this protein has two tryptophan residues (Trp19 and Trp61) for monitoring intrinsic fluorescence emission, Trp61 is mostly quenched by two cysteine residues (Cys66-Cys160) nearby. Tryptophan19 is located at the central hydrophobic calyx of the protein [36]. The fluorescence spectra of β-Lg in the presence of different PG concentrations showed the quenching effect of the ligand to intrinsic fluorescence of the protein without changing the maximum emission wavelength and shape of the peaks. This behavior has been reported during interaction of curcumin (as an anti-inflammatory, anticancer drug) with β-Lg [37]. Quenching profile of β-Lg-PG complex in various temperatures (290 K, 300 K and 310 K) revealed the straight 11
(kJmol )
-26.53
-25.12 -27.76
correlation between Ksv and temperature, which demonstrates that the quenching speed and β-Lg-PG complex formation is elevated by increase of temperature. In contrast to the βLg-curcumin complex formation, in β-Lg-PG interaction at high temperature (310 K), decrease of fluorescence emission intensity of the protein was more visible in lower PG/HSA ratios and sloped off at higher ones in comparison to fluorescence intensity alterations at 290 and 300 K. As shown in Fig. 2c-d, at 310 K, around 90% of the quenching was observed at HSA/PG molar ratios of 4.0, although at 290 and 300 K around 78% and 82% of quenching were observed at the molar ratio of 7.0. As illustrated in Table 1, the estimated Kq values in different temperatures (3.92×1012, 5.69×1012 and 8.28×1012) are much higher than diffusion-controlled process (2×1010 M-1s-1) of a dynamic quenching in the binding process. Subsequently, the main mechanism of quenching should be static. Estimated Kb values represent the moderate binding affinity of the PG to β-Lg as protein carrier (see Table 1). Moreover, the estimated number of binding sites of PG on β-Lg (1.32, 1.45 and 1.68) at the three different temperatures showed binding of about 1.5 PG molecules per each β-Lg which suggests its dimer form dominancy in the mixture. As reported in the literature, β-Lg exists at the equilibrium ratio of monomer and dimer at neutral pH and the proportion of dimer is 29% when the protein concentration is 10 µM [38]. Although the estimated number of binding molecules per protein is calculated to be 3:2, at low concentrations the dominant form of the β-Lg is monomer and the oligomer formation can be ignored [39].
3.2. Thermodynamic parameters and binding forces Thermodynamic parameters at different temperatures represent the driving forces of complex formation of molecules. Van′t Hoff plots showed the values of enthalpy (ΔH˚), entropy (ΔS˚) and free energy (ΔG˚) of binding processes. According to the data from both enthalpy (ΔH) and entropy (ΔS) changes, the model of drug-protein interaction can be explained as follows: 1) ΔH>0 and ΔS>0, hydrophobic forces are significant, 2) ΔH
