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Aggregates of a cationic porphyrin as supramolecular probes for biopolymers
JIB-09814; No of Pages 6 Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
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Aggregates of a cationic porphyrin as supramolecular probes for biopolymers Ilaria Giuseppina Occhiuto a, Mario Samperi b, Mariachiara Trapani c, Giovanna De Luca d, Andrea Romeo b, Robert F. Pasternack e, Luigi Monsù Scolaro b,c,⁎ a
Dipartimento di Scienze Chimiche, Via Marzolo 1, 35131 Padova, Italy Dipartimento di Scienze Chimiche and C.I.R.C.M.S.B., V.le F. Stagno d'Alcontres 31, 98166 Messina, Italy Istituto per lo Studio dei Materiali Nanostrutturati ISMN-CNR c/o Dipartimento di Scienze Chimiche, University of Messina, V.le F. Stagno D'Alcontres, 31-98166 Messina, Italy d Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, V.le Annunziata, 98168 Messina, Italy e Department of Chemistry & Biochemistry, Swarthmore College, 500 College Avenue, Swarthmore, PA 19081, USA b c
a r t i c l e
i n f o
Article history: Received 15 June 2015 Received in revised form 11 September 2015 Accepted 30 September 2015 Available online xxxx Keywords: Porphyrin Supramolecular aggregate Chiroptical sensor Chirality Circular dichroism
a b s t r a c t The copper(II) derivative of the dicationic trans-bis(N-methylpyridinium-4-yl)diphenylporphyrin (t-CuPagg) forms large fractal aggregates in aqueous solution under moderate ionic strength conditions. A kinetic investigation of the aggregation process allows for a choice of experimental conditions to quickly obtain stable assemblies in solution. These positively charged aggregates are able to interact efficiently with negatively charged chiral species, (including bacterial spores) leading to induced circular dichroism signals in the Soret region of the porphyrin, now acting as a sensitive chiroptical probe. © 2015 Elsevier Inc. All rights reserved.
1. Introduction The design and synthesis of supramolecular optical sensors for the detection of chiral molecules/biomolecules is actively investigated due to their implications both in fundamental and applicative fields [1]. In particular, it is important to find systems able to quickly report information on very diluted samples. Porphyrins have been extensively used to this purpose, due to their rich spectroscopic features, e.g., high extinction coefficients and resonant light scattering effects [2] that allow their use as sensitive probes. Hetero-aggregates based on water soluble porphyrins have been successfully exploited to detect and amplify traces of aggregated amino acids [3] and polypeptides [4–6]. The large circular dichroism (CD) signals observed for J-aggregates of the anionic tetrakis(4-sulfonatophenyl)porphyrin has been ascribed to the presence of optically active species (i.e. bacterial membranes) present as traces in ultrapure water [7], or to added chiral templates [8–12]. Trans-bis(N-methylpyridinium-4-yl)diphenylporphyrin and its copper(II) derivative (t-CuPagg, Fig. 1) are dicationic water soluble porphyrins that have been shown to form extended supramolecular aggregates on nucleic acids [13,14]. This species as well as its parent unmetalated porphyrin (t-H2Pagg) form fractal assemblies whose structural features are modulated by the ionic strength [15–18]. These aggregates are quite large ⁎ Corresponding author. E-mail address: [email protected] (L.M. Scolaro).
(sizes ranging from 100 nm up to 1–2 μm), flexible and porous. The strong electronic coupling among the neighboring chromophores, along with the extended dimensions of the aggregates, is responsible for large resonant light scattering signals in their aqueous solutions [2]. Quite recently, t-CuPagg porphyrin in its dimeric [19] and largely aggregated form [20] has been proposed as chiroptical sensors for conformations and chirality of poly(glutamate). In particular, t-CuPagg studies have shown the presence of a left handed helix (poly-proline-II) and the coexistence of different conformations for this simple polypeptide [19]. Here we report an investigation on the kinetics of formation of aggregates of t-CuPagg and their use as chiroptical probes for a variety of chiral negatively charged biopolymers, i.e. calf thymus DNA, polyglutamate and human serum albumin (HSA), as well as bacterial spores. 2. Experimental 2.1. Materials and methods 5,15-bis(N-methylpyridinium-4-yl)-10,15-bis-diphenylporphine (t-H2Pagg, chloride salt) was purchased from Frontier Scientific and used as received. Its copper(II) derivative (t-CuPagg) was prepared by literature procedures [21]. Porphyrins stock solutions were prepared dissolving the solids in dust-free water (Galenica Senese) and stored in the dark. Solution concentrations were determined from known molar extinction coefficient at the Soret maximum (t-CuPagg: 2.34 × 105 M− 1 cm− 1) [13].
http://dx.doi.org/10.1016/j.jinorgbio.2015.09.013 0162-0134/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: I.G. Occhiuto, et al., Aggregates of a cationic porphyrin as supramolecular probes for biopolymers, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.09.013
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I.G. Occhiuto et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Fig. 1. Structure of the copper(II) derivative of trans-bis(N-methylpyridinium-4yl)diphenylporphyrin (t-CuPagg, chloride salt).
Calf thymus DNA (ct-DNA, type I) was obtained from Sigma and was purified according to literature methods [22]. The concentration of a ct-DNA stock solution in 1 mM phosphate buffer, pH 7, containing 10 mM NaCl, was determined in molar base-pairs using ε260 = 13,200 M− 1 cm − 1 [23]. Sodium salts of poly-L -glutamic acid (FW ~ 17 kD and ~ 1.5 kD) were obtained from Aldrich Chemicals Co. Stock solution of the polypeptides were prepared by dissolving the solids in acetate buffer at pH 4.2 (ionic strength 5 mM). Concentrations of the peptide solutions were determined spectrophotometrically, using ε205 = 2150 M− 1 cm− 1 [24]. Human Serum Albumin (HSA) was purchased from Sigma and stock solutions were prepared by dust-free Millipore water in phosphate buffer 0.01 M pH = 7.4. Aqueous suspensions of spores of Bacillus clausii were prepared from commercial samples (8 × 108 units/mL, Sanofi), removing the excipients through dialysis against Millipore water. All other reagents were obtained from Aldrich Chemicals Co. and used as received without further purification. Fractal aggregates of t-CuPagg were freshly prepared by adding a concentrated sodium chloride solution (0.06–0.1 M) to an aqueous solution of the porphyrin (5 μM). When needed, an appropriate buffer was added to set the pH. The chiral biopolymers were added as last reagents to these preformed aggregates. UV–Vis absorption and extinction spectra were measured on an Agilent mod. 8453 diode-array spectrophotometer using 1 cm path length quartz cells (Hellma). Resonance light scattering (RLS) experiments were performed on a Jasco model FP-750 spectrofluorimeter using a synchronous scan protocol with right angle geometry [2]. The circular dichroism (CD) spectra were recorded on a JASCO J-720 spectropolarimeter, equipped with a 450 W xenon lamp. CD spectra were corrected both for the cell and solvent contributions. The dissymmetry g-factor has been calculated as (ΔA/A) [25]. The kinetic measurements (extinction) were followed at fixed wavelength by mixing the reagents in a silica cell in the thermostated compartment of the instrument, with a temperature accuracy of 0.1 K. The average aggregation time, τE, defined according to the equation τ E = ∫ I(t)dt/I(0) was evaluated by numerical integration of the observable I(t) = Ext(t) — Ext(∞) versus time, divided by I(0) = Ext(0) — Ext(∞) (where Ext(t), Ext(0) and Ext(∞) are the extinction at time t, at t = 0 and after completion of the reaction, respectively) [18].
bands at 545 and 590 nm. Under these conditions, the presence of extended aggregates can be ruled out by the low intensity in the resonance light scattering (RLS) spectra, that show only a minimum at 418 nm, due to photon loss by absorbance of the sample. Previous investigations have pointed out the dimeric nature of this porphyrin even at very low concentrations [19]. When the ionic strength of the solution is increased, large spectral changes are observed in the extinction and RLS spectra. Fig. 2 shows a typical UV–Vis spectral change observed in time after adding sodium chloride (0.1 M) to a 3 μM solution of t-CuPagg. The decrease of the B band is paralleled by an increase of a new band at 444 nm, that is ascribed to extended porphyrin aggregates, as confirmed by the large enhancement in the RLS spectra (strong peaks between 350 and 650 nm, Fig. 3). In order to find a range of experimental conditions to form these species, we investigated the effect of added sodium chloride in the range of 2–150 mM on the kinetics of aggregation and the final distribution between non-aggregated and aggregated porphyrin for a 5 μM aqueous solution of t-CuPagg. When [NaCl] b 15 mM, the UV–Vis extinction spectra display only a slight decrease of the 418 nm band. Above this concentration, the band at 444 nm, corresponding to the aggregated porphyrin, begins to appear, first as a shoulder, then for [NaCl] N 50 mM as the main spectral feature in this region (Fig. 4a–b). The corresponding aggregation kinetics have been monitored and, as expected, they become faster on increasing the ionic strength. The extinction traces follow a typical sigmoidal profile with an initial lag time [26,27]. The mean aggregation times have been determined and the values are reported in Fig. 5. On the bases of these experimental findings, in order to have a quite rapid (b 60 s) and complete formation of aggregates in solution, we chose to use [NaCl] = 0.06–0.1 M. A higher concentration of salt is to be avoided due to the instability of the colloidal system, leading to precipitation of the assemblies. The behavior of this porphyrin is similar to that of the parent t-H2Pagg, although the latter species requires higher ionic strength conditions to achieve a complete aggregated state [18]. This difference can be ascribed to the coordination of copper(II) ion into the macrocycle core, that makes it more rigid and less flexible [26,27]. An analogous pattern could be found in the case of tetrakis(N-methylpyridinium-4-yl)porphyrin and its copper(II) derivative, the latter being much more prone to aggregation than the metal free derivative [28]. 3.2. t-CuPagg@ct-DNA The supramolecular assembling of t-CuPagg onto nucleic acids has been reported in literature, using a completely different protocol [13]. Since porphyrins are able to intercalate into the double stranded helix
3. Results and discussion 3.1. t-CuPagg The UV–Vis extinction spectra of t-CuPagg at micromolar concentration in neat water exhibit a B band at 418 nm, together with two Q
Fig. 2. UV/Vis extinction spectral changes after adding NaCl (final concentration: 0.1 M) to a solution containing 5 μM t-CuPagg (T = 298 K, scanning time 1 s).
Please cite this article as: I.G. Occhiuto, et al., Aggregates of a cationic porphyrin as supramolecular probes for biopolymers, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.09.013
I.G. Occhiuto et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Fig. 3. RLS spectra of a) t-CuPagg 5 μM in water (solid), b) after aggregation with NaCl 0.1 M (dashed) at 298 K.
of DNA under low ionic strength conditions, in the earlier investigations the intercalated porphyrin well-dispersed in the nucleic acid was the starting state for this system. The supramolecular assembly formation was initiated by adding a consistent amount of salt, that leads to the porphyrin de-intercalating and self-assembling using the nucleic acid
Fig. 4. a) UV/Vis extinction spectra in samples equilibrated at 298 K containing t-CuPagg 5 μM after adding increasing amount of NaCl (2 × 10−3 M up to 0.15 M, the arrows mark the increase in concentration); b) plot of the final extinction values at 418 and 444 nm as function of increasing [NaCl].
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Fig. 5. Mean aggregation time τE measured for the kinetics of aggregation of t-CuPagg 5 μM at 298 K as function of increasing NaCl concentration.
as a chiral scaffold. In this case, the assembling process exhibits rates strongly dependent on the porphyrin and DNA concentration as well as the final ionic strength. Recent studies have demonstrated that the mixing protocol has a deep impact on the final aggregated state [19, 20,29]. Therefore in the present investigations, we adopted a protocol based on mixing porphyrin aggregates at the selected ionic strength and pH with the chiral target. It is worth to note that: i) being the porphyrin achiral, the initial assemblies do not exhibit optical activity. Therefore, the observation of induced circular dichroism (ICD) signals in the UV–Vis region of the B-band of the porphyrin is a clear indication of the transmission of chirality from the target species to the aggregates, and ii) the interaction between the positively charged porphyrin assemblies and the negatively charged biopolymers is primarily electrostatic and very fast, leading to a rapid transmission of the chirality. No transfer of chirality to the porphyrin aggregates is observed when positively charged biopolymers are employed (data not shown). The addition of a small concentration of calf thymus DNA (ct-DNA; 0.1 μM) to a solution of the preformed t-CuPagg aggregates at pH 7.2 (1 mM phosphate buffer) leads to quite modest changes in the UV–Vis spectral features of the aggregated porphyrin. The band at 444 nm undergoes a slight hypochromicity (b10%), with no detectable shift in its position (Fig. 6a). The corresponding RLS spectra exhibit a small increase in the intensity of the feature at 474 nm (Fig. 6b), pointing to a moderate increase of the overall dimension of the aggregated species. Most importantly, a conservative CD spectrum is almost instantaneously induced at 444 nm with a negative Cotton effect (Fig. 6c). It is interesting to point that the ICD spectra observed in the present investigation are more symmetric and lower in intensity with respect to those previously reported in the literature for a different mixing protocol (pre-intercalated porphyrin followed by ionic strength jump) [13]. These spectral features undergo only very slight changes on aging the solution. When a higher concentration of ct-DNA (30 μM) is added to the t-CuPagg aggregates, the general pattern of behavior is similar (Fig. 7a–c): i) the hypochromicity of the B-band is higher (N20%), ii) the intensity of RLS is larger and iii) the ICD is about 5 times larger. An important difference can be observed in aging the samples. In this case, after the initial instantaneous changes, the 444 nm band slowly interconverts to a new feature at 428 nm in the UV–Vis extinction spectra. This slow process can be monitored also by a decrease of the RLS intensity at 474 nm (ca 30%), accompanied by a very small reduction of the ICD intensity. These experimental findings point to a partial disruption of the fractal assemblies due to a slow intercalation of the t-CuPagg monomers into double helical DNA. The intensity of the instantaneous ICD signal depends on the concentration of ct-DNA. It increases very
Please cite this article as: I.G. Occhiuto, et al., Aggregates of a cationic porphyrin as supramolecular probes for biopolymers, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.09.013
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Fig. 6. UV/Vis extinction (a), RLS (b) and CD (c) spectra of t-CuPagg in water (black), after aggregation with NaCl (red), soon after addition of 0.1 μM ct-DNA (green) and after 1 h (blue). Experimental conditions: [t-CuPagg] = 5 μM; [NaCl] = 0.1 M; [phosphate buffer] = 1 mM, pH 7.2.
steeply for low DNA concentration, then levels-off evidencing a very slight decrease at high loads (Fig. 8). The electrostatic interaction between the two species is very efficient at low level of added nucleic acids, saturating the aggregates at higher concentrations. 3.3. t-CuPagg@L-PGA A solution of the preformed t-CuPagg aggregates has been treated with increasing amount of poly-L-glutamic acid (L-PGA) having two different molecular weights (MW 17 kD and 1.5 kD) at pH 4 (acetate buffer). Under these experimental conditions, L-PGA is predominantly in the form of a right-handed α-helix and the glutamate residues are half-protonated [30]. As previously reported for this system [20], all the extinction spectra retain the spectral features of an aggregated
Fig. 7. UV/Vis extinction (a), RLS (b) and CD (c) spectra of t-CuPagg in water (solid), after aggregation with NaCl (dotted), soon after addition of 30 μM ct-DNA (dashed) and after 1 h (dash–dotted). Experimental conditions: [t-CuPagg] = 5 μM; [NaCl] = 0.1 M; [phosphate buffer] = 1 mM, pH 7.2.
species with a B-band at 444 nm, together with a higher energy band at 380 nm. RLS spectra display a resonant signal at 475 nm, whose intensity increases on increasing the PGA concentration (data not shown). A CD signal is induced in the region of the porphyrin B-band (Fig. 9a), showing a bisignate profile with a positive Cotton effect centered at 444 nm and a much weaker one centered at 390 nm with a negative Cotton effect. The intensity of ICD increases almost linearly for very small PGA concentration (up to 10 μM), then it tends to level off and the behavior is reported in Fig. 9b. The two polymers behave similarly, being the total intensity larger in the case of PGA having a lower molecular weight. 3.4. t-CuPagg@HSA Human serum albumin (HSA) is a globular protein with a molecular weight of 66.4 kD and an isoelectric point of 4.8; i.e., it is negatively
Please cite this article as: I.G. Occhiuto, et al., Aggregates of a cationic porphyrin as supramolecular probes for biopolymers, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.09.013
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Fig. 8. Dissymmetry g-factor for a batch titration experiment, where increasing amounts of ct-DNA have been added to fully-aggregated solutions of t-CuPagg. Experimental conditions: [t-CuPagg] = 5 μM; [NaCl] = 0.1 M; [phosphate buffer] = 1 mM, pH 7.2.
charged at neutral pH [31]. Similarly to PGA, addiction of increasing amount of HSA to preformed fractal aggregates at pH 7.4 (1 mM phosphate buffer) leads to a rapid appearance of an induced conservative CD signal having a negative Cotton effect in the region of t-CuPagg
Fig. 10. a) CD spectra of t-CuPagg aggregates after adding HSA 300 μM. b) dissymmetry g-factor for a batch titration experiment, where increasing amounts of HSA have been added to fully-aggregated solutions of t-CuPagg. Experimental conditions: [t-CuPagg] = 5 μM, [NaCl] = 60 mM, phosphate buffer 1 mM pH = 7.4, T = 298 K.
aggregates (Fig.10a). These spectral features suggest that porphyrin aggregates are affected by the presence of the protein at very low concentration, responding excellently as a chiroptical probe. Fig. 10 shows the increase of the total ICD signal as a function of HSA concentration, displaying an almost linear behavior up to 10 μM. 3.5. t-CuPagg@Bacillus clausii Spores of Bacillus clausii provide a simple check for detecting chiral contamination. Bacillus clausii is a Gram-positive microorganism whose external membrane carries a modest negative charge [32]. The addition of small quantities of a suspension of the spores of this bacterium to the fractal assemblies leads to CD spectra with quite intense positive bisignate bands centered at 440 nm (Fig. 11). 3.6. Concluding remarks
Fig. 9. a) ICD spectrum of pre-aggregated t-CuPagg after adding [L-PGA] = 50 μM; b) dissymmetry g-factor for a batch titration experiment, where increasing amounts of L-PGA (MW 17 kD (squares) and 1.5 kD (circles) have been added to fully-aggregated solutions of t-CuPagg. Experimental conditions: [t-CuPagg] = 5 μM; [NaCl] = 0.1 M; [acetate buffer] = 5 mM, pH 4.0.
The porphyrin t-CuPagg exhibits a marked propensity to selfaggregate even at rather low ionic strength conditions, leading to extended fractal aggregates. This property is more pronounced with respect to the parent unmetalated t-H2Pagg, probably due to a structural rigidity due to the presence of the copper(II) ion that imposes a square planar coordination. These assemblies are positively charged and interact quite efficiently with negatively chiral species, giving a
Please cite this article as: I.G. Occhiuto, et al., Aggregates of a cationic porphyrin as supramolecular probes for biopolymers, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.09.013
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Fig. 11. ICD spectrum of a sample of aggregated t-CuPagg after adding spores of Bacillus clausii. Experimental conditions: [t-CuPagg] = 5 μM, [NaCl] = 60 mM, [Bacillus clausii] = 107 units/mL, phosphate buffer 1 mM pH = 7.2, T = 298 K.
linear response in of ICD signals in a range of low biopolymer concentration. On increasing the concentration of the chiral inducers, the transmission of the chirality levels off, probably due to a rapid saturation of the interaction sites. Previous investigations have shown that the observed chirality is a mesoscopic and not a local property [20]. An important feature is the flexibility and porosity of the fractal aggregates, that explains their ability to quickly interact with the external chiral bias [20]. Finally, as a word of caution, Fig. 12 shows a comparison between samples of t-CuPagg aggregates prepared using water from two different sources. When high purity water from sealed glass vials (for injectable preparations, Galenica Senese) is used, the CD spectra do not reveal any detectable signals (solid trace). On the contrary, even if we are not able to determine definitely the source of chiral contamination, for samples of normal doubly distilled water stored in plastic (polyethylene) containers, weak to moderate dichroic signals are always present in the CD spectra (dashed trace). Our experimental findings suggest that the t-CuPagg system can be exploited not only as a chiroptical probe for nucleic acids and proteins, but also for efficient detection of chiral contaminants. Abbreviations CD circular dichroism ICD induced circular dichroism RLS resonance light scattering t-H2Pagg 5,15-bis(N-methylpyridinium-4-yl)-10,15-bisdiphenylporphine t-CuPagg 5,15-bis(N-methylpyridinium-4-yl)-10,15-bisdiphenylporphirinato copper(II) ct-DNA calf thymus DNA PGA poly-glutamic acid L-PGA poly-L-glutamic acid HSA human serum albumin
Acknowledgments The authors thank MIUR (PRIN 2010-2011), C.I.R.C.M.S.B. and CNR for financial support.
Fig. 12. CD spectra of samples of preformed t-CuPagg aggregates in pure water (for injectable preparations, Galenica Senese) (solid trace) and in doubly distilled water from a plastic (polyethylene) bottle (dashed trace). Experimental conditions: [t-CuPagg] = 5 μM, [NaCl] = 60 mM, T = 298 K.
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Please cite this article as: I.G. Occhiuto, et al., Aggregates of a cationic porphyrin as supramolecular probes for biopolymers, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.09.013
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Aggregates of a cationic porphyrin as supramolecular probes for biopolymers Ilaria Giuseppina Occhiuto a, Mario Samperi b, Mariachiara Trapani c, Giovanna De Luca d, Andrea Romeo b, Robert F. Pasternack e, Luigi Monsù Scolaro b,c,⁎ a
Dipartimento di Scienze Chimiche, Via Marzolo 1, 35131 Padova, Italy Dipartimento di Scienze Chimiche and C.I.R.C.M.S.B., V.le F. Stagno d'Alcontres 31, 98166 Messina, Italy Istituto per lo Studio dei Materiali Nanostrutturati ISMN-CNR c/o Dipartimento di Scienze Chimiche, University of Messina, V.le F. Stagno D'Alcontres, 31-98166 Messina, Italy d Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, V.le Annunziata, 98168 Messina, Italy e Department of Chemistry & Biochemistry, Swarthmore College, 500 College Avenue, Swarthmore, PA 19081, USA b c
a r t i c l e
i n f o
Article history: Received 15 June 2015 Received in revised form 11 September 2015 Accepted 30 September 2015 Available online xxxx Keywords: Porphyrin Supramolecular aggregate Chiroptical sensor Chirality Circular dichroism
a b s t r a c t The copper(II) derivative of the dicationic trans-bis(N-methylpyridinium-4-yl)diphenylporphyrin (t-CuPagg) forms large fractal aggregates in aqueous solution under moderate ionic strength conditions. A kinetic investigation of the aggregation process allows for a choice of experimental conditions to quickly obtain stable assemblies in solution. These positively charged aggregates are able to interact efficiently with negatively charged chiral species, (including bacterial spores) leading to induced circular dichroism signals in the Soret region of the porphyrin, now acting as a sensitive chiroptical probe. © 2015 Elsevier Inc. All rights reserved.
1. Introduction The design and synthesis of supramolecular optical sensors for the detection of chiral molecules/biomolecules is actively investigated due to their implications both in fundamental and applicative fields [1]. In particular, it is important to find systems able to quickly report information on very diluted samples. Porphyrins have been extensively used to this purpose, due to their rich spectroscopic features, e.g., high extinction coefficients and resonant light scattering effects [2] that allow their use as sensitive probes. Hetero-aggregates based on water soluble porphyrins have been successfully exploited to detect and amplify traces of aggregated amino acids [3] and polypeptides [4–6]. The large circular dichroism (CD) signals observed for J-aggregates of the anionic tetrakis(4-sulfonatophenyl)porphyrin has been ascribed to the presence of optically active species (i.e. bacterial membranes) present as traces in ultrapure water [7], or to added chiral templates [8–12]. Trans-bis(N-methylpyridinium-4-yl)diphenylporphyrin and its copper(II) derivative (t-CuPagg, Fig. 1) are dicationic water soluble porphyrins that have been shown to form extended supramolecular aggregates on nucleic acids [13,14]. This species as well as its parent unmetalated porphyrin (t-H2Pagg) form fractal assemblies whose structural features are modulated by the ionic strength [15–18]. These aggregates are quite large ⁎ Corresponding author. E-mail address: [email protected] (L.M. Scolaro).
(sizes ranging from 100 nm up to 1–2 μm), flexible and porous. The strong electronic coupling among the neighboring chromophores, along with the extended dimensions of the aggregates, is responsible for large resonant light scattering signals in their aqueous solutions [2]. Quite recently, t-CuPagg porphyrin in its dimeric [19] and largely aggregated form [20] has been proposed as chiroptical sensors for conformations and chirality of poly(glutamate). In particular, t-CuPagg studies have shown the presence of a left handed helix (poly-proline-II) and the coexistence of different conformations for this simple polypeptide [19]. Here we report an investigation on the kinetics of formation of aggregates of t-CuPagg and their use as chiroptical probes for a variety of chiral negatively charged biopolymers, i.e. calf thymus DNA, polyglutamate and human serum albumin (HSA), as well as bacterial spores. 2. Experimental 2.1. Materials and methods 5,15-bis(N-methylpyridinium-4-yl)-10,15-bis-diphenylporphine (t-H2Pagg, chloride salt) was purchased from Frontier Scientific and used as received. Its copper(II) derivative (t-CuPagg) was prepared by literature procedures [21]. Porphyrins stock solutions were prepared dissolving the solids in dust-free water (Galenica Senese) and stored in the dark. Solution concentrations were determined from known molar extinction coefficient at the Soret maximum (t-CuPagg: 2.34 × 105 M− 1 cm− 1) [13].
http://dx.doi.org/10.1016/j.jinorgbio.2015.09.013 0162-0134/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: I.G. Occhiuto, et al., Aggregates of a cationic porphyrin as supramolecular probes for biopolymers, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.09.013
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Fig. 1. Structure of the copper(II) derivative of trans-bis(N-methylpyridinium-4yl)diphenylporphyrin (t-CuPagg, chloride salt).
Calf thymus DNA (ct-DNA, type I) was obtained from Sigma and was purified according to literature methods [22]. The concentration of a ct-DNA stock solution in 1 mM phosphate buffer, pH 7, containing 10 mM NaCl, was determined in molar base-pairs using ε260 = 13,200 M− 1 cm − 1 [23]. Sodium salts of poly-L -glutamic acid (FW ~ 17 kD and ~ 1.5 kD) were obtained from Aldrich Chemicals Co. Stock solution of the polypeptides were prepared by dissolving the solids in acetate buffer at pH 4.2 (ionic strength 5 mM). Concentrations of the peptide solutions were determined spectrophotometrically, using ε205 = 2150 M− 1 cm− 1 [24]. Human Serum Albumin (HSA) was purchased from Sigma and stock solutions were prepared by dust-free Millipore water in phosphate buffer 0.01 M pH = 7.4. Aqueous suspensions of spores of Bacillus clausii were prepared from commercial samples (8 × 108 units/mL, Sanofi), removing the excipients through dialysis against Millipore water. All other reagents were obtained from Aldrich Chemicals Co. and used as received without further purification. Fractal aggregates of t-CuPagg were freshly prepared by adding a concentrated sodium chloride solution (0.06–0.1 M) to an aqueous solution of the porphyrin (5 μM). When needed, an appropriate buffer was added to set the pH. The chiral biopolymers were added as last reagents to these preformed aggregates. UV–Vis absorption and extinction spectra were measured on an Agilent mod. 8453 diode-array spectrophotometer using 1 cm path length quartz cells (Hellma). Resonance light scattering (RLS) experiments were performed on a Jasco model FP-750 spectrofluorimeter using a synchronous scan protocol with right angle geometry [2]. The circular dichroism (CD) spectra were recorded on a JASCO J-720 spectropolarimeter, equipped with a 450 W xenon lamp. CD spectra were corrected both for the cell and solvent contributions. The dissymmetry g-factor has been calculated as (ΔA/A) [25]. The kinetic measurements (extinction) were followed at fixed wavelength by mixing the reagents in a silica cell in the thermostated compartment of the instrument, with a temperature accuracy of 0.1 K. The average aggregation time, τE, defined according to the equation τ E = ∫ I(t)dt/I(0) was evaluated by numerical integration of the observable I(t) = Ext(t) — Ext(∞) versus time, divided by I(0) = Ext(0) — Ext(∞) (where Ext(t), Ext(0) and Ext(∞) are the extinction at time t, at t = 0 and after completion of the reaction, respectively) [18].
bands at 545 and 590 nm. Under these conditions, the presence of extended aggregates can be ruled out by the low intensity in the resonance light scattering (RLS) spectra, that show only a minimum at 418 nm, due to photon loss by absorbance of the sample. Previous investigations have pointed out the dimeric nature of this porphyrin even at very low concentrations [19]. When the ionic strength of the solution is increased, large spectral changes are observed in the extinction and RLS spectra. Fig. 2 shows a typical UV–Vis spectral change observed in time after adding sodium chloride (0.1 M) to a 3 μM solution of t-CuPagg. The decrease of the B band is paralleled by an increase of a new band at 444 nm, that is ascribed to extended porphyrin aggregates, as confirmed by the large enhancement in the RLS spectra (strong peaks between 350 and 650 nm, Fig. 3). In order to find a range of experimental conditions to form these species, we investigated the effect of added sodium chloride in the range of 2–150 mM on the kinetics of aggregation and the final distribution between non-aggregated and aggregated porphyrin for a 5 μM aqueous solution of t-CuPagg. When [NaCl] b 15 mM, the UV–Vis extinction spectra display only a slight decrease of the 418 nm band. Above this concentration, the band at 444 nm, corresponding to the aggregated porphyrin, begins to appear, first as a shoulder, then for [NaCl] N 50 mM as the main spectral feature in this region (Fig. 4a–b). The corresponding aggregation kinetics have been monitored and, as expected, they become faster on increasing the ionic strength. The extinction traces follow a typical sigmoidal profile with an initial lag time [26,27]. The mean aggregation times have been determined and the values are reported in Fig. 5. On the bases of these experimental findings, in order to have a quite rapid (b 60 s) and complete formation of aggregates in solution, we chose to use [NaCl] = 0.06–0.1 M. A higher concentration of salt is to be avoided due to the instability of the colloidal system, leading to precipitation of the assemblies. The behavior of this porphyrin is similar to that of the parent t-H2Pagg, although the latter species requires higher ionic strength conditions to achieve a complete aggregated state [18]. This difference can be ascribed to the coordination of copper(II) ion into the macrocycle core, that makes it more rigid and less flexible [26,27]. An analogous pattern could be found in the case of tetrakis(N-methylpyridinium-4-yl)porphyrin and its copper(II) derivative, the latter being much more prone to aggregation than the metal free derivative [28]. 3.2. t-CuPagg@ct-DNA The supramolecular assembling of t-CuPagg onto nucleic acids has been reported in literature, using a completely different protocol [13]. Since porphyrins are able to intercalate into the double stranded helix
3. Results and discussion 3.1. t-CuPagg The UV–Vis extinction spectra of t-CuPagg at micromolar concentration in neat water exhibit a B band at 418 nm, together with two Q
Fig. 2. UV/Vis extinction spectral changes after adding NaCl (final concentration: 0.1 M) to a solution containing 5 μM t-CuPagg (T = 298 K, scanning time 1 s).
Please cite this article as: I.G. Occhiuto, et al., Aggregates of a cationic porphyrin as supramolecular probes for biopolymers, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.09.013
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Fig. 3. RLS spectra of a) t-CuPagg 5 μM in water (solid), b) after aggregation with NaCl 0.1 M (dashed) at 298 K.
of DNA under low ionic strength conditions, in the earlier investigations the intercalated porphyrin well-dispersed in the nucleic acid was the starting state for this system. The supramolecular assembly formation was initiated by adding a consistent amount of salt, that leads to the porphyrin de-intercalating and self-assembling using the nucleic acid
Fig. 4. a) UV/Vis extinction spectra in samples equilibrated at 298 K containing t-CuPagg 5 μM after adding increasing amount of NaCl (2 × 10−3 M up to 0.15 M, the arrows mark the increase in concentration); b) plot of the final extinction values at 418 and 444 nm as function of increasing [NaCl].
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Fig. 5. Mean aggregation time τE measured for the kinetics of aggregation of t-CuPagg 5 μM at 298 K as function of increasing NaCl concentration.
as a chiral scaffold. In this case, the assembling process exhibits rates strongly dependent on the porphyrin and DNA concentration as well as the final ionic strength. Recent studies have demonstrated that the mixing protocol has a deep impact on the final aggregated state [19, 20,29]. Therefore in the present investigations, we adopted a protocol based on mixing porphyrin aggregates at the selected ionic strength and pH with the chiral target. It is worth to note that: i) being the porphyrin achiral, the initial assemblies do not exhibit optical activity. Therefore, the observation of induced circular dichroism (ICD) signals in the UV–Vis region of the B-band of the porphyrin is a clear indication of the transmission of chirality from the target species to the aggregates, and ii) the interaction between the positively charged porphyrin assemblies and the negatively charged biopolymers is primarily electrostatic and very fast, leading to a rapid transmission of the chirality. No transfer of chirality to the porphyrin aggregates is observed when positively charged biopolymers are employed (data not shown). The addition of a small concentration of calf thymus DNA (ct-DNA; 0.1 μM) to a solution of the preformed t-CuPagg aggregates at pH 7.2 (1 mM phosphate buffer) leads to quite modest changes in the UV–Vis spectral features of the aggregated porphyrin. The band at 444 nm undergoes a slight hypochromicity (b10%), with no detectable shift in its position (Fig. 6a). The corresponding RLS spectra exhibit a small increase in the intensity of the feature at 474 nm (Fig. 6b), pointing to a moderate increase of the overall dimension of the aggregated species. Most importantly, a conservative CD spectrum is almost instantaneously induced at 444 nm with a negative Cotton effect (Fig. 6c). It is interesting to point that the ICD spectra observed in the present investigation are more symmetric and lower in intensity with respect to those previously reported in the literature for a different mixing protocol (pre-intercalated porphyrin followed by ionic strength jump) [13]. These spectral features undergo only very slight changes on aging the solution. When a higher concentration of ct-DNA (30 μM) is added to the t-CuPagg aggregates, the general pattern of behavior is similar (Fig. 7a–c): i) the hypochromicity of the B-band is higher (N20%), ii) the intensity of RLS is larger and iii) the ICD is about 5 times larger. An important difference can be observed in aging the samples. In this case, after the initial instantaneous changes, the 444 nm band slowly interconverts to a new feature at 428 nm in the UV–Vis extinction spectra. This slow process can be monitored also by a decrease of the RLS intensity at 474 nm (ca 30%), accompanied by a very small reduction of the ICD intensity. These experimental findings point to a partial disruption of the fractal assemblies due to a slow intercalation of the t-CuPagg monomers into double helical DNA. The intensity of the instantaneous ICD signal depends on the concentration of ct-DNA. It increases very
Please cite this article as: I.G. Occhiuto, et al., Aggregates of a cationic porphyrin as supramolecular probes for biopolymers, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.09.013
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Fig. 6. UV/Vis extinction (a), RLS (b) and CD (c) spectra of t-CuPagg in water (black), after aggregation with NaCl (red), soon after addition of 0.1 μM ct-DNA (green) and after 1 h (blue). Experimental conditions: [t-CuPagg] = 5 μM; [NaCl] = 0.1 M; [phosphate buffer] = 1 mM, pH 7.2.
steeply for low DNA concentration, then levels-off evidencing a very slight decrease at high loads (Fig. 8). The electrostatic interaction between the two species is very efficient at low level of added nucleic acids, saturating the aggregates at higher concentrations. 3.3. t-CuPagg@L-PGA A solution of the preformed t-CuPagg aggregates has been treated with increasing amount of poly-L-glutamic acid (L-PGA) having two different molecular weights (MW 17 kD and 1.5 kD) at pH 4 (acetate buffer). Under these experimental conditions, L-PGA is predominantly in the form of a right-handed α-helix and the glutamate residues are half-protonated [30]. As previously reported for this system [20], all the extinction spectra retain the spectral features of an aggregated
Fig. 7. UV/Vis extinction (a), RLS (b) and CD (c) spectra of t-CuPagg in water (solid), after aggregation with NaCl (dotted), soon after addition of 30 μM ct-DNA (dashed) and after 1 h (dash–dotted). Experimental conditions: [t-CuPagg] = 5 μM; [NaCl] = 0.1 M; [phosphate buffer] = 1 mM, pH 7.2.
species with a B-band at 444 nm, together with a higher energy band at 380 nm. RLS spectra display a resonant signal at 475 nm, whose intensity increases on increasing the PGA concentration (data not shown). A CD signal is induced in the region of the porphyrin B-band (Fig. 9a), showing a bisignate profile with a positive Cotton effect centered at 444 nm and a much weaker one centered at 390 nm with a negative Cotton effect. The intensity of ICD increases almost linearly for very small PGA concentration (up to 10 μM), then it tends to level off and the behavior is reported in Fig. 9b. The two polymers behave similarly, being the total intensity larger in the case of PGA having a lower molecular weight. 3.4. t-CuPagg@HSA Human serum albumin (HSA) is a globular protein with a molecular weight of 66.4 kD and an isoelectric point of 4.8; i.e., it is negatively
Please cite this article as: I.G. Occhiuto, et al., Aggregates of a cationic porphyrin as supramolecular probes for biopolymers, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.09.013
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Fig. 8. Dissymmetry g-factor for a batch titration experiment, where increasing amounts of ct-DNA have been added to fully-aggregated solutions of t-CuPagg. Experimental conditions: [t-CuPagg] = 5 μM; [NaCl] = 0.1 M; [phosphate buffer] = 1 mM, pH 7.2.
charged at neutral pH [31]. Similarly to PGA, addiction of increasing amount of HSA to preformed fractal aggregates at pH 7.4 (1 mM phosphate buffer) leads to a rapid appearance of an induced conservative CD signal having a negative Cotton effect in the region of t-CuPagg
Fig. 10. a) CD spectra of t-CuPagg aggregates after adding HSA 300 μM. b) dissymmetry g-factor for a batch titration experiment, where increasing amounts of HSA have been added to fully-aggregated solutions of t-CuPagg. Experimental conditions: [t-CuPagg] = 5 μM, [NaCl] = 60 mM, phosphate buffer 1 mM pH = 7.4, T = 298 K.
aggregates (Fig.10a). These spectral features suggest that porphyrin aggregates are affected by the presence of the protein at very low concentration, responding excellently as a chiroptical probe. Fig. 10 shows the increase of the total ICD signal as a function of HSA concentration, displaying an almost linear behavior up to 10 μM. 3.5. t-CuPagg@Bacillus clausii Spores of Bacillus clausii provide a simple check for detecting chiral contamination. Bacillus clausii is a Gram-positive microorganism whose external membrane carries a modest negative charge [32]. The addition of small quantities of a suspension of the spores of this bacterium to the fractal assemblies leads to CD spectra with quite intense positive bisignate bands centered at 440 nm (Fig. 11). 3.6. Concluding remarks
Fig. 9. a) ICD spectrum of pre-aggregated t-CuPagg after adding [L-PGA] = 50 μM; b) dissymmetry g-factor for a batch titration experiment, where increasing amounts of L-PGA (MW 17 kD (squares) and 1.5 kD (circles) have been added to fully-aggregated solutions of t-CuPagg. Experimental conditions: [t-CuPagg] = 5 μM; [NaCl] = 0.1 M; [acetate buffer] = 5 mM, pH 4.0.
The porphyrin t-CuPagg exhibits a marked propensity to selfaggregate even at rather low ionic strength conditions, leading to extended fractal aggregates. This property is more pronounced with respect to the parent unmetalated t-H2Pagg, probably due to a structural rigidity due to the presence of the copper(II) ion that imposes a square planar coordination. These assemblies are positively charged and interact quite efficiently with negatively chiral species, giving a
Please cite this article as: I.G. Occhiuto, et al., Aggregates of a cationic porphyrin as supramolecular probes for biopolymers, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.09.013
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Fig. 11. ICD spectrum of a sample of aggregated t-CuPagg after adding spores of Bacillus clausii. Experimental conditions: [t-CuPagg] = 5 μM, [NaCl] = 60 mM, [Bacillus clausii] = 107 units/mL, phosphate buffer 1 mM pH = 7.2, T = 298 K.
linear response in of ICD signals in a range of low biopolymer concentration. On increasing the concentration of the chiral inducers, the transmission of the chirality levels off, probably due to a rapid saturation of the interaction sites. Previous investigations have shown that the observed chirality is a mesoscopic and not a local property [20]. An important feature is the flexibility and porosity of the fractal aggregates, that explains their ability to quickly interact with the external chiral bias [20]. Finally, as a word of caution, Fig. 12 shows a comparison between samples of t-CuPagg aggregates prepared using water from two different sources. When high purity water from sealed glass vials (for injectable preparations, Galenica Senese) is used, the CD spectra do not reveal any detectable signals (solid trace). On the contrary, even if we are not able to determine definitely the source of chiral contamination, for samples of normal doubly distilled water stored in plastic (polyethylene) containers, weak to moderate dichroic signals are always present in the CD spectra (dashed trace). Our experimental findings suggest that the t-CuPagg system can be exploited not only as a chiroptical probe for nucleic acids and proteins, but also for efficient detection of chiral contaminants. Abbreviations CD circular dichroism ICD induced circular dichroism RLS resonance light scattering t-H2Pagg 5,15-bis(N-methylpyridinium-4-yl)-10,15-bisdiphenylporphine t-CuPagg 5,15-bis(N-methylpyridinium-4-yl)-10,15-bisdiphenylporphirinato copper(II) ct-DNA calf thymus DNA PGA poly-glutamic acid L-PGA poly-L-glutamic acid HSA human serum albumin
Acknowledgments The authors thank MIUR (PRIN 2010-2011), C.I.R.C.M.S.B. and CNR for financial support.
Fig. 12. CD spectra of samples of preformed t-CuPagg aggregates in pure water (for injectable preparations, Galenica Senese) (solid trace) and in doubly distilled water from a plastic (polyethylene) bottle (dashed trace). Experimental conditions: [t-CuPagg] = 5 μM, [NaCl] = 60 mM, T = 298 K.
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Please cite this article as: I.G. Occhiuto, et al., Aggregates of a cationic porphyrin as supramolecular probes for biopolymers, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.09.013