Article Cite This: J. Phys. Chem. B 2019, 123, 5832−5840
pubs.acs.org/JPCB
Characterization of Protoporphyrin IX Species in Vitro Using Fluorescence Spectroscopy and Polar Plot Analysis Kai Wen Teng*,†,‡ and Sang Hak Lee*,‡,§ †
Center for Biophysics and Computational Biology and ‡Department of Physics, University of Illinois of Urbana-Champaign, Urbana, Illinois 61801, United States § Department of Chemistry, Pusan National University, Busan 46241, Korea
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ABSTRACT: Protoporphyrin IX (PPIX) is a photodynamic therapy (PDT) agent for the treatment of various types of cancer. The effectiveness of PDT is believed to be associated with aggregation of PPIX in cells. However, the aggregation equilibrium of PPIX in the cellular environment and in solution is still poorly understood. This is attributed by the lack of a method that allows for controllable generation of PPIX aggregates and robust analysis technique for measuring their photophysical properties. In this study, the dynamics of PPIX aggregation were investigated under high pressure and different solvent conditions using time-resolved fluorescence spectroscopy. The data were analyzed on a polar plot, a model-free analysis method that has become increasingly popular for fluorescence lifetime studies. We discovered that increasing hydrostatic pressure enhanced the formation of J-type aggregates based on measured absorbance, spectra, and lifetime features. Formation of large aggregates, which have a subnanosecond lifetime in the excited state, was observed under the increasing concentration of divalent cations as well as under a solvent of around neutral pH. PPIX monomerizes from the aggregate as pH becomes more basic, not dimerization as proposed by previous studies. Here, we demonstrate that the combination of time-resolved measurement and polar plot analysis is very robust for monitoring the presence of different types of PPIX aggregates formed in various chemical environments.
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INTRODUCTION Protoporphyrin IX (PPIX) is an endogenously produced molecule in cells as part of the heme-biosynthesis pathway. PPIX preferentially accumulates in various cancer cell lines when exogenous 5-aminolevulinic acids are applied.1−3 The long-lived excited state of PPIX allows it to undergo an intersystem crossing to the triplet state and reacts with oxygen, producing reactive oxygen species (ROS), which will cause damage to the surrounding cellular compartments.4 The selective accumulation in cancer cells and the photophysical property make PPIX an ideal candidate for photodynamic therapy (PDT). PPIX is prone to ionization and aggregation in physiological relevant pH,5−7 and whether these factors will influence the efficiency of PDT is still unclear. Previous studies have shown that different PPIX species (ions and aggregates) are distributed across various cellular compartments.8,9 However, since the photophysical properties of PPIX are not very well characterized in vitro, it is difficult to directly correlate the species observed in cells to either PPIX ions or aggregates. Spectroscopic approaches are often used to characterize ionized PPIX and its aggregates based on their photophysical properties. Porphyrins are known to form either the J-type aggregate or the H-type aggregates, depending on the alignment of individual molecules in the aggregates,10−14 and the photophysical properties of these aggregates are very © 2019 American Chemical Society
different. Furthermore, despite what is known about the types of the aggregate that porphyrins can form, it is still difficult to generate these different aggregates of PPIX to better study them. The aggregation of PPIX is known to be dependent on concentration, types of ions present, pH, and solvents used,13−15 and these factors can also shift the ionization status of the PPIX. Therefore, to controllably induce aggregation, we incorporated the use of high pressure to directly observe shifts in equilibrium between the monomer and aggregate of PPIX in a step-wise fashion.16,17 Due to a spectral overlap between the PPIX monomer, aggregate, and ionized species, it is hard to draw conclusion regarding which of these are present using solely steady-state techniques. Fluorescence lifetime measurements have been used to reliably differentiate molecular aggregates from monomers.18−20 Lifetime measurements in combination with other spectroscopy methods have revealed that the PPIX monomer has an excited state lifetime of 16−20 ns.21,22 The contribution of shorter lifetimes was often discovered in samples containing PPIX both in vivo and in vitro that were proposed to have come from a variety of factors such as the formation of photoproducts, protein binding, or aggregaReceived: February 27, 2019 Revised: June 12, 2019 Published: June 14, 2019 5832
DOI: 10.1021/acs.jpcb.9b01913 J. Phys. Chem. B 2019, 123, 5832−5840
Article
The Journal of Physical Chemistry B tion.23−25 The fluorescence lifetime of the PPIX aggregate is expected to be shorter than the monomer. This is based on the theoretical framework that the intrinsic fluorescence lifetime is related to the number of molecules that are coherently excited in the aggregate; the greater the number of coherently excited monomer in the aggregate, the faster the radiative or nonradiative decay rate, which shortens the excited state lifetime.26 Therefore, the lifetime in the excited state provides a clue to the relative sizes of the aggregate. The recent analysis of lifetime data in the frequency domain as well as the time domain has been focused on using a modelfree analysis method called the polar plot.27−29 A more complete picture of the aggregation dynamics can be visualized by using polar plot analysis; the lifetime in the excited state changes due to any perturbations applied that will move the measured polar coordinate linearly between two states: the aggregate and the monomer. Such analysis can also be applied to spectral lifetime measurement30 where lifetime measurements are performed at several increments of wavelength. This is useful for studying molecular aggregates because the emission of the aggregate is often mixed in with the monomer spectrum. Using both time-resolved measurement and polar plot analysis, we deduce a complete picture of the aggregation dynamics of PPIX under the effects of different pH values, ionic strength, types of ions, and under high pressure. PPIX species with overlapping emission spectra are resolved by performing spectral lifetime measurements. Our technique reveals that aggregation of PPIX and dissociation of PPIX from the aggregate can be observed across acidic to basic pH; PPIXs exist as a monomer in the cases of both extreme acidic and basic conditions and form aggregates at around neutral pH. Aggregates induced by both changing pH and ions are the large type H-aggregate. In addition, a large fraction of smaller J-aggregate was first observed in vitro by introducing high pressure to PPIX dissolved in DMSO.
introduced to the sample from the PPIX stock solution; however, its concentration did not exceed 4% by volume, except for the PPIX monomer solution in which DMSO itself is the solvent. Fluorescence Measurements under High Pressure. Fluorescence emission, excitation, and lifetime measurements under high pressure were recorded using a SLM 8000C fluorometer (SLM Aminco) with ISS Phoenix upgrade. A 405 nm laser diode (ISS) was used as the excitation source. Frequency domain lifetime measurement was achieved using a heterodyne detection method by modulating the laser diode at 40 MHz and the detector at 40 MHz plus 400 Hz. A highpressure cell (NOVA Swiss) was mounted to the sample chamber of the SLM fluorometer, and hydrostatic pressure was applied to the sample by pumping ethanol into the highpressure cell. The sample containing solution of PPIX was loaded into a custom build quartz cylindrical cuvette that is sealed with a piston. The cylindrical cuvette was placed inside the high-pressure cell, and when ethanol is introduced into the high-pressure cell, the piston compresses, exerting pressure on the PPIX sample. Whenever the pressure is increased, the sample is allowed to equilibrate at the new pressure for 10 min before the measurement takes place. At the end of the experiment, the pressure is brought back to standard pressure, and final measurement is taken after 20 min of equilibrating to standard pressure. In most cases, at least 60% of the original monomer intensity at 630 nm has returned. The recovering percentage varies depending on how tight the piston fits with the custom-made pressure cuvette and the amount of photobleaching that took place. In some cases, the intensity recovery of the monomer was as high as 93%. Scatter of the excitation light was used for the reference lifetime measurements with 0 ns as its fluorescence lifetime. To collect modulation and phase at different emission wavelengths for lifetime measurement under high pressure, the monochromator of the fluorometer was set to move from 625 to 705 nm at 10 nm increments. The excitation light was only unblocked during the time of measurements to avoid photobleaching. Steady-State and Time-Dependent Fluorescence Measurements under Different Solvent Conditions. Absorbance measurement was taken with an Agilent 8453E UV−visible spectroscopy system. Fluorescence emission spectra for PPIX in DMSO and Triton X-100 as well as solutions with different pH values were recorded with the ISS PC1 fluorescence spectrophotometer with an excitation wavelength set to 400 nm. Lifetime measurements for experiments under different solvents were all performed using a custom build homodyne full field fluorescence lifetime imaging microscope (FLIM).31,32 The excitation source for the FLIM is a 532 nm laser diode (World Star Tech.), and fluorescence emission was collected over the wavelengths of 560−640 nm. An image intensifier (Kentech Instruments Ltd.) and CCD (QImaging) were used for full-field detection. The intensity of the excitation source was modulated by a Pockels cell (Conoptics), and the intensity of the fluorescence emission was modulated by modulating the detector gain of the image intensifier. For homodyne detection, both excitation and emission were modulated at 40 MHz. The waveform that modulates the intensifier gain was phase shifted over the whole period (2π) during acquisition, and eight phase-shifted intensity images were collected along with an intensity image before and after each phase shifted to correct for photobleaching. To rebuild the fluorescence response containing
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EXPERIMENTAL SECTION Sample Preparation. A 0.5 mM PPIX stock solution was prepared by dissolving protoporphyrin IX (Frontier Scientific, Inc., Logan, UT) in dimethyl sulfoxide (DMSO). The solution was stored in the dark and was used within 2 weeks. To prepare PPIX solution that contains predominantly PPIX monomers, the calculated amount of stock solution was added to DMSO or 0.6% Triton X-100 in 5 mM disodium phosphate at pH 8.0 to a final concentration of 10 μM. The high-pressure experiments were performed at a slightly lower PPIX concentration of 8.2 μM. To test the influence of pH on the aggregation of PPIX, solutions with pH ranging from 0.7 to 10.4 were prepared by adjusting the pH of distilled water with stock acid solution containing 0.1 or 1 N HCl or stock base solution containing 0.1 or 1 N NaOH. The pH of each solution was measured with an electronic pH meter (Fisher Scientific), and the sample was mixed thoroughly and was measured immediately after preparation. To study the effect of salt concentration on PPIX aggregation, NaCl was dissolved in solutions of pH 1.5, 4.5, and 10.5 with a final salt concentration of 0.5 M. To examine the effect of different types of ions, final concentrations of MgCl2, CaCl2, and NaCl in concentrations of 0.08, 0.4, 2 , and 10 mM were prepared in 50 mM Tris-HCl buffer and were adjusted to pH 9.0. The final PPIX concentration in these solutions was 10 μM. Trace amounts of DMSO are present in the above solution that were 5833
DOI: 10.1021/acs.jpcb.9b01913 J. Phys. Chem. B 2019, 123, 5832−5840
Article
The Journal of Physical Chemistry B
acquisition to improve the signal-to-noise ratio of mainly the poorly fluorescent PPIX aggregates. The average polar coordinate as well as standard deviation from all the pixels in the image was projected onto the polar plot.
phase delay and demodulation due to the presence of fluorophores, digital Fourier transform algorithm was applied at each pixel post binning. Instrumental parameters such as the inherent phase and modulation of the instrument were solved using a known reference (Rhodamine 6G in water), which has a 4.11 ns lifetime. Polar Plot Representation of Time-Resolved Data. We use the polar plot to represent the modulation and phase data collected in the frequency domain to analyze multicomponent lifetimes.27−29 This analysis displays frequency domain multicomponent lifetime signals by graphing directly (without further analysis) modulation and phase parameters as vectors in the x−y plane. The polar plot is especially convenient and straightforward for following the kinetics of PPIX aggregation. If the measured frequency domain data are a contribution of two distinct lifetime pools, the intensity and species fractions from each contribution can be evaluated quantitatively. Derivation of polar plot analysis for frequency domain lifetime data has been reported previously.27 In a frequency domain lifetime measurement, the demodulation (Mmeas) and phase (ϕmeas) of the fluorescence relative to the excitation are measured. The measured modulation (Mmeas) and phase (ϕmeas) are transformed into Cartesian space by defining the x and y coordinates as follows:
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RESULTS Steady-State and Time-Dependent Measurement of PPIX Monomers. Absorbance and fluorescence spectra of PPIX in DMSO and Triton X-100 as well as its polar coordinate were recorded as shown in Figure 1. Figure 1A,B shows that the absorbance and emission spectra in both solvents are identical. The strongest absorbance band of the PPIX monomer in DMSO and Triton X-100 was in the Soret region at 407 nm. The emission spectra of PPIX in DMSO and Triton-X showed two bands, with the first band at 637 nm and the second bands at 705 and 707 nm for DMSO and Triton-X, respectively. The measured lifetimes, as shown in Figure 1C, were very close between the two samples, with PPIX in DMSO displaying a slightly shorter lifetime. Nonetheless, both polar coordinates were very close to being on the semicircle plot that represents the lifetime of the PPIX monomer to be ∼16 ns, which is in agreement with the literature value. Measurement of the Steady-State Spectra and Spectral Lifetime of PPIX in DMSO and Triton X-100 under High Pressure. To investigate the photophysical properties of PPIX aggregates, we measured the emission spectra and monitored the changes in modulation and phase in terms of polar plot coordinates as the pressure increases. Figure 2A shows that the intensity of fluorescence emission at 675 nm increased as pressure increased from 1 to 3000 bar. On the other hand, the band at 630 nm that represents the PPIX monomer decreased as pressure increased. Excitation spectra were measured with an emission window fixed at 630 and 670 nm, which would collect the fluorescence emission from the monomer and aggregate, respectively. The excitation spectra revealed that the absorbance band in the Soret region of the pressure-induced aggregate is redshifted by 50 nm compared to the PPIX monomer (Figure 2B). Figure 2C shows the polar plot coordinates for the measured modulation and phase as a function of emission wavelengths from 625 to 705 nm at two different pressures (1 and 3000 bar). Although PPIX in DMSO was previously shown to display a predominant single component of the lifetime on the FLIM system, which collects over a range of wavelength, the spectrally resolved lifetime measurement revealed a second species with a shorter lifetime at 675 nm even before pressure was applied (Figure 2C). The polar plot shows that the short lifetime component becomes increasingly dominant as pressure increased from 1 to 3000 bar. Extrapolation of the measured polar coordinates up to 655 nm on the polar plot appears to be a straight line, which would intercept with the 3−4 ns mark on the semicircle. This indicates that the aggregate species formed under high pressure in the wavelength region of 625−655 nm has a lifetime of 3−4 ns. The polar plot trajectory deviates from linear as the emission wavelength increase beyond 655 nm, and the measured polar coordinate now lies in an area formed by 16, 6, and 3−4 ns. This suggests that multiple aggregated species with lifetimes ranging from 3 to 6 ns may be present under high pressure at these wavelengths (625−655 nm). The polar plot also shows that under 3000 bar pressure, the PPIX aggregate is the predominant fluorescent species at wavelengths of 665 and 675 nm (indicated by arrows) based on the proximity of polar coordinates to the semicircle. This
x = M meas cos ϕmeas y = M meas sin ϕmeas
Polar coordinates from only a single lifetime will lie on a semicircle with a diameter of 1, centered at (0 and 0.5). Multiple lifetime data will lie inside of this semicircle. Polar coordinates from fluorescence with two lifetime components will lie inside the semicircle and on a straight line connecting the two single-component lifetimes on the semicircle. That is, the measured polar coordinate containing two-component lifetime is the vector sum of the x and y components from each of the two lifetime species weighed by their intensity contributions α1 and α2. For a two-component lifetime system, the measured polar coordinates in relation to its constituents are M meas cos ϕmeas = α1M1 cos ϕ1 + α2M 2 cos ϕ2 M meas sin ϕmeas = α1M1 sin ϕ1 + α2M 2 sin ϕ2
where M1, M2, ϕ1, and ϕ2 are modulation and phase of the two contributing lifetimes in the sample, respectively. The sample may contain more than two lifetimes since there may be a distribution of differently sized aggregates formed in the sample. However, the measured polar plot coordinate is weighed by both the monomer lifetime and the average lifetime of the aggregates, providing that the distribution of the aggregates did not change (we will see that this is the case in our experiment). Therefore, the measured points would still lie on a straight line connecting the monomer and average lifetime of the aggregates, making it a case of two-component lifetime distribution. In our high-pressure study, the modulation and phase were measured by a single photomultiplier tube (PMT) channel. In the effect of solvent studies, modulation and phase were measured using an intensifier and CCD where each pixel contains a measured modulation and phase value. The modulation and phase were homogeneous throughout the image, and since the spatial resolution is not important for our measurement, we binned the image with 2 × 2 pixels post5834
DOI: 10.1021/acs.jpcb.9b01913 J. Phys. Chem. B 2019, 123, 5832−5840
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The Journal of Physical Chemistry B
The same experiment was conducted by adding PPIX to 0.6% Triton X-100, which is another solvent known to induce monomerization of PPIX. Applying up to 3000 bar pressure to the PPIX monomer sample containing 0.6% Triton X-100 did not appear to enhance the formation of the PPIX aggregate (Figure S1) since a band was not observed at 675 nm nor did the intensity at 630 nm decreases. This is likely due to different mechanisms of solubilization in these different solvents. Steady-State and Time-Dependent Measurements of PPIX at Various pH. As shown in Figure 3A, absorption spectra of PPIX (10 μM) were measured at different pH conditions (pH 0.7, 1.5, 5.0, and 10.4). A narrow and intense absorbance band is observed at 408 nm as the pH decreases from 5.0 to 0.7. At pH 5.0, there was a broadband absorption in the Soret region and an appearance of band at 354 nm, while the 408 nm band disappeared. As pH becomes more basic from pH 5.0 to 10.4, an absorbance band located at 379 nm is detected, and the broadband absorption lessened. Emission spectra of PPIX (10 μM) in pH 0.7 and 10.4 were plotted to compare the two species as shown in Figure 3B. At pH 0.7, the emission spectra of PPIX show two emission bands at 608 and 668 nm with a roughly equal intensity. For the pH 10.4 sample, the emission bands are located at 626 and 691 nm with an intense first band and a weak second band. The fluorescence intensity of samples at intermediate pH was very low, and the emission spectrum was not measured due to its low quantum yield. This implies that the PPIX monomer is likely to be a dominant species at both pH 10.4 and 0.7. Comparing the absorbance spectra at pH 0.7 to the PPIX monomer spectra (DMSO) reveals that the four Q bands in the visible region has been reduced to two Q bands (Figure S2), which suggests an increase in molecular symmetry due to protonation at lower pH. Fluorescence lifetime measurements of 10 μM PPIX solutions ranging from pH 0.7 to 10.4 were carried out in the frequency domain. The measured modulation and phase of each sample were projected on the polar plot (Figure 3C). At pH 10.4, the intensity contribution was dominated by mostly the 16 ns species, and as pH decreased, the intensity contribution from subnanosecond species increased gradually until pH 2.6. This is shown on the polar plot where at pH 10.4, the polar coordinate was closer to the 16 ns mark on the semicircle, and as pH decreased, the polar coordinate was pulled more and more inside toward the subnanosecond lifetime. The measured polar coordinates from samples with pH between 10.4 and 2.6 lie on a straight line connecting the nearly 16 ns and subnanosecond marks on the semicircle, resembling a shift in the intensity contributions of two lifetime fractions as a function of pH. The goodness of fit to a straight line indicates that the two-component approximation is apparently reasonable. As the pH decreased from 2.6 to 1.5, the polar coordinate of the sample went toward 7 ns on the semicircle of the polar plot. The least-square polynomial fit for data from pH 2.6 to 1.5 also showed good two-component approximation, and an intercept that is near 7 ns on the semicircle. Further decrease in pH from 1.5 to 0.7 shifted the lifetime from around 7 to 5 ns. The polar coordinates of both pH 1.5 and 0.7 samples are very close to the semicircle, indicating the two single-component lifetimes. Effect of Ionic Strength on the Aggregation of PPIX at Various pH. The polar coordinates of PPIX in pH 10.5 solution show predominantly intensity contribution from the PPIX monomer since the measured polar coordinate is close to
Figure 1. Spectroscopic Measurements of PPIX in DMSO and Triton X-100. (A) Absorbance spectra of PPIX in DMSO and Triton X-100. PPIX dissolved in these two solvents are monomerized and therefore has identical spectra. (B) Emission spectra of PPIX in DMSO and Triton X-100. The emission spectra are also identical; the tall and short bands are the signature spectra of the PPIX monomer. (C) Polar plot analysis of PPIX in DMSO and Triton X-100. The measured polar coordinates lie on the semicircle, which is an indication of single-component lifetime around 16 ns.
wavelength region corresponds to the 675 nm band on the fluorescence emission spectra in Figure 2A. As the emission wavelength moves past 675 to 705 nm, the polar plot trajectory backtrack toward a 16 ns lifetime, which is expected since the intensity contribution of the PPIX monomer relative to the aggregate is higher due to the presence of a weaker second emission band from the PPIX monomer located between 680 and 730 nm. 5835
DOI: 10.1021/acs.jpcb.9b01913 J. Phys. Chem. B 2019, 123, 5832−5840
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The Journal of Physical Chemistry B
Figure 2. Fluorescence emission spectra and polar coordinates of PPIX in DMSO under various pressures. (A) Fluorescence emission spectra of PPIX in DMSO under 1 to 3000 bar pressure in 500 bar increments. As pressure increases, the fluorescence intensity of the 630 nm band diminishes with exception of going from 500 to 1000 bar. The fluorescence intensity of 675 nm band increases as pressure increases. (B) Excitation spectra of PPIX with emission fixed at 630 and 670 nm. The excitation spectrum of the 630 nm band resembles the Soret band absorbance of the PPIX monomer. Excitation spectrum of the 670 nm band resembles that of the aggregate, which has a redshifted absorbance spectrum compared to the excitation spectrum of the monomer. (C) Polar plot analysis of modulation and phase data collected from frequency domain lifetime measurement under standard pressure and 3000 bar pressure, while emission wavelength collected increases from 625 to 705 nm in 10 nm increments. At a standard pressure of 1 bar, the polar plot coordinates collected at each wavelength moved toward 3−4 ns and then turns back to 16 ns. Under 3000 bar pressure, the measured polar coordinates migrated further toward 3−4 ns before curving toward 5−6 ns and back toward the original trajectory to 16 ns.
concentration of 10 mM NaCl. A gradual increase in concentration of the divalent cation Mg2+ caused the measured polar coordinate to shift toward a shorter lifetime. The effect of divalent cation is the most pronounce when a larger ion such as Ca2+ was introduced. The measured polar coordinates shifted farther to the right toward the subnanosecond region compared to Mg2+ measurements. The trajectories of the polar plot indicate that both ions will induce the formation of an aggregate with a lifetime of 1 to 0.5 ns.
the location of the 16 ns lifetime on the semicircle. As 0.5 M NaCl was introduced in the solution, the measured lifetime on the polar coordinate shifted to the right that indicated the formation of the PPIX aggregate (Figure 4A). In addition, the Soret band in the absorption spectrum becomes broader and less intense as shown in Figure 4B that implies that the formation of aggregates occurred at this condition (pH 0.5 and 0.5 M NaCl). As mentioned earlier, at pH 1.5, the measured polar coordinate is dominated by the species with around 7 ns lifetime. After the addition of 0.5 M NaCl to this solution, the polar coordinate shifted toward 5 ns, indicating the protonation of nitrogen atoms in the center with increasing ionic strength (Figure 4A). The change in ionization of PPIX is evident in absorption spectra as well, with the appearance of a more intense band at 407 nm (Figure 4C), but not broadening or shifting of the band, which suggests that this is not aggregation. At pH 4.5 where PPIX is predominantly aggregated, addition of 0.5 M NaCl only changed the polar coordinate slightly toward the 7 ns lifetime on the semicircle but not as dramatic as the effect seen under pH 1.5 and 10.5. Type of Aggregates Formed under the Influence of Divalent Cation at Basic pH. Polar plot representation of measured modulation and phase data for PPIX under the effects of different concentrations of various cations is shown in Figure 5. The polar coordinates of PPIX measurements showed almost no change in PPIX aggregation at a
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DISCUSSION The polar plot projection of measured modulation and phase from PPIX in DMSO and Triton-X 100 showed that the PPIX monomer has a fluorescence lifetime of ∼16 ns. This is consistent with the lifetime of the monomer as reported by previous studies.21,22 However, this value is the average lifetime of all species present in the sample over a wide range of wavelength. In spectrally resolved lifetime measurement, we were able to detect a trace amount of PPIX aggregate at 675 nm that has a drastically shorter lifetime (∼4 ns). When pressure is applied to the sample using a hydrostatic compressor, we observed an increase in formation of the PPIX aggregate that has a redshifted absorbance spectrum and a shorter lifetime between 3 and 6 ns as compared with that of the monomer. This enhancement in the formation of the aggregate due to increasing pressure is expected, as pressure 5836
DOI: 10.1021/acs.jpcb.9b01913 J. Phys. Chem. B 2019, 123, 5832−5840
Article
The Journal of Physical Chemistry B
Figure 4. Effect of ionic strength on PPIX aggregation and ionization. (A) Polar plot analysis of PPIX with the addition of 0.5 M NaCl. The polar coordinate of PPIX species at pH 10.5 shifted toward the right in the direction of the the subnanosecond species as 0.5 M NaCl was introduced. At pH 1.5, the addition of 0.5 M NaCl shifted the measured polar coordinate toward 5 ns species. Addition of 0.5 M NaCl did not introduce a large effect on the already aggregated PPIX at pH 4.5. (B) Absorbance spectra of PPIX at pH 10.5 under the influence of 0.5 M NaCl. The absorbance band in the Soret region decreased with the addition of 0.5 M NaCl, which is consistent with the change detected on the polar plot. (C) Absorbance spectra of PPIX in pH 1.5 under the effect of 0.5 M NaCl. The 407 nm absorbance band increased as NaCl was introduced, that is, also consistent with the change observed on the polar plot.
Figure 3. Absorbance and fluorescence spectra of PPIX in aqueous solution of varied pH. (A) Absorbance spectra of PPIX in pH 0.7, 1.5, 5, and 10.4. The four absorbance spectra are drastically different, indicating four different PPIX species at these pH values. (B) The fluorescence emission spectra of PPIX at pH 10.4 and 0.7. The emission spectra at pH 10.4 showed similar spectra to that of PPIX measured in DMSO and Triton X-100. PPIX in pH 0.7 showed two bands that are equal in the intensity, and the emission spectra is also blueshifted compared to the one measured in DMSO and Triton X100. (C) Polar plot analysis of PPIX in different pH values. Leastsquare fitting to a straight line was applied to the measured polar coordinate at pH ranging from 10 to 3. Intersection between the straight line and polar plot was also shown (empty square). From pH 10 to 3, PPIX undergoes equilibrium from a lifetime around 16 ns to aggregate with subnanosecond. From pH 3 to 1.5, PPIX undergoes transition from the aggregate to PPIX species with a roughly 7 ns lifetime (empty square). At the most acidic pH, the measured polar coordinate lies on the semicircle indicating a lifetime of 5 ns.
further confirmed that the appearance of the new band in the PPIX-DMSO under high pressure did not come from the formation of photoproducts. Comparing the emission spectra and lifetime data of PPIX at pH 10.4 to PPIX in DMSO and Triton-X 100 showed that formation of the PPIX monomer is favored at basic pH. This contradicts observations from the previous literature, which stated that dimerization of PPIX is favored at basic pH.13,35 The polar plot coordinates between measurements of PPIX in DMSO, Triton-X, and pH 10.4 solution clearly showed that disaggregation is favored as pH becomes more basic. The discrepancy in studies based on solely steady-state techniques could come from the fact that although disaggregation is favored at basic pH, a fraction of the PPIX is still aggregated. This is evident in our study since the polar coordinate of the pH 10.4 sample is slightly inside the semicircle. The increase in aggregation as the pH approaches 3 is likely due to the protonation of the carboxyl group on the side chain, the porphine core, or both, changing the molecular charge from negative to neutral. Scolaro et al. proposed that aggregation of PPIX is due to hydrogen bonding between the carboxylic side chains and the porphine base as well as stacking interaction.13 In order to satisfy the proposed model of the PPIX aggregate, at least one of the carboxylic acid has to be protonated, and the porphine core has to be neutral. Therefore, as pH becomes more basic, either the porphine core is deprotonated, or both carboxylic side chains are deprotonated, preventing hydrogen
would shift the equilibrium toward the species that occupies a smaller volume. We propose this species to be a small J-type aggregate of PPIX. Formation of the J-type aggregate would imply that the initial aggregation step starts laterally rather than axially, as the H-type aggregate would be formed if the aggregates were first formed by stacking. The edge to edge hydrophobic interaction proposed by Scolaro et al. is seen to be the driving force for the formation of the aggregate under pressure.13 Previous in vivo studies have reported aggregates with lifetimes ranging from 3 to 6 ns being the predominant species in the intracellular regions.33,34 However, in solution, we could see high yield of this species only with the aid of high pressure, as ionic strength or divalent cations appeared to only drive the formation of a larger aggregate with
