Co-reporter:Ujjal Bhattacharjee, Catherine Graham, Stefanie Czub, Sandor Dudas, Mark A. Rasmussen, Thomas A. Casey, and Jacob W. Petrich
Journal of Agricultural and Food Chemistry 2016 Volume 64(Issue 1) pp:320-325
Publication Date(Web):December 1, 2015
DOI:10.1021/acs.jafc.5b04218
Transmissible spongiform encephalopathies (TSE) are progressive, neurodegenerative disorders, of which bovine spongiform encephalopathy (BSE) is of special concern because it is infectious and debilitating to humans. The possibility of using fluorescence spectroscopy to screen for BSE in cattle was explored. Fluorescence spectra from the retinas of experimentally infected BSE-positive cattle with clinical disease were compared with those from both sham-inoculated and non-inoculated BSE-negative cattle. The distinct intensity difference of about 4–10-fold between the spectra of the BSE-positive and the BSE-negative (sham-inoculated and non-inoculated) eyes suggests the basis for a means of developing a rapid, noninvasive examination of BSE in particular and TSEs in general.
Co-reporter:Ujjal Bhattacharjee;Moneim Elshobaki;Kalyan Santra;Jonathan M. Bobbitt;Sumit Chaudhary;Emily A. Smith
Macromolecular Chemistry and Physics 2016 Volume 217( Issue 16) pp:1801-1809
Publication Date(Web):
DOI:10.1002/macp.201600113
Co-reporter:Kalyan Santra, Jinchun Zhan, Xueyu Song, Emily A. Smith, Namrata Vaswani, and Jacob W. Petrich
The Journal of Physical Chemistry B 2016 Volume 120(Issue 9) pp:2484-2490
Publication Date(Web):February 10, 2016
DOI:10.1021/acs.jpcb.6b00154
The need for measuring fluorescence lifetimes of species in subdiffraction-limited volumes in, for example, stimulated emission depletion (STED) microscopy, entails the dual challenge of probing a small number of fluorophores and fitting the concomitant sparse data set to the appropriate excited-state decay function. This need has stimulated a further investigation into the relative merits of two fitting techniques commonly referred to as “residual minimization” (RM) and “maximum likelihood” (ML). Fluorescence decays of the well-characterized standard, rose bengal in methanol at room temperature (530 ± 10 ps), were acquired in a set of five experiments in which the total number of “photon counts” was approximately 20, 200, 1000, 3000, and 6000 and there were about 2–200 counts at the maxima of the respective decays. Each set of experiments was repeated 50 times to generate the appropriate statistics. Each of the 250 data sets was analyzed by ML and two different RM methods (differing in the weighting of residuals) using in-house routines and compared with a frequently used commercial RM routine. Convolution with a real instrument response function was always included in the fitting. While RM using Pearson’s weighting of residuals can recover the correct mean result with a total number of counts of 1000 or more, ML distinguishes itself by yielding, in all cases, the same mean lifetime within 2% of the accepted value. For 200 total counts and greater, ML always provides a standard deviation of <10% of the mean lifetime, and even at 20 total counts there is only 20% error in the mean lifetime. The robustness of ML advocates its use for sparse data sets such as those acquired in some subdiffraction-limited microscopies, such as STED, and, more importantly, provides greater motivation for exploiting the time-resolved capacities of this technique to acquire and analyze fluorescence lifetime data.
Co-reporter:Feng Zhu, Long Men, Yijun Guo, Qiaochu Zhu, Ujjal Bhattacharjee, Peter M. Goodwin, Jacob W. Petrich, Emily A. Smith, and Javier Vela
ACS Nano 2015 Volume 9(Issue 3) pp:2948
Publication Date(Web):February 9, 2015
DOI:10.1021/nn507020s
Organometallic halide perovskites CH3NH3PbX3 (X = I, Br, Cl) have quickly become one of the most promising semiconductors for solar cells, with photovoltaics made of these materials reaching power conversion efficiencies of near 20%. Improving our ability to harness the full potential of organometal halide perovskites will require more controllable syntheses that permit a detailed understanding of their fundamental chemistry and photophysics. In this manuscript, we systematically synthesize CH3NH3PbX3 (X = I, Br) nanocrystals with different morphologies (dots, rods, plates or sheets) by using different solvents and capping ligands. CH3NH3PbX3 nanowires and nanorods capped with octylammonium halides show relatively higher photoluminescence (PL) quantum yields and long PL lifetimes. CH3NH3PbI3 nanowires monitored at the single particle level show shape-correlated PL emission across whole particles, with little photobleaching observed and very few off periods. This work highlights the potential of low-dimensional organometal halide perovskite semiconductors in constructing new porous and nanostructured solar cell architectures, as well as in applying these materials to other fields such as light-emitting devices and single particle imaging and tracking.Keywords: morphology control; nanocrystals; organometal halide perovskites; preferred orientation; single particle photoluminescence; size control;
Co-reporter:Aleem Syed;Michael D. Lesoine;Ujjal Bhattacharjee;Emily A. Smith
Photochemistry and Photobiology 2014 Volume 90( Issue 4) pp:767-772
Publication Date(Web):
DOI:10.1111/php.12248
Abstract
Time binning is used to increase the number of photon counts in the peak channel of stimulated emission depletion fluorescence lifetime decay curves to determine how it affects the resulting lifetime image. The fluorescence lifetime of the fluorophore, Alexa Fluor 594 phalloidin, bound to F-actin is probed in cultured S2 cells at a spatial resolution of ~40 nm. This corresponds to a 10-fold smaller probe volume compared to confocal imaging, and a reduced number of photons contributing to the signal. Pixel-by-pixel fluorescence lifetime measurements and error analysis show that an average of 40 ± 30 photon counts in the peak channel with a signal-to-noise ratio of 20 is enough to calculate a reliable fluorescence lifetime from a single exponential fluorescence decay. No heterogeneity in the actin cytoskeleton in different regions of the cultured cells was measured in the 40–400 nm spatial regime.
Co-reporter:Sayantan Bose;Holger Schönenbrücher;Jürgen A. Richt;Thomas A. Casey;Mark A. Rasmussen;Marcus E. Kehrli Jr
Photochemistry and Photobiology 2013 Volume 89( Issue 4) pp:864-868
Publication Date(Web):
DOI:10.1111/php.12056
Abstract
Recently, we have proposed that the fluorescence spectra of sheep retina can be well correlated with the presence or absence of scrapie. Scrapie is the most widespread TSE (transmissible spongiform encephalopathy) affecting sheep and goats worldwide. Mice eyes have been previously reported as a model system to study age-related accumulation of lipofuscin, which has been investigated by monitoring the increasing fluorescence with age covering its entire life span. The current work aims at developing mice retina as a convenient model system to diagnose scrapie and other fatal TSE diseases in animals such as sheep and cows. The objective of the research reported here was to determine whether the spectral features are conserved between two different species namely mice and sheep, and whether an appropriate small animal model system could be identified for diagnosis of scrapie based on the fluorescence intensity in retina. The results were consistent with the previous reports on fluorescence studies of healthy and scrapie-infected retina of sheep. The fluorescence from the retinas of scrapie-infected sheep was significantly more intense and showed more heterogeneity than that from the retinas of uninfected mice. Although the structural characteristics of fluorescence spectra of scrapie-infected sheep and mice eyes are slightly different, more importantly, murine retinas reflect the enhancement of fluorescence intensity upon infecting the mice with scrapie, which is consistent with the observations in sheep eyes.
Co-reporter:Michael D. Lesoine ; Ujjal Bhattacharjee ; Yijun Guo ; Javier Vela ; Jacob W. Petrich ;Emily A. Smith
The Journal of Physical Chemistry C 2013 Volume 117(Issue 7) pp:3662-3667
Publication Date(Web):January 18, 2013
DOI:10.1021/jp312231k
Subdiffraction spatial resolution luminescence depletion imaging was performed with giant CdSe/14CdS nanocrystal quantum dots (g-NQDs) dispersed on a glass slide. Luminescence depletion imaging used a Gaussian shaped excitation laser pulse overlapped with a depletion pulse, shaped into a doughnut profile, with zero intensity in the center. Luminescence from a subdiffraction volume is collected from the central portion of the excitation spot, where no depletion takes place. Up to 92% depletion of the luminescence signal was achieved. An average full width at half-maximum of 40 ± 10 nm was measured in the lateral direction for isolated g-NQDs at an air interface using luminescence depletion imaging, whereas the average full width at half-maximum was 450 ± 90 nm using diffraction-limited, confocal luminescence imaging. Time-gating of the luminescence depletion data was required to achieve the stated spatial resolution. No observable photobleaching of the g-NQDs was present in the measurements, which allowed imaging with a dwell time of 250 ms per pixel to obtain images with a high signal-to-noise ratio. The mechanism for luminescence depletion is likely stimulated emission, stimulated absorption, or a combination of the two. The g-NQDs fulfill a need for versatile, photostable tags for subdiffraction imaging schemes where high laser powers or long exposure times are used.
Co-reporter:Michael D. Lesoine, Sayantan Bose, Jacob W. Petrich, and Emily A. Smith
The Journal of Physical Chemistry B 2012 Volume 116(Issue 27) pp:7821-7826
Publication Date(Web):June 13, 2012
DOI:10.1021/jp303912p
Supercontinuum (SC) stimulated emission depletion (STED) fluorescence lifetime imaging is demonstrated by using time-correlated single-photon counting (TCSPC) detection. The spatial resolution of the developed STED instrument was measured by imaging monodispersed 40-nm fluorescent beads and then determining their fwhm, and was 36 ± 9 and 40 ± 10 nm in the X and Y coordinates, respectively. The same beads measured by confocal microscopy were 450 ± 50 and 430 ± 30 nm, which is larger than the diffraction limit of light due to underfilling the microscope objective. Underfilling the objective and time gating the signal were necessary to achieve the stated STED spatial resolution. The same fluorescence lifetime (2.0 ± 0.1 ns) was measured for the fluorescent beads by using confocal or STED lifetime imaging. The instrument has been applied to study Alexa Fluor 594-phalloidin labeled F-actin-rich projections with dimensions smaller than the diffraction limit of light in cultured cells. Fluorescence lifetimes of the actin-rich projections range from 2.2 to 2.9 ns as measured by STED lifetime imaging.
Co-reporter:Philip J. Carlson, Sayantan Bose, Daniel W. Armstrong, Tommy Hawkins, Mark S. Gordon, and Jacob W. Petrich
The Journal of Physical Chemistry B 2012 Volume 116(Issue 1) pp:503-512
Publication Date(Web):November 29, 2011
DOI:10.1021/jp207840q
An investigation of the structure and dynamics of the high-energy ionic liquid, 1-hydroxyethyl-4-amino-1,2,4-triazolium nitrate (HEATN), was undertaken. Both experimental and computational methods were employed to understand the fundamental properties, characteristics, and behavior of HEATN. The charge separation, according to the electrostatic potential derived charges, was assessed. The MP2 (second-order perturbation theory) geometry optimizations find six dimer and five tetramer structures and allow one to see the significant highly hydrogen bonded network predicted within the HEATN system. Due to the prohibitive scaling of ab initio methods, the fragment molecular orbital (FMO) method was employed and assessed for feasibility with highly energetic ionic liquids using HEATN as a model system. The FMO method was found to adequately treat the HEATN ionic liquid system as evidenced by the small relative error obtained. The experimental studies involved the investigation of the solvation dynamics of the HEATN system via the coumarin 153 (C153) probe at five different temperatures. The rotational dynamics through the HEATN liquid were also measured using C153. Comparisons with previously studied imidazolium and phosphonium ionic liquids show surprising similarity. To the authors’ knowledge, this is the first experimental study of solvation dynamics in a triazolium-based ionic liquid.
Co-reporter:Sayantan Bose, Ramkrishna Adhikary, Charles A. Barnes, D. Bruce Fulton, Mark S. Hargrove, Xueyu Song, and Jacob W. Petrich
The Journal of Physical Chemistry A 2011 Volume 115(Issue 16) pp:3630-3641
Publication Date(Web):May 6, 2010
DOI:10.1021/jp1008225
We present a comparison of the dielectric response obtained from fluorescence upconversion experiments and from molecular dynamics simulations of the complexes of coumarin 153 with five apomyoglobins (apoMbs): wild-type horse heart (HH-WT) and those of wild-type sperm whale (SW-WT); its two triple mutants, L29F/H64Q/V68F and H64L/V68F/P88A; and its double mutant, L29F/V68L. Comparisons between experimental and simulated solvation relaxation functions, C(t)s, for the wild-type proteins range from very good to excellent. For the three mutants we investigated, however, agreement between experiment and simulation was considerably inferior. Thus, an NMR study of the complex of the HH-WT complex apoMb, and fluorescence energy transfer and anisotropy studies of the five complexes, were performed to investigate the structures upon which the simulations were based. The NMR measurements confirm our earlier conclusions that the C153 lies in the heme pocket of the HH-WT apoMb. For the wild-type complexes, fluorescence energy transfer measurements provide two rise times, suggesting a definite spatial relationship between the two Trp donors and the C153 acceptor. These results confirm the structural integrity of the wild-type complexes and validate the initial structures used for the molecular dynamics simulations. On the other hand, the three mutants provided single exponential rise times for energy transfer, suggesting that the position of the C153 used in the simulations may have been in error or that the C153 is mobile on the time scale of the energy transfer experiment. Fluorescence anisotropy studies also suggest that the double mutant was not structurally intact. Furthermore, examination of these systems demonstrates the sensitivity of C153 to its environment and permits the observation of differences in the heme pockets. These results point to the importance of structural characterization of modified proteins used in studies of the dielectric response and suggest strategies for performing molecular dynamics simulations of modified proteins.
Co-reporter:Ramkrishna Adhikary, Charles A. Barnes, Rachel L. Trampel, Samuel J. Wallace, Tak W. Kee, and Jacob W. Petrich
The Journal of Physical Chemistry B 2011 Volume 115(Issue 36) pp:10707-10714
Publication Date(Web):August 4, 2011
DOI:10.1021/jp200080s
The photophysical properties of cyclocurcumin in various solvents are studied for the first time to shed light on the nonradiative processes of the parent compound, curcumin, which has a range of medicinal properties. Steady-state fluorescence and fluorescence–excitation spectra of cyclocurcumin in polar aprotic solvents are strongly dependent on excitation (λex) and emission (λem) wavelength, respectively. The fluorescence quantum yield also depends on λex and increases with the viscosity of the medium. Time-resolved studies show nonexponential fluorescence decay in all solvents studied. The two fluorescence decay components of cyclocurcumin in alcohols exhibit a strong dependence on viscosity and temperature. NMR spectroscopy indicates that cyclocurcumin is entirely in the trans form with respect to the C6–C7 double bond in methanol, chloroform, and acetone. It is suggested that at least two conformational isomers about another single bond (C5–C6 or C7–C1″ or both) and that trans-to-cis isomerization about the C6–C7 double bond of these isomers provide the most likely means of rationalizing the steady-state spectra and the nonradiative decay mechanisms in both protic and aprotic polar solvents.
Co-reporter:Ramkrishna Adhikary, Prasun Mukherjee, Govindarajan Krishnamoorthy, Robert A. Kunkle, Thomas A. Casey, Mark A. Rasmussen and Jacob W. Petrich
Analytical Chemistry 2010 Volume 82(Issue 10) pp:4097
Publication Date(Web):April 22, 2010
DOI:10.1021/ac100179u
The feasibility of exploiting fluorescence spectra of the eye for diagnosis of transmissible spongiform encephalopathies (TSEs) was examined. Retinas from scrapie-positive sheep were compared with scrapie-negative sheep using fluorescence spectroscopy, and distinct differences in the fluorescence intensity and spectroscopic signatures were observed. The characteristic fluorescent signatures are thought to be the result of an accumulation of lipofuscin in the retina. It appears that the eye, in particular the retina, is a useful tissue for noninvasive examination of some neurological pathologies such as scrapie. The development of procedures based on examinations of the eye that permit the detection of neurological disorders in animals holds great promise.
Co-reporter:Sayantan Bose, Daniel W. Armstrong and Jacob W. Petrich
The Journal of Physical Chemistry B 2010 Volume 114(Issue 24) pp:8221-8227
Publication Date(Web):May 28, 2010
DOI:10.1021/jp9120518
We investigated the reactivity and stability of a commercial mixture of cellulases in eight ionic liquids by optical and calorimetric techniques. First, hydrolysis by cellulases from Tricoderma reesei in these ionic liquids was benchmarked against that in aqueous buffer. Only 1-methylimidazolium chloride (mim Cl) and tris-(2-hydroxyethyl)methylammonium methylsulfate (HEMA) provided a medium in which hydrolysis could occur. While hydrolysis at 65 °C is initially much faster in buffer than in these two liquids, it reaches a plateau after 2 h, whereas the reaction progresses monotonically in the two ionic liquids. This difference in the rate of hydrolysis is largely attributed to two factors: (1) the higher viscosity of the ionic liquids and (2) the enzymes are irreversibly denatured at 50 °C in buffer while they are stable to temperatures as high as 115 °C in HEMA. We explored whether fluorescence quenching of aromatic amino acids of the enzymes was indeed a signature of protein denaturation, as has been suggested in the literature, and concluded that quenching is not necessarily associated with denaturation. When it does occur, for example, in the presence of ionic liquids formed from imidazolium cations and chloride anions, it arises from the imidazolium rather than the chloride. Finally, we conclude that HEMA is a promising, novel, green medium for performing cellulose hydrolysis reactions to convert biomass into biofuels. Because of the thermal stability it imparts to enzymes, its ability to solubilize biomass, and the fact that it does not quench tryptophyl fluorescence (thus permitting monitoring of the enzymes by fluorescence spectroscopy), HEMA provides an ideal starting point for the design of ionic liquids, not only for the hydrolysis of biomass, but also for use with a wide spectrum of enzymatic reactions.
Co-reporter:Ramkrishna Adhikary, Philip J. Carlson, Tak W. Kee and Jacob W. Petrich
The Journal of Physical Chemistry B 2010 Volume 114(Issue 8) pp:2997-3004
Publication Date(Web):February 5, 2010
DOI:10.1021/jp9101527
Femtosecond fluorescence upconversion experiments were performed on the naturally occurring medicinal pigment, curcumin, in anionic, cationic, and neutral micelles. In our studies, the micelles are composed of sodium dodecyl sulfate (SDS), dodecyl trimethyl ammonium bromide (DTAB), and triton X-100 (TX-100). We demonstrate that the excited-state kinetics of curcumin in micelles have a fast (3−8 ps) and slow (50−80 ps) component. While deuteration of curcumin has a negligible effect on the fast component, the slow component exhibits a pronounced isotope effect of ∼1.6, indicating that micelle-captured curcumin undergoes excited-state intramolecular hydrogen atom transfer. Studies of solvation dynamics of curcumin in a 10 ps time window reveal a fast component (≤300 fs) followed by a 8, 6, and 3 ps component in the solvation correlation function for the TX-100, DTAB, and SDS micelles, respectively.
Co-reporter:Prasun Mukherjee;Sayantan Bose;Alyse A. Hurd;Ramkrishna Adhikary;Holger Schönenbrücher;Amir N. Hamir;Jürgen A. Richt;Thomas A. Casey;Mark A. Rasmussen
Photochemistry and Photobiology 2009 Volume 85( Issue 1) pp:234-238
Publication Date(Web):
DOI:10.1111/j.1751-1097.2008.00425.x
Abstract
The integrated fluorescence of murine eyes is collected as a function of age. This fluorescence is attributed to pigments generally referred to as lipofuscin and is observed to increase with age. No difference in fluorescence intensity is observed between the eyes of males or females. This work provides a benchmark for further studies that are planned in order to use such signatures as markers of central nervous system (CNS) tissue or even of diseased CNS tissue and provides a basis for determining the age of a healthy animal.
Co-reporter:Ramkrishna Adhikary;Holger Schönenbrücher;Mark A. Rasmussen;Thomas A. Casey;Amir N. Hamir;Marcus E. Kehrli;Jürgen A. Richt
Photochemistry and Photobiology 2009 Volume 85( Issue 6) pp:1322-1326
Publication Date(Web):
DOI:10.1111/j.1751-1097.2009.00593.x
Abstract
We describe a comparison of the fluorescence spectra of bovine tissues with murine tissues in order to determine whether spectral features are conserved and whether an appropriate and practical laboratory small animal model system could be identified to be used for investigation of tissue- and age-related fluorescence signal patterns. Recently it has been shown that spectral signatures of lipofuscin have enabled the detection of bovine central nervous system (CNS) tissue in meat products with high sensitivity (Schönenbrücher, H., Adhikary, R., Mukherjee, P., Casey, T.A., Rasmussen, M.A., Maistrovich, F.D., Hamir, A.N., Kehrli, M.J., Richt, J., Petrich, J.W. [2008] J Agric Food Chem56, 6220–6226). We report that brain and spinal cord of mice provide fluorescence spectra similar to those of bovine brain and spinal cord. It is concluded that murine CNS tissue is an appropriate model system for bovine CNS tissue for the development of fluorometric CNS detection assays.
Co-reporter:Ramkrishna Adhikary, Prasun Mukherjee, Tak W. Kee and Jacob W. Petrich
The Journal of Physical Chemistry B 2009 Volume 113(Issue 15) pp:5255-5261
Publication Date(Web):March 24, 2009
DOI:10.1021/jp901234z
The potential use of the naturally occurring yellow-orange pigment curcumin as a photodynamic therapy agent is one of the most exciting applications of this medicinal compound. Although subnanosecond spectroscopy has been used to investigate the photophysical processes of curcumin, the time resolution is insufficient to detect important and faster photoinduced processes, including solvation and excited-state intramolecular hydrogen atom transfer (ESIHT). In this study, the excited-state photophysics of curcumin is studied by means of ultrafast fluorescence upconversion spectroscopy. The results show two decay components in the excited-state kinetics with time scales of 12−20 ps and ∼100 ps in methanol and ethylene glycol. The resulting prominent isotope effect in the long component upon deuteration indicates that curcumin undergoes ESIHT in these solvents. The short component (12−20 ps) is insensitive to deuteration, and multiwavelength fluorescence upconversion results show that this decay component is due to solvation of excited-state curcumin.
Co-reporter:Holger Schönenbrücher, Ramkrishna Adhikary, Prasun Mukherjee, Thomas A. Casey, Mark A. Rasmussen, Frank D. Maistrovich, Amir N. Hamir, Marcus E. Kehrli Jr., Jürgen A. Richt and Jacob W. Petrich
Journal of Agricultural and Food Chemistry 2008 Volume 56(Issue 15) pp:6220-6226
Publication Date(Web):July 12, 2008
DOI:10.1021/jf0734368
The removal of central nervous system (CNS) tissues as part of bovine spongiform encephalopathy (BSE) risk material is one of the highest priority tasks to avoid contamination of the human food chain with BSE. No currently available method enables the real-time detection of possible CNS tissue contamination on carcasses during slaughter. The fluorescent pigment lipofuscin is a heterogeneous, high-molecular weight material that has been shown to be enriched in high concentrations in neuronal tissues. In this study, lipofuscin fluorescence was investigated as a marker for real-time detection of CNS contamination. Front-faced fluorescence spectra of brain and spinal cord samples from 11 cattle gave identical, reproducible fluorescence signal patterns with high intensities. The specificity of these spectra was assessed by investigating 13 different non-CNS tissues enabling the differentiation of brain and spinal cord by signal intensity and structure of the spectra, respectively. Small quantities of bovine spinal cord were reliably detected in the presence of raw bovine skeletal muscle, fat, and vertebrae. The presented data are a fundamental basis for the development of a prototype device allowing real-time monitoring of CNS tissue contamination on bovine carcasses and meat cuts.
Co-reporter:Prasun Mukherjee;Ramkrishna Adhikary;Mintu Halder;Pavol Miskovsky
Photochemistry and Photobiology 2008 Volume 84( Issue 3) pp:706-712
Publication Date(Web):
DOI:10.1111/j.1751-1097.2007.00234.x
Abstract
The accumulation and interaction of hypericin with the biologically important macromolecule, low-density lipoprotein (LDL), is investigated using various steady-state and time-resolved fluorescence measurements. It is concluded that multiple hypericins can penetrate considerably deeply into the LDL molecule. Up to ∼20 nonaggregated hypericin molecules can enter LDL; but upon increasing the hypericin concentration, the fluorescence lifetime of hypericin decreases drastically, suggesting most likely the self-quenching of aggregated hypericin. There is also evidence of energy transfer from tryptophans of the constituent protein, apoB-100, to hypericin in LDL. The results demonstrate the ability of LDL to solubilize hypericin (a known photosensitizer) in nonaggregated form, which has implications for the construction of drug delivery systems.
Co-reporter:Christopher S. Lobban;Steven J. Hallam;Prasun Mukherjee
Photochemistry and Photobiology 2007 Volume 83(Issue 5) pp:1074-1094
Publication Date(Web):6 AUG 2007
DOI:10.1111/j.1751-1097.2007.00191.x
In this paper, we review the literature and present some new data to examine the occurrence and photophysics of the diverse hypericin-like chromophores in heterotrichs, the photoresponses of the cells, the various roles of the pigments and the taxa that might be studied to advance our understanding of these pigments. Hypericin-like chromophores are known chemically and spectrally so far only from the stentorids and Fabrea, the latter now seen to be sister to stentorids in the phylogenetic tree. For three hypericin-like pigments, the structures are known but these probably do not account for all the colors seen in stentorids. At least eight physiological groups of Stentor exist depending on pigment color and presence/absence of zoochlorellae, and some species can be bleached, leading to many opportunities for comparison of pigment chemistry and cell behavior. Several different responses to light are exhibited among heterotrichs, sometimes by the same cell; in particular, cells with algal symbionts are photophilic in contrast to the well-studied sciaphilous (shade-loving) species. Hypericin-like pigments are involved in some well-known photophobic reactions but other pigments (rhodopsin and flavins) are also involved in photoresponses in heterotrichs and other protists. The best characterized role of hypericin-like pigments in heterotrichs is in photoresponses and they have at least twice evolved a role as photoreceptors. However, hypericin and hypericin-like pigments in diverse organisms more commonly serve as predator defense and the pigments are multifunctional in heterotrichs. A direct role for the pigments in UV protection is possible but evidence is equivocal. New observations are presented on a folliculinid from deep water, including physical characterization of its hypericin-like pigment and its phylogenetic position based on SSU rRNA sequences. The photophysics of hypericin and hypericin-like pigments is reviewed. Particular attention is given to how their excited-state properties are modified by the environment. Dramatic changes in excited-state behavior are observed as hypericin is moved from the homogeneous environment of organic solvents to the much more structured surroundings provided by the complexes it forms with proteins. Among these complexes, it is useful to consider the differences between environments where hypericin is not found naturally and those where it is, notably, for example, in heterotrichs. It is clear that interaction with a protein modifies the photophysics of hypericin and understanding the molecular basis of this interaction is one of the outstanding problems in elucidating the function of hypericin and hypericin-like chromophores.
Co-reporter:Prasun Mukherjee;Mintu Halder;Mark S. Hargrove
Photochemistry and Photobiology 2006 Volume 82(Issue 6) pp:1586-1590
Publication Date(Web):28 JUN 2008
DOI:10.1111/j.1751-1097.2006.tb09815.x
In order to provide a thorough characterization of a system with which to study the dielectric response of a protein, a well-defined system complex of a fluorescent probe and protein is required. We have argued that such a system is provided by coumarin 153 and apomyoglobin (Photochem. Photobiol. 79, 440–446 [2004]). In order to demonstrate further that coumarin 153 exhibits negligible nonspecific binding to the surface of apomyoglobin, we study its interactions with both the apo and holo proteins. We further make a similar comparison with 8-anilino-l-naphthalenesulfonic acid, for which an NMR structure with apomyoglobin has been obtained. Our results confirm the appropriateness of the system of coumarin 153 and apomyoglobin for the investigation of solvation by the protein matrix.
Co-reporter:Lindsay Sers;Mintu Halder;Tom Ling Xiao;Jie Ding;Daniel W. Armstrong
Photochemistry and Photobiology 2005 Volume 81(Issue 1) pp:183-186
Publication Date(Web):23 MAY 2007
DOI:10.1111/j.1751-1097.2005.tb01539.x
We report the first separation of the enantiomers of hypericin. Their steady-state optical spectra and ultrafast primary photoprocesses are investigated in chiral environments. Within experimental error, there is no difference between the two enantiomers in any of the systems considered. This is consistent with the emerging picture that the rich and extended absorption spectrum of hypericin is not a result of ground-state heterogeneity. It is also consistent with the observation that the spectra and photophysics of hypericin are generally insensitive to environments in which it does not aggregate.
Co-reporter:L. Sers;M. Halder;Y. Liu;R. A. Arnold;P. K. Chowdhury;D. W. Armstrong;S. Kundu;X. Song;M. S. Hargrove;J. W. Petrich
Photochemistry and Photobiology 2004 Volume 79(Issue 5) pp:440-446
Publication Date(Web):1 MAY 2007
DOI:10.1111/j.1751-1097.2004.tb00032.x
Understanding a protein's dielectric response requires both a theoretical model and a well-defined experimental system. The former has already been proposed by Song (J. Chem. Phys. 116, 9359 [2002]). We suggest that the latter is provided by the complex of coumarin 153 (C153) with apomyoglobin (ApoMb). C153 has been exhaustively studied and has proven to be an excellent probe of the solvation dynamics of polar solvents. Myoglobin is one of the most thoroughly studied proteins. Myoglobins from a wide range of species have been subject to X-ray structural analysis and site-directed mutagenesis. Here, we demonstrate the existence of a robust C153-apomyglobin system by means of molecular dynamics simulations, equilibrium binding studies using a Job's plot and capillary electrophoresis, circular dichroism and time-resolved fluorescence. The reorganization energy of C153 bound to ApoMb is compared with that of C153 in bulk solvent using the method of Jordanides et al. (J. Phys. Chem. B 103, 7995 [1999]).
Co-reporter:D. B. Fulton;J. W. Petrich;P. Chowdhury;A. H. Andreotti;J. Wen;K. Das;A. Datta
Photochemistry and Photobiology 2003 Volume 77(Issue 1) pp:5-9
Publication Date(Web):1 MAY 2007
DOI:10.1562/0031-8655(2003)0770005DSITGS2.0.CO2
Nuclear magnetic resonance measurements indicate that hypericin exists in the same “normal” tautomeric form irrespective of whether the solvent is dimethyl sulfoxide or tetrahydrofuran. This result is discussed in the context of previous experimental and theoretical work. It is concluded that solvent perturbations cannot induce tautomerization in hypericin.
Co-reporter:J. Park;A. Datta;P. K. Chowdhury;J. W. Petrich
Photochemistry and Photobiology 2001 Volume 73(Issue 2) pp:105-109
Publication Date(Web):1 MAY 2007
DOI:10.1562/0031-8655(2001)0730105ITESHA2.0.CO2
The excited-state intramolecular H-atom transfer of hypericin (Hyp) was investigated as a function of pH in monodispersed reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate/heptane/water and in complexes with Tb3+ under conditions in which one of the two carbonyl groups of Hyp is incapable of accepting a hydrogen atom. The results of pump-probe transient absorption experiments provide no evidence for a concerted H-atom transfer mechanism.
Co-reporter:Scott W. Nelson;Richard B. Honzatko;Jin Wen;Herbert J. Fromm
Photochemistry and Photobiology 2001 Volume 74(Issue 5) pp:679-685
Publication Date(Web):1 MAY 2007
DOI:10.1562/0031-8655(2001)0740679EOTIPF2.0.CO2
The environment of Trp57, introduced by the mutation of a tyrosine in the dynamic loop of porcine liver fructose-1,6-bisphosphatase (FBPase), was examined using time-resolved fluorescence and directed mutation. The Trp57 enzyme was studied previously by X-ray crystallography and steady-state fluorescence, the latter revealing an unexpected redshift in the wavelength of maximum fluorescence emission for the R-state conformer. The redshift was attributed to the negative charge of Asp127 in contact with the indole side chain of Trp57. Time-resolved fluorescence experiments here reveal an indole side chain less solvent exposed and more rigid in the R-state, than in the T-state of the enzyme, consistent with X-ray crystal structures. Replacement of Asp127 with an asparagine causes a 6 nm blueshift in the wavelength of maximum fluorescence emission for the R-state conformer, with little effect on the emission maximum of the T-state enzyme. The data here support the direct correspondence between X-ray crystal structures of FBPase and conformational states of the enzyme in solution, and provide a clear example of the influence of microenvironment on the fluorescence properties of tryptophan.
Co-reporter:Anindya Datta;Alexre V. Smirnov;George Chumanov;Jin Wen
Photochemistry and Photobiology 2000 Volume 71(Issue 2) pp:166-172
Publication Date(Web):1 MAY 2007
DOI:10.1562/0031-8655(2000)0710166MRCFTE2.0.CO2
The excited-state intramolecular H-atom transfer reactions of hypocrellins B and A are compared by using time-resolved absorption and fluorescence upconversion techniques. The hypocrellin B photophysics are well described by a simple model involving one ground-state species and excited-state forward and reverse H-atom transfer with a nonfluorescent excited state. We suggest that excited-state conformational changes are coupled to the H-atom transfer in hypocrellin B just as gauche/anti changes are coupled to the H-atom transfer in hypocrellin A.
Co-reporter:P. K. Chowdhury;K. D. Ashby;A. Datta;J. W. Petrich
Photochemistry and Photobiology 2000 Volume 72(Issue 5) pp:612-618
Publication Date(Web):1 MAY 2007
DOI:10.1562/0031-8655(2000)0720612EOPOTF2.0.CO2
The well-characterized, monodispersed nature of reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate/heptane and their usefulness in approximating a membrane-like environment have been exploited to investigate the effect of pH and water pool size on the photophysical properties of hypericin (Hyp). Our measurements reveal two titratable groups of pKa∼1.5 and ∼12.5. These are assigned to the HypH+/Hyp equilibrium (the deprotonation of a carbonyl group) and the Hyp−/Hyp2− equilibrium (the deprotonation of a peri hydroxyl group). The low-energy absorbance maxima of HypH+, of Hyp and Hyp− and of Hyp2− are 583, 594 and 613 nm, respectively. Neither at pH 13 nor at 1 M HCl is the system entirely in the Hyp2− or the HypH+ forms. Ours is the first study of Hyp in reverse micelles as well as the first time-resolved study of Hyp as a function of pH.
Co-reporter:Philip G. Haydon;Doug. S. English;Robert T. Doyle
Photochemistry and Photobiology 1999 Volume 69(Issue 3) pp:301-305
Publication Date(Web):2 JAN 2008
DOI:10.1111/j.1751-1097.1999.tb03290.x
Abstract— The photodynamic drug, hypericin, is studied in fetal rat neurons using fluorescence microscopy. Hypericin has an extremely high affinity for the cell membrane and is found to a smaller extent in the nucleus. Fluorescent excitation of hypericin is shown to cause irreversible damage to the cell membranes of living neurons. Fixed cells were used to make ultrafast time-resolved measurements to avoid the deleterious effects of long-term exposure to intense light and room temperatures. To our knowledge, these are the first ultrafast time-resolved measurements of the fluorescence lifetime of hypericin in a subcellular environment. Nonexponential fluorescence decay is observed in hypericin in the neurons. This nonexponential decay is discussed in terms of other examples where non-exponential decay is induced in hypericin upon its binding to biomolecules. The nonaradiative processes giving rise to the nonexponential hypericin decay are attributed to excited-state electron transfer, excited-state proton transfer or both.
Co-reporter:Kaustuv Das;Alexra V. Smirnov;Jin Wen;Pavol Miskovsky
Photochemistry and Photobiology 1999 Volume 69(Issue 6) pp:633-645
Publication Date(Web):2 JAN 2008
DOI:10.1111/j.1751-1097.1999.tb03339.x
Abstract— Time-resolved fluorescence and absorption measurements are performed on hypericin complexed with human serum albumin, HSA (1:4, 1:1 and ∼5:1 hypericin: HSA complexes). Detailed comparisons with hypocrellin A/HSA complexes (1:4 and 1:1) are made. Our results are consistent with the conclusions of previous studies indicating that hypericin binds to HSA by means of a specific hydrogen-bonded interaction between its carbon-yl oxygen and the N1-H of the tryptophan residue in the HA subdomain of HSA. (They also indicate that some hypericin binds nonspecifically to the surface of the protein.) A single-exponential rotational diffusion time of 31 ns is measured for hypericin bound to HSA, indicating that it is very rigidly held. Energy transfer from the tryptophan residue of HSA to hypericin is very efficient and is characterized by a critical distance of 94 Å, from which we estimate a time constant for energy transfer of ∼3 × 10–15 s. Although it is tightly bound to HSA, hypericin is still capable of executing excited-state intramolecular proton (or hydrogen atom) transfer in the ∼5:1 complex, albeit to a lesser extent than when it is free in solution. It appears that the proton transfer process is completely impeded in the 1:1 complex. The implications of these results for hypericin (and hypocrellin A) are discussed in terms of the mechanism of intramolecular excited-state proton transfer, the mode of binding to HSA and the light-induced antiviral and antitumor activity.
Co-reporter:R. L. Rich;D. S. English;J. W. Petricht
Photochemistry and Photobiology 1998 Volume 67(Issue 1) pp:76-83
Publication Date(Web):2 JAN 2008
DOI:10.1111/j.1751-1097.1998.tb05167.x
7-Azatryptophan is an alternative to tryptophan as an optical probe of protein structure and dynamics. 7-Azatryptophan is synthetically incorporated into an octapeptide (NAc-Lys-Ala-Cys-Pro-7-azatryptophan-Asn-Cys-Asp-NH2) that mimics the active site of potato chymotrypsin inhibitor II, which is known to be a strong inhibitor of α-chymotrypsin. The synthetic octapeptide retains some of this inhibitory activity. This is the first compound containing the 7-azaindole chromophore to display a nonexponential fluorescence decay (well fit to two exponentials) in water when fluorescence is collected over the entire emission band. The effect of external quenchers on the fluorescence decay is monitored and seen to differ markedly for the two components. These results are discussed in terms of the solvation of the 7-azaindole chromophore itself, which promotes or impedes excited-state tautomerization. The fluorescence quenching of free indole and 7-azaindole are compared. The fluorescence quenching of octapeptides containing both chromophores is also compared. It is the thesis of this article that the nonexponential fluorescence decay of the 7-azatryptophan-containing octapeptide is a consequence of excited-state tautomerization of the 7-azaindole chromophore. This tautomerization is suggested to be promoted by solvent reorganization induced by the peptide backbone or by direct interactions of the 7-azaindole with neighboring amino acid side chains.
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