MODEL STUDIES ON THE PHOTOCHEMICAL PRODUCTION OF LENTICULAR FLUOROPHORES

Abstract With aging, human lens proteins accumulate fluorophores having blue and green emissions. Model studies were undertaken to determine the role of 3-hydroxykynurenine (3-HK) and its glucoside (3-HKG) in the photochemical production of those fluorophores. Experiments were carried out using 10⁻³ M 3-HK solutions in the presence or absence of glycine (1 M ), which was used to mimic the environment of the lens. The solutions were photolyzed (transmission above 295 nm) for various periods of time while the loss of starting material and the formation of fluorescent photoproducts (blue emission at 470 nm, and green emission at 520 nm) were monitored using fluorescence and UV-visible spectroscopy and thin-layer and high-pressure liquid chromatography analysis. Several parameters were varied such as oxygen tension and the addition of the free radical scavenger, penicillamine. The photolytic loss of 3-HK in the absence of glycine occurred approximately 5-10 times faster than in its presence. Conversely, blue and green fluorophores formed in irradiated solutions containing glycine but not with the photolysis of 3-HK alone. The blue fluorophore was formed first and appeared then to be photochemically converted to the green one, with the rate of formation of the latter increasing with an increase in UV dosage or oxidizing conditions. The addition of penicillamine drastically reduced the photochemical formation of both fluorophores.
Both the blue and green fluorophores appear to result from the photochemically induced covalent attachment of 3-HK to glycine. In the human lens, these reactions can explain the age-related loss of 3-HKG with the concomitant formation of fluorophores covalently attached to lens proteins, probably via the amino group of lysine.     

The photochemical attachment of the O-glucoside of 3-hydroxykynurenine to alpha-crystallin: a model for lenticular aging

The young human lens contains a small metabolite from tryptophan called the O-glucoside of 3-hydroxykynurenine (3-HKG). Its function is to absorb most radiation between 295 and 400 nm, preventing it from reaching the retina. With age the concentration of this component decreases while the lens crystallins acquire covalently attached chromophores. This study investigates the photochemical attachment of 3-HKG to lens alpha-crystallin. Initial studies showed that alpha-crystallin photolyzed in the presence of 3-HKG developed a fluorescence (emission, 440 nm) and UV-visible spectrum similar to that found in aged human lens proteins. Extensive studies were then performed on the tryptic HPLC maps as monitored by photodiode array and fluorescent detection. Numerous photoproducts with either blue (emission, > 400 nm) or green (emission, > 500 nm) fluorescence were formed in addition to nonfluorescent compounds with absorption maxima above 300 nm. Comparisons were made between these model photoproducts and peptide maps from alpha-crystallin isolated from old human lenses. In terms of retention time and UV-visible spectra at least two of the peptides that appear in the model system are also present in the human samples. It is concluded that one of the aging processes in the human lens is the photochemically induced attachment of 3-HKG to lens proteins.     

THE PHOTOREACTIONS OF 8-METHOXYPSORALEN WITH TRYPTOPHAN AND LENS PROTEINS*

Abstract— We have previously demonstrated that 8-methoxypsoralen (8-MOP) can be found in the lenses of rats injected (i.p.) with this drug, and that its presence can lead to a photosensitized enhancement of lenticular fluorescence. The cutaneous photosensitizing properties of psoralens are thought to be mediated via their excited triplet states, resulting in photoaddition cyclobutane products between pyri-midine bases and 8-MOP. We have now investigated the possibility that similar types of photoadducts could be generated between 8-MOP and the aromatic amino acid residues in lens proteins. Our experiments involved in vitro irradiation (at 360 nm) of aqueous solutions of 0.1 mM 8-MOP plus purified alpha, beta, or gamma crystallins from calf or normal human (under 20 years of age) lenses. UV absorption and fluorescence emission spectra were measured before and after radiation, and aliquots from all experiments were frozen and kept in the dark for subsequent phosphorescence and EPR spectroscopy. Similar experiments were performed with irradiated aqueous solutions of tryptophan or thymine plus 8-MOP. All controls consisted of solutions kept in the dark. NMR spectra demonstrated that the hydrogen atoms at the 3,4 and 4',5' positions of the 8-MOP molecule were lost following irradiation, suggesting that these two sites were involved in the photoproduct formed between tryptophan and 8-MOP. These studies strongly suggest that 8-MOP is capable of forming photoaddition products with tryptophan and with lens proteins as well as DNA in vivo, resulting in its permanent retention within the ocular lens.     

STUDIES ON HUMAN LENSES: II. DISTRIBUTION AND SOLUBILITY OF FLUORESCENT PIGMENTS IN CATARACTOUS AND NON-CATARACTOUS LENSES OF INDIAN ORIGIN

Abstract— Fluorometric studies of cataractous and non-cataractous human lenses were carried out to study the emission characteristics and the distribution and solubility of lenticular pigments. Most of the detected fluorophores were well distributed over the cortical and nuclear portion of the lens. The decrease in solubility of proteins with aging and cataract formation is concomitant with increasing photolysis of tryptophan. However, this is likely a phenomenon independent of the photochemical transformations of the lens proteins. The number of emitting species in the diseased lenses are higher than in the normal mature lenses. A species emitting around 375 or 388 nm is of particular interest (λ_cx, 330 nm) in that the emission characteristics of this fluorophore resemble kynurenic acid which has a high photosensitizing efficiency. The concentration of fluorescent pigments in the lenses of Indian origin is significantly high. The intense pigmentation could be attributed largely to the formation of photoproducts in the absence of normal endogenous antioxidant accumulation that is dependent on nutrition standard. If, indeed, any of these fluorescent pigments, because of their photosensitizing ability, are responsible for lenticular opacity, it is not the abundance of sunlight alone but also malnutrition that could account for the high incidence of cataract in India.     

STUDIES ON HUMAN LENSES: II. DISTRIBUTION AND SOLUBILITY OF FLUORESCENT PIGMENTS IN CATARACTOUS AND NON-CATARACTOUS LENSES OF INDIAN ORIGIN

Fluorometric studies of cataractous and non-cataractous human lenses were carried out to study the emission characteristics and the distribution and solubility of lenticular pigments. Most of the detected fluorophores were well distributed over the cortical and nuclear portion of the lens. The decrease in solubility of proteins with aging and cataract formation is concomitant with increasing photolysis of tryptophan. However, this is likely a phenomenon independent of the photochemical transformations of the lens proteins. The number of emitting species in the diseased lenses are higher than in the normal mature lenses. A species emitting around 375 or 388 nm is of particular interest (lambda cx 330 nm) in that the emission characteristics of this fluorophore resemble kynurenic acid which has a high photosensitizing efficiency. The concentration of fluorescent pigments in the lenses of Indian origin is significantly high. The intense pigmentation could be attributed largely to the formation of photoproducts in the absence of normal endogenous antioxidant accumulation that is dependent on nutrition standard. If, indeed, any of these fluorescent pigments, because of their photosensitizing ability, are responsible for lenticular opacity, it is not the abundance of sunlight alone but also malnutrition that could account for the high incidence of cataract in India.     

IN VITRO STUDIES ON THE PHOTOSENSITIZED OXIDATION OF LENS PROTEINS BY PORPHYRINS

Abstract Porphyrins, which may be introduced into the eye as a result of abnormal porphyrin metabolism (uroporphyrin–Uro) or when used in the diagnosis or photodynamic therapy of certain tumors, including intraocular tumors (hematoporphyrin–Hp and'hematoporphyrin derivative'–Hpd and mesotetra( P -sulfonatophenyl)porphyrin–TPPS) are efficient photosensitizers in biological systems. We have been studying the potential phototoxic side effects of these drugs in the lens of the eye. Encapsulated in the human lens is a mixture of soluble protein crystallins. With little turnover of protein in the lens, any photosensitized modifications will accumulate and may result in an opacification of the lens. To evaluate the potential of different porphyrins to induce such damage, a series of porphyrins were photolyzed (transmission above 295 nm) in the presence of calf lens protein (2 mg m⁻¹). Marked photopolymerization and histidine destruction were observed for the lens protein photolyzed in the presence of all of the drugs. We have found that the relative effectiveness of the following porphyrins to induce that damage is: Uro = TPPS Hpd = Hp. Both the singlet oxygen quencher, azide, and the free radical scavenger, penicillamine, decrease this photosensitized oxidative damage to lens protein. TPPS binds significantly to lens protein and this binding leads to conformational changes in that protein.     

Ratiometric fluorescent sensor based on inhibition of resonance for detection of cadmium in aqueous solution and living cells

Although cadmium has been recognized as a highly toxic heavy metal and poses many detrimental effects on human health, the Cd(2+)-uptake and nosogenesis mechanisms are still insufficiently understood, mainly because of the lack of facile analytical methods for monitoring changes in the environmental and intracellular Cd(2+) concentrations with high spatial and temporal reliability. To this end, we present the design, synthesis, and photophysical properties of a cadmium sensor, DQCd1 based on the fluorophore 4-isobutoxy-6-(dimethylamino)-8-methoxyquinaldine (model compound 1). Preliminary investigations indicate that 1 could be protonated under neutral media and yield a resonance process over the quinoline fluorophore. Upon excitation at 405 nm, 1 shows a strong fluorescence emission at 554 nm with a quantum yield of 0.17. Similarly, DQCd1 bears properties comparable to its precursor. It exhibits fluorescence emission at 558 nm (Φ(f) = 0.15) originating from the monocationic species under physiological conditions. Coordination with Cd(2+) causes quenching of the emission at 558 nm and simultaneously yields a significant hypsochromic shift of the emission maximum to 495 nm (Φ(f) = 0.11) due to inhibition of the resonance process. Thus, a single-excitation, dual-emission ratiometric measurement with a large blue shift in emission (Δλ = 63 nm) and remarkable changes in the ratio (F(495 nm)/F(558 nm)) of the emission intensity (R/R(0) up to 15-fold) is established. Moreover, the sensor DQCd1 exhibits very high sensitivity for Cd(2+) (K(d) = 41 pM) and excellent selectivity response for Cd(2+) over other heavy- and transition-metal ions and Na(+), K(+), Mg(2+), and Ca(2+) at the millimolar level. Therefore, DQCd1 can act as a ratiometric fluorescent sensor for Cd(2+) through inhibition of the resonance process. Confocal microscopy and cytotoxicity experiments indicate that DQCd1 is cell-permeable and noncytotoxic under our experimental conditions. It can indeed visualize the changes of intracellular Cd(2+) in living cells using dual-emission ratiometry.     

Photophysics and biological applications of the environment-sensitive fluorophore 6-N,N-dimethylamino-2,3-naphthalimide

We have synthesized a new environment-sensitive fluorophore, 6-N,N-dimethylamino-2,3-naphthalimide (6DMN). This chromophore exhibits valuable fluorescent properties as a biological probe with emission in the 500-600 nm range and a marked response to changes in the environment polarity. The 6DMN fluorescence is red-shifted in polar protic environments, with the maximum emission intensity shifting more than 100 nm from 491 nm in toluene to 592 nm in water. Additionally, the fluorescence quantum yield decreases more than 100-fold from chloroform (Phi = 0.225) to water (Phi = 0.002). The scope and applications of the 6DMN probe are expanded with the synthesis of an Fmoc-protected amino acid derivative (5), which contains the fluorophore. This unnatural amino acid has been introduced into several peptides, demonstrating that it can be manipulated under standard solid-phase peptide synthesis conditions. Peptides incorporating the new residue can be implemented for monitoring protein-protein interactions as exemplified in studies with Src homology 2 (SH2) phosphotyrosine binding domains. The designed peptides exhibit a significant increase in the quantum yield of the long wavelength fluorescence emission band (596 nm) upon binding to selected SH2 domains (e.g., Crk SH2, Abl SH2, and PI3K SH2). The peptides can be used as ratiometric sensors, since the short wavelength band (460 nm) was found almost invariable throughout the titrations.     

FLUORESCENCE LIFETIMES OF OCULAR LENS CHROMOPHORES

Abstract— Fluorescence lifetimes are reported for intact human lenses in vitro. Two spectral regions were investigated: The first was excited at 296nm and detected at 332 or 370nm and corresponds to emission from tryptophan residues in the lens proteins. The second spectral region was excited at 359 nm and detected at 435 nm and corresponds to non-tryptophan 'fluorogen' fluorescence. The latter displayed a constant lifetime, 3.8 ns, independent of the anatomical part of the lens excited. This value was compared with measured lifetimes for some model fluorogens. The tryptophan fluorescence lifetime (332 nm detection) was found to vary from 1.8 to 2.8 ns depending on the anatomical part of the lens excited.     

Fluorescence Properties of Isolated Intact Normal Human Corneas

Abstract— We have examined the fluorescence properties of excised intact normal human corneas from over a hundred donors, using synchronous excitation fluorescence spectroscopy. In some of the corneas from the donors, a fluoro-phore with an excitation band centered at 330 nm was observed. This fluorophore does not seem to correspond to the dityrosine moiety or to any photoproducts of tryptophan. Isolated corneas irradiated with light of 295 nm wavelength do not produce any fluorescent photoproducts, suggesting that the intact tissue has endogenous quenchers, radical scavengers and antioxidants that inhibit its photodamage. The non-tryptophan fluorophores that accumulate in some corneas thus appear to arise largely from the nonenzymatic glycosylation (glycation) of the constituent proteins as similar fluorophores are detected in the corneas of rats in which diabetes is induced.     

Environment-sensitive fluorophores with benzothiadiazole and benzoselenadiazole structures as candidate components of a fluorescent polymeric thermometer

An environment-sensitive fluorophore can change its maximum emission wavelength (λ(em)), fluorescence quantum yield (Φ(f)), and fluorescence lifetime in response to the surrounding environment. We have developed two new intramolecular charge-transfer-type environment-sensitive fluorophores, DBThD-IA and DBSeD-IA, in which the oxygen atom of a well-established 2,1,3-benzoxadiazole environment-sensitive fluorophore, DBD-IA, has been replaced by a sulfur and selenium atom, respectively. DBThD-IA is highly fluorescent in n-hexane (Φ(f) =0.81, λ(em) =537?nm) with excitation at 449?nm, but is almost nonfluorescent in water (Φ(f) =0.037, λ(em) =616?nm), similarly to DBD-IA (Φ(f) =0.91, λ(em) =520?nm in n-hexane; Φ(f) =0.027, λ(em) =616?nm in water). A similar variation in fluorescence properties was also observed for DBSeD-IA (Φ(f) =0.24, λ(em) =591?nm in n-hexane; Φ(f) =0.0046, λ(em) =672?nm in water). An intensive study of the solvent effects on the fluorescence properties of these fluorophores revealed that both the polarity of the environment and hydrogen bonding with solvent molecules accelerate the nonradiative relaxation of the excited fluorophores. Time-resolved optoacoustic and phosphorescence measurements clarified that both intersystem crossing and internal conversion are involved in the nonradiative relaxation processes of DBThD-IA and DBSeD-IA. In addition, DBThD-IA exhibits a 10-fold higher photostability in aqueous solution than the original fluorophore DBD-IA, which allowed us to create a new robust molecular nanogel thermometer for intracellular thermometry.     

Nanosecond relaxation dynamics of protein GB1 identified by the time-dependent red shift in the fluorescence of tryptophan and 5-fluorotryptophan

The B1 domain of Streptococcal protein G (GB1) is a small, thermostable protein containing a single tryptophan residue. We recorded time-resolved fluorescence of the wild-type GB1 and its 5-fluorotryptophan (5FTrp) variant at more than 30 emission wavelengths between 300 and 470 nm. The time-resolved emission spectra reveal no signs of heterogeneity, but show a time-dependent red shift characteristic of microscopic dielectric relaxation. This is true for both 5FTrp and unmodified Trp in GB1. The time-dependent red shifts in the fluorescence of 5FTrp and unmodified Trp are essentially identical, confirming that the shift is caused by the relaxation of the protein matrix rather than by the fluorophore itself. The total amplitude (but not the rate) of the time-dependent red shift depends on the fluorophore, specifically, on the magnitude of the vector difference between its excited state and ground state electric dipole moments; for 5FTrp this is estimated to be about 88% of that for the unmodified Trp. The decay of the excited state fluorophore population is not monoexponential for either fluorophore; however, the deviation from the monoexponential decay law is larger in the case of unmodified Trp. The relaxation dynamics of GB1 was found to be considerably faster than that of other proteins studied previously, consistent with the small size, tightly packed core, and high thermodynamic stability of GB1.     

Fluorescence properties of domperidone and its determination in pharmaceutical preparations

The native fluorescence characteristics of domperidone in various solvents and at different pH values are reported. The fluorophore is the benzo-1,3-diazolin-2-one group which is also present in the analogous benperidol, droperidol and pimozide molecules from which domperidone is derived; the substituent may therefore be considered as a relatively strong fluorophore; its excitation and emission maxima are little influenced by pH variation. Domperidone can be determined in pharmaceutical preparations by its native fluorescence in ethanol (λ_ex = 283 nm ;λ_em = 324 nm) and in 0.01 M HCl (λ_ex = 284 nm;λ_em = 329 mn) with a detection limit of 0.01 μg ml^-1.     

New fluorophores with rod-shaped polycyano pi-conjugated structures: synthesis and photophysical properties

[reaction: see text] Novel rod-shaped polycyano-oligo(phenyleneethynylene)s were synthesized by Pd cross-coupling reaction. Polycyano groups were found to greatly improve the emission efficiency (Phi(f)) of OPEs. By the end donor modification, we achieved the creation of very intense blue light-emitting fluorophore with the SMe group (Phi(f) = 0.972, log epsilon 4.89, lambda(em) 455 nm) and very intense yellow light-emitting fluorophore with the NMe(2) group (Phi(f) = 0.999, log epsilon 4.75, lambda(em) 555 nm). Contrasting Phi(f) solvent dependency of 6 and 7 and a linear relationship between Phi(f) and sigma(p)-X over the whole region of sigma(p)-X were also found.     

Spectroscopic study of visible-light effects on hypericin-lens proteins systems

Molecular interactions between hypericin and alpha-, beta- and gamma-crystallin proteins have been studied by means of absorption and steady-state fluorescence spectroscopy, aiming to clarify if and how the pigment binds to the proteins and to investigate the effects of visible-light irradiation on these molecular systems. Such a study is a prerequisite for assessing the possibility of using hypericin as a mild antidepressant and/or as a photodynamic agent for the treatment of eye tumors and eye viral and bacterial diseases without side injuries to the lens. We have shown that in dark-kept samples, with increasing alpha-crystallin concentration, both the fluorescence emission intensity and the ratio of the absorption maxima around 590 and 550 nm of hypericin increase. These effects have been attributed to the monomerization of nonfluorescent hypericin aggregates caused by the binding of the pigment to alpha-crystallin. The binding constant of hypericin has been evaluated to be of the order of 3.0 (mg/mL)-1, corresponding to a dissociation constant of the order of 0.3 mg/mL. Following irradiation with light of wavelengths over 400 nm, at an irradiance of 20 mW/cm2, both tryptophan and hypericin fluorescence emission intensities decrease. These effects are suggested to be the consequence of a spatial rearrangement of the protein framework which takes place following the alpha-crystallin photopolymerization sensitized by hypericin itself described in the literature. For the sake of comparison hypericin has been studied also in the presence of beta H-, beta L- and gamma-crystallins at the same concentration.     

A novel two-fluorophore approach to ratiometric sensing of Zn(2+)

We present a small molecule ratiometric Zn2+-sensing system based on two fluorophores excited by visible light, a Zn2+-insensitive reporter fluorophore, coumarin 343, and a Zn2+-sensitive fluorescein-based compound, ZPA-1. The two fluorophores are linked by an ester to give Coumazin-1, a membrane-permeable, essentially nonfluorescent compound. Upon exposure to esterases, Coumazin-1 is hydrolyzed to its constituent fluorophores. Measurement of the ratio of coumarin emission at 488 nm (lambdaexc = 445 nm) and comparison with ZPA-1 emission at 534 nm (lambdaexc = 505 nm) affords information about the amount of sensor present as well as the amount of Zn2+ present. A generally applicable synthetic route to amide-functionalized ZP1 sensors is also described. The Zn2+-sensing properties of one member of this class are similar to those of the parent ZP1 sensor, with slightly tighter binding and lower background signal.     

RED EDGE EXCITATION EFFECT IN INTACT EYE LENS

Shift in the wavelength of emission upon shift in the excitation wavelength towards the red edge of the absorption band is termed Red Edge Excitation Shift (REES). This effect is observed only in situation where the fluorophore mobility with respect to the surrounding matrix is considerably reduced. We have observed such red edge excitation effect in the intact eye lens. The REES observed for a normal lens is different from that seen in a photodamaged lens and hence appears to be a potential tool to monitor the changes in the state of the lens. Photodamage experiments with tryptophan in polyethylene glycol (PEG) and intact eye lens indicate that the red edge photon can also cause photodamage.     

Interstitial fluorescence spectroscopy in the human prostate during motexafin lutetium-mediated photodynamic therapy

The in vivo fluorescence emission from human prostates was measured before and after motexafin lutetium (MLu)-mediated photodynamic therapy (PDT). A single side-firing optical fiber was used for both the delivery of 465 nm light-emitting diode excitation light and the collection of emitted fluorescence. It was placed interstitially within the prostate via a closed transparent plastic catheter. Fitting of the collected fluorescence emission spectra using the known fluorescence spectrum of 1 mg/kg MLu in an intralipid phantom yields a quantitative measure of the local MLu concentration. We found that an additional correction factor is needed to account for the reduction of the MLu fluorescence intensity measured in vivo due to strong optical absorption in the prostate. We have adopted an empirical correction formula given by C = (3.1 cm(-1)/micro's) exp (microeff x 0.97 cm), which ranges from approximately 3 to 16, with a mean of 9.3 +/-4.8. Using a computer-controlled step motor to move the probe incrementally along parallel tracks within the prostate we can determine one-dimensional profiles of the MLu concentration. The absolute MLu concentration and the shape of its distribution are confirmed by ex vivo assay and by diffuse absorption measurements, respectively. We find significant heterogeneity in photosensitizer concentration within and among five patients. These variations occur over large enough spatial scales compared with the sampling volume of the fluorescence emission that mapping the distribution in three dimensions is possible.     

Photodynamics of cataract: an update on endogenous chromophores and antioxidants

Cataract of the eye lens is characterized by (1) progressive opacification or loss of transparency; (2) accumulation of molecules that absorb in the UV-visible range of the spectrum; and (3) formation of fluid-filled vacuoles, particularly in diabetics when sugar levels in the lens are high. Of the various causative factors for cataract, light is an important one. Because the level of light below 320 nm reaching the lens is quite low, photodynamic effects of endogenous compounds absorbing in the UV-A-visible region become important in this connection. In this update we summarize a list of accumulant chromophores that have been identified in aging and cataract human lenses and their roles as potential sensitizers, antioxidants or as benign filters. Because such photodynamic cataractogenesis is oxidative in nature, we also cite examples where administration of antioxidants could help delay cataract progression.     

TIME RESOLVED SPECTROSCOPIC STUDIES ON THE INTACT HUMAN LENS

Abstract— The human lens is continually under photooxidative stress from ambient radiation. In the young lens the major absorbing (between300–400 nm) species is the glucoside of 3-hydroxy kynurenine. Using time resolved fluorescence spectroscopy on both the isolated compound and the intact human lens, the first excited singlet state of this compound is shown to have fast (ps) decay processes. This would tend to minimize damage to lens constituents because there would be little time for energy transfer into more harmful channels. Thus, this compound appears to act as a protection for the retina. With aging, human lens proteins become yellow with absorptions out to 450 nm. Time resolved diffuse reflectance spectroscopic studies on intact older human lenses showed that excitation (355 nm) resulted in the formation of long lived (microseconds) transient species with an absorption maximum at ca 490 nm. Similar spectra were obtained from two model systems used to explain age related changes in human lens proteins.