Room Temperature Phosphorescence from Tryptophan and Halogenated Tryptophan Analogs in Amorphous Sucrose

Abstract. Tryptophan phosphorescence lifetime and quantum yield are sensitive to the local environment. The phosphorescence from tryptophan analogs, however, has not been studied. We report here data on the room temperature phosphorescence of tryptophan, 4-, 5- and 6-fluoro-DL-tryptophan (4-F-trp, 5-F-trp and 6-F-trp) and 5-bromo-DL-tryptophan (5-Br-trp) embedded in glassy powders of freeze-dried sucrose. In aqueous solution, the absorption of the analogs was either blue-shifted (4-F-trp), red-shifted (5-F-trp and 5-Br-trp) or not shifted (6-F-trp) with respect to tryptophan. The phosphorescence emission spectra of all analogs were red-shifted compared to trp (442 nm) with maxima at 446 nm (5-F-trp), 451 mn (6-F-trp), 452 nm (5-Br-trp) and 469 nm (4-F-trp). The 5-F-trp and 6-F-trp analogs had emission intensities similar to tryptophan (relative quantum yields of 0.68 and 0.91, respectively, compared to tryptophan), while the intensities of the 4-F and 5-Br analogs were lower (relative quantum yields of 0.039 and 0.022, respectively). All analogs exhibited complex decay behavior requiring several exponentials for an adequate fit; the average lifetimes were all lower than that of trp (1039 ms). The average lifetimes of the fluorinated analogs (5-F, 721 ms; 6-F, 482 ms and 4-F, 35 ms) scaled approximately with the relative quantum yields while that of 5-Br (0.53 ms) was significantly lower. Analysis of the individual lifetimes suggested that the fluorinated analogs differ in their sensitivity to environmental interactions, with 5-F- and 6-F-trp quenched 1.5-2-fold and 4-F-trp about 23-fold more efficiently than tryptophan. The red-shifted 5-F-trypto-phan analog, which has been incorporated into proteins, may provide an alternative phosphorescence probe for selective phosphorescence detection of a specific protein in a complex mixture.     

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.     

PHOSPHORESCENCE LIFETIME STUDIES OF INTERACTIONS BETWEEN SERUM ALBUMINS AND SODIUM DODECYL SULFATE

Binding of sodium dodecyl sulfate (SDS) to bovine serum albumin (BSA) and human serum albumin (HSA) in aqueous solutions at room temperature induces significant changes in the phosphorescence lifetime of tryptophan (Trp) residues. A steep rise of the phosphorescence lifetime from 1.9 ms to 10.0 ms for BSA and from 1.9 ms to 5.5 ms for HSA is observed when the total SDS concentration increased from 0.0 mM to 0.22 mM at 1 mg/mL protein concentration. As the total SDS concentrationis further inccreased to 2.2 mM, a slower increase in the phosphorescence lifetime is observed, from 10.0 ms to 19.5 ms for BSA and from 5.5 ms to 7.2 ms for HSA. It appears that the phosphorescence lifetime modifications are mainly due to an increase of protein matrix rigidity around Trp residues. The observed differences (between HSA and BSA) allow us to distinguish the contribution of the two Trp residues to the BSA phosphorescence.     

Photophysical and photochemical studies of 2-phenylbenzimidazole and UVB sunscreen 2-phenylbenzimidazole-5-sulfonic acid

The sunscreen agent 2-phenylbenzimidazole-5-sulfonic acid (PBSA) and its parent 2-phenylbenzimidazole (PBI) cause DNA photodamage via both Type-I and Type-II mechanisms when UVB irradiated. We have studied the photophysical and photochemical properties of these compounds and their ability to photogenerate reactive oxygen species including free radicals. PBI and PBSA exhibit both oxidizing and reducing properties in their excited state. The absorption and fluorescence properties of PBSA depend strongly upon pH, and hence the photochemistry of PBSA was studied in both neutral and alkaline solutions. PBSA showed strong oxidizing properties when UV irradiated in neutral aqueous solution (pH 7.4) in the presence of cysteine, glutathione and azide, as evidenced by the detection of the corresponding S-cysteinyl, glutathiyl and azidyl radicals with the aid of the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). However, when an aqueous anaerobic solution (pH 10) of PBSA and either nitromethane (NM) or 4-nitrobenzoic acid (4-NBA) were irradiated, the corresponding nitro anion radicals were observed. This finding suggests that both NM and 4-NBA are reduced by direct electron transfer from the excited state PBSA. During UV irradiation of an aerobic solution of PBSA, O2*- and *OH radical were generated and trapped by DMPO. Further, PBI (in ethanol) and PBSA (in ethylene glycol : water 2: 1 mixture) showed low temperature (77 K) phosphorescence (lambdamax = 443, 476 and 509 nm) and also an electron paramagnetic resonance half-field transition (deltaMs = +/-2), which is evidence for a triplet state. This triplet produced singlet oxygen (1O2) with quantum yields 0.07 and 0.04 in MeCN for PBI and PBSA, respectively. These studies demonstrate that UV irradiation of PBSA and PBI generates a variety of free radicals and active oxygen species that may be involved in the photodamage of DNA.     

The triplet-state lifetime of indole derivatives in aqueous solution

An important feature of tryptophan phosphorescence, crucial for probing protein structure and dynamics, is the drastic reduction of the lifetime (tau) in fluid solutions. Initial reports of indole and derivatives showed that tau decreases from 6 s in rigid glasses to about 1 ms in aqueous solutions at ambient temperature. Recently a report by Fischer et al. questioned the validity of the millisecond lifetime, claiming that in millimolar electrolyte solutions tau is about 40 micros, similar to the 12-30 micros of earlier determinations based on flash photolysis. Longer lived phosphorescence was detected in pure water but because it exhibited an initial growing phase and an anomalously large triplet yield, the emission was attributed to an artifact arising from the slow, first-order, geminate recombination of the radical cation and electron generated by photochemistry. In this study, we reexamine both the phosphorescence lifetime and the triplet quantum yield of indole, N-acetyl tryptophanamide (NATA), N-methyl tryptophan and the tryptophan-glycine-glycine tripeptide under the same conditions adopted by Fischer et al. as well as over a wider range of electrolyte and buffering salts concentrations, pH, solvent and temperature. Throughout, the results show that the phosphorescence decay is slow and uniform down to the 12 micros resolution of the instrument, with no evidence of short-lived, 40 micros-like components. Most compelling was the similarity between the fluorescence-normalized triplet yield of indole derivatives in water and that of W59 in the protein ribonuclease T1 or of NATA in rigid glasses. Its invariance over experimental conditions that varied the production of photoproducts several fold and the characteristic susceptibility of the triplet lifetime to O2, proton and ground state quenching demonstrated that the triplet state was formed predominantly through normal intersystem crossing and that its unquenched lifetime was at least 9 ms.     

PHOSPHORESCENCE MAXIMA AND TRIPLET STATE LIFETIMES OF NAD + AND ε-NAD+ IN TERNARY COMPLEXES WITH HORSE LIVER ALCOHOL DEHYDROGENASE

This paper describes the phosphorescence emission and decay times of NAD+ and its fluorescent etheno derivative, epsilon-NAD+, in the pyrazole ternary complex with horse liver alcohol dehydrogenase (ADH). We show that the epsilon-NAD+ triplet state, as well as the tryptophan triplet state, can be utilized to monitor the coenzyme-enzyme interaction. The decays of NAD+ and AMP are single exponential, and the lifetimes are the same within experimental error. The phosphorescence lifetimes, evaluated as single exponentials, are slightly shorter in epsilon-NAD+ than they are in epsilon-AMP. Whereas the decay of epsilon-AMP was adequately fit by a single exponential with a time constant of very close to 0.5 s, it was necessary to fit the decay of epsilon-NAD+ to a double exponential. Ternary complexes with NAD+ excited at 297 nm exhibit decay kinetics nearly identical to those of ADH by itself. On the other hand, when excitation of the epsilon-NAD+ ternary complex is provided at 313 nm, where there is very little absorption by either tryptophan residue, the decay law of the ternary complex is similar to that of epsilon-NAD+ in solution. Our results demonstrate that NAD+ and epsilon-NAD+ quench tryptophan phosphorescence in ADH. Normalizing the phosphorescence intensity to the 0-0 vibronic band assigned to Trp-15 (blue-edge), we calculate a 21% decrease in the phosphorescence associated with Trp-314 at stoichiometric saturation of the coenzyme binding sites with NAD+ in the ternary complex. When the active sites are saturated with epsilon-NAD+, the relative phosphorescence due to Trp-314 decreases by 63%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lens alpha-crystallin and hypericin: a photophysical mechanism explains observed lens

Determining whether alpha-crystallin (the major lens protein) affects the photophysics of hypericin, a photosensitizing agent found in various plants, such as St. John's Wort, is important. Hypericin shows promise in cancer and human immunodeficiency virus therapy but may harm individuals taking St. John's Wort extracts (for mild to moderate depression). Hypericin causes hypericism, which is characterized by cellular damage in light-exposed areas. Ocular tissues are at risk for photosensitized damage; thus, we investigated the effects on hypericin photophysics by alpha-crystallin. We measured the transient absorption spectra and the 1270 nm luminescence of singlet (1Deltag) oxygen produced from hypericin in the presence of alpha-crystallin. alpha-Crystallin complexes hypericin, extending the lifetime of its triplet excited state; the Stern-Volmer slope is negative, but not linear, after a saturation curve. Damage to the lens protein by hypericin is known to occur via singlet oxygen, which oxidizes methionine, tryptophan and histidine residues. Binding to alpha-crystallin does not inhibit singlet oxygen formation by hypericin. alpha-Crystallin reacts with singlet oxygen with a rate constant of 1.3 x 10(8) M(-1) s(-1). Thus, we anticipate that hypericin will be an effective photosensitizer in the lens.     

Photochemically modified alpha-crystallin: a model system for aging in the primate lens.

The purpose of this study was to quantitatively study the changes that occur upon irradiation of 3-hydroxykynurenine (3-HK) in the presence of alpha-crystallin under conditions similar to those in the lens. The samples were prepared in 10 mM phosphate buffer at pH 7.4, bubbled with O2 or Ar and irradiated with 300-400 nm light. The amount of light absorbed by the samples (Iabs) was measured using azobenzene as an actinometer. Modifications to alpha-crystallin were monitored by ultraviolet-visible and fluorescence spectroscopy. Aerobic samples had increased absorption around 320 nm and above 400 nm while the 3-HK maximum at 368 nm decreased. The isolated modified protein showed that there was increased absorption throughout the spectrum. Changes in the anaerobic samples were similar to those of the aerobic but occurred more slowly. As irradiation time increased fluorescence emission of the isolated protein red shifted and quantum yields of fluorescence (phi f) were calculated at different irradiation time intervals by comparison to 3-HK. By comparing OD320/OD365 for the model system to values from primate lenses, Iabs can be correlated with age and transmission of the sample in the blue region of the spectrum and thus allows lenticular aging to be quantitated.     

ROOM-TEMPERATURE PHOSPHORESCENCE FROM AZURIN DERIVATIVES. PHOSPHORESCENCE QUENCHING IN OXIDIZED NATIVE AZURIN

The tryptophan phosphorescence from a series of derivatives of Pseudomonas aeruginosa azurin has been monitored at 30 degrees C in pH 8.5 buffer solution. The phosphorescence lifetimes fall in the range of 230-270 ms for deoxygenated solutions of derivatives containing Cd(II), Cu(I), Co(II), Ni(II), Hg(II) or apoazurin. A weak signal with a lifetime of ca 130 ms is observed from solutions of oxidized native azurin, but this component is ascribed to a modified form of azurin in solution, i.e. protein heterogeneity, on the basis of the unique sensitivity to quenching by dioxygen. Aside from this minor component, the tryptophan phosphorescence in the Cu(II) protein appears to be fully quenched. The quenching is assigned an electron-transfer mechanism involving transient reduction of the metal center. The same mechanism is deemed to be responsible for fluorescence quenching in oxidized native azurin as well. These observations are of interest because aromatic groups like tryptophan may be conduits for physiological electron-transfer processes involving the copper center.     

THE ANAEROBIC PHOTOLYSIS OF TRYPTOPHAN CONTAINING PEPTIDES—II

Abstract A number of trp–containing peptides (gly- l -trp, l -ala- l -trp, l -trp-gly and N-acetyl-trp) have been photolyzed under anaerobic conditions. Analysis of the resultant photoproduct mixtures suggests the intermediacy of an hydrated electron in the case of trp-gly and N-acetyl-trp. Amino acid trp peptides deaminate with a concomitant cyclization to the 4-position of the indole. This appears to occur via a short-lived radical indole complex. Little difference was detected between photoproduct mixtures using either vycor or a Corning CS-0-54 (cut off ca. 295 nm) as filters. Thus these reactions can occur both above and below the ionization point of trp.     

Photooxidation of lens alpha-crystallin by hypericin (active ingredient in St. John's Wort)

Hypericin is the active ingredient in the over the counter antidepressant medication St. John's Wort. Hypericin produces singlet oxygen and other excited state intermediates that indicate it should be a very efficient phototoxic agent in the eye. Furthermore it absorbs in the UV and visible range, which means it can potentially damage both the lens and the retina. Lens alpha-crystallin, isolated from calf lenses, was irradiated in the presence of hypericin (5 x 10(-5) M, 10 mM ammonium bicarbonate, pH 7.0) and in the presence and absence of light (> 300 nm, 24 mW/cm2). Hypericin-induced photosensitized photopolymerization as assessed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Further analysis of the oxidative changes occurring in alpha-crystallin using mass spectrometry showed specific oxidation of methionine, tryptophan and histidine residues, which increased with irradiation time. Hypericin did not damage the lens protein in the dark. Damage to alpha-crystallin could undermine the integrity of the lens directly by protein denaturation and indirectly by disturbing chaperone function. Therefore, in the presence of light, hypericin can induce changes in lens protein that could lead to the formation of cataracts. Appropriate precautions should be taken to protect the eye from intense sunlight while on this antidepressant medication.     

2+2] Photocycloaddition reaction dynamics of triplet pyrimidines

Taking the 266 nm excited pyrimidine (uracil or thymine) with cyclopentene as model reaction systems, we have examined the photoproduct formation dynamics from the [2 + 2] photocycloaddition reactions of triplet pyrimidines in solution and provided mechanistic insights into this important DNA photodamage reaction. By combining two compliment methods of nanosecond time-resolved transient IR and UV-vis laser flash-photolysis spectroscopy, the photoproduct formation dynamics as well as the triplet quenching kinetics are measured. Characteristic IR absorption bands due to photoproduct formation have been observed and product quantum yields are determined to be ～0.91% for uracil and ～0.41% for thymine. Compared to the measured large quenching rate constants of triplet uracil (1.5 × 10(9) M(-1)s(-1)) or thymine (0.6 × 10(9) M(-1)s(-1)) by cyclopentene, the inefficiency in formation of photoproducts indicates competitive physical quenching processes may exist on the route leading to photoproducts, resulting in very small product yields eventually. Such an energy wasting process is found to be resulted from T(1)/S(0) surface crossings by the hybrid density functional calculations, which compliments the experiments and reveals the reaction mechanism.     

TEMPERATURE DEPENDENCE OF THE PHOSPHORESCENCE LIFETIMES OF HETEROGENEOUS TRYPTOPHAN RESIDUES IN GLOBULAR PROTEINS BETWEEN 293 AND 77 K

Abstract— The phosphorescence of five globular proteins containing tryptophan residues was observed in deoxygenated neutral ethylene glycol-phosphate buffer (1:1 by volume) at 293 and 77 K. Their spectral features at 293 K are closely identical to those at 77 K apart from a lack of tyrosine phosphorescence at 293 K. and are independent of the excitation wavelength between 250 and 310 nm. From the present results. it can be concluded that the buried tryptophan residues are the only phosphorescing centers at room temperature. Their phosphorescence lifetimes were measured as a function of temperature in the range from 77 to 293 K. At room temperature, their phosphorescence lifetimes are between about 1 and 500 ms. On the basis of their temperature dependence, the heterogeneous tryptophan environments are discussed in terms of a temperature-activated nonradiative rate. We suggest that the observation of the phosphorescence characteristics of globular proteins containing tryptophan residues buried in the interior of the protein molecule at room temperature is likely to prove useful in probing the protein structure in solution.     

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.     

Comparison of the Time-resolved Absorption and Phosphorescence from the Tryptophan Triplet State in Proteins in Solution

Measurement of the room temperature Trp triplet state lifetime in proteins by time-resolved phosphorescence can provide valuable information on the structure and dynamics of proteins in solution. Our time-resolved absorption measurements on the long-lived states resulting from electronic excitation of the chromophore demonstrate the presence of more complex behavior than revealed by time-resolved phosphorescence. To provide additional insight into this behavior, a comparative study of time-resolved transient absorption and time-resolved phosphorescence of proteins in solution was carried out. The results show that the time evolution of the long-lived states observed through transient absorption often differs considerably from that observed in time-resolved phosphorescence. In some proteins, the presence of competing reactions complicates the interpretation of the transient absorption measurements (which may affect the phosphorescence yield). A more complete characterization of these processes will likely prove useful in the study of protein structure and dynamics in solution.     

The primary photophysics of the Avena sativa phototropin 1 LOV2 domain observed with time-resolved emission spectroscopy

The phototropins are blue-light receptors that base their light-dependent action on the reversible formation of a covalent bond between a flavin mononucleotide (FMN) cofactor and a conserved cysteine in light, oxygen or voltage (LOV) domains. The primary reactions of the Avena sativa phototropin 1 LOV2 domain were investigated by means of time-resolved and low-temperature fluorescence spectroscopy. Synchroscan streak camera experiments revealed a fluorescence lifetime of 2.2 ns in LOV2. A weak long-lived component with emission intensity from 600 to 650 nm was assigned to phosphorescence from the reactive FMN triplet state. This observation allowed determination of the LOV2 triplet state energy level at physiological temperature at 16600 cm(-1). FMN dissolved in aqueous solution showed pH-dependent fluorescence lifetimes of 2.7 ns at pH 2 and 3.9-4.1 ns at pH 3-8. Here, too, a weak phosphorescence band was observed. The fluorescence quantum yield of LOV2 increased from 0.13 to 0.41 upon cooling the sample from 293 to 77 K. A pronounced phosphorescence emission around 600 nm was observed in the LOV2 domain between 77 and 120 K in the steady-state emission.     

The triplet-state lifetime of indole in aqueous and viscous environments: significance to the interpretation of room temperature phosphorescence in proteins

The interpretation of room temperature phosphorescence studies of proteins requires an understanding of the mechanisms governing the tryptophan triplet-state lifetimes of residues fully exposed to solvent and those deeply buried in the hydrophobic core of proteins. Since solvents exposed tryptophans are expected to behave similarly to indole free in solution, it is important to have an accurate measure of the triplet state lifetime of indole in aqueous solution. Using photon counting techniques and low optical fluence (J/cm(2)), we observed the triplet-state lifetime of aqueous, deoxygenated indole and several indole derivatives to be approximately 40 micros, closely matching the previous reports by Bent and Hayon based on flash photolysis (12 micros; Bent, D. V.; Hayon, E. J. Am. Chem. Soc. 1975, 97, 2612-2619) but much shorter than the 1.2 ms lifetime observed more recently (Strambini, G. B.; Gonnelli, M. J. Am. Chem. Soc. 1995, 117, 7646-7651). However, we have now been able to reproduce the long lifetime reported by the latter workers for aqueous indole solutions and show that it likely arises from geminate recombination of the indole radical cation and solvated electron, a conclusion based on studies of the indole radical cation in water (Bent and Hayon, 1975). The evidence for this comes from a fast rise in the phosphorescence emission and measurements of a corresponding enhanced quantum yield in unbuffered solutions. This species can be readily quenched, and the corresponding fast rise disappears, leaving a monoexponential 40 micros decay, which we argue is the true indole triplet lifetime. The work is put in the context of room temperature phosphorescence studies of proteins.     

Absorption and emission spectroscopic characterization of Ir(ppy)3

The absorption and emission spectroscopic behaviour of cyclometalated fac-tris(2-phenylpyridine) iridium(III) [Ir(ppy)₃] is studied at room temperature. Liquid solutions, doped films, and neat films are investigated. The absorption cross-section spectra including singlet–triplet absorption, the triplet–singlet stimulated emission cross-section spectra, the phosphorescence quantum distributions, the phosphorescence quantum yields and the phosphorescence signal decays are determined. In neat films fluorescence self-quenching occurs, in diluted solid solution (polystyrene and dicarbazole-biphenyl films) as well as deaerated liquid solution (toluene) high phosphorescence quantum yields are obtained, and in air-saturated liquid solutions (chloroform, toluene, tetrahydrofuran) the phosphorescence efficiency is reduced by triplet oxygen quenching. At intense short-pulse laser excitation the phosphorescence lifetime is shortened by triplet–triplet annihilation. No amplification of spontaneous emission in the phosphorescence spectral region was observed indicating higher excited-state absorption than stimulated emission.     

Nanosecond laser photolysis studies of chlorosomes and artificial aggregates containing bacteriochlorophyll e: evidence for the proximity of carotenoids and bacteriochlorophyll a in chlorosomes from Chlorobium phaeobacteroides strain CL1401

Time-resolved, laser-induced changes in absorbance, delta A(lambda; t), have been recorded with a view to probing pigment-pigment interactions in chlorosomes (control as well as carotenoid-depleted) and artificial aggregates of bacteriochlorophyll e (BChle). Control chlorosomes were isolated from Chlorobium phaeobacteroides strain CL1401, whose chromophores comprise BChle, bacteriochlorophyll a (BChla) and several carotenoid (Car) pigments; Car-depleted chlorosomes, from cells grown in cultures containing 2-hydroxybiphenyl. Artificial aggregates were prepared by dispersing BChle in aqueous phase in the presence of monogalactosyl diglyceride. In chlorosomes delta A(lambda; t) shows, besides a signal attributable to triplet Car (with a half-life of about 4 microseconds), signals in the Qy regions of both BChl. The BChla signal decays at the same rate as the Car signal, which is explained by postulating that some Car are in intimate contact with some baseplate BChla pigments, and that when a ground-state Car changes into a triplet Car, the absorption spectrum of its BChla neighbors undergoes a concomitant change (termed transient environment-induced perturbation). The signal in the Qy-region of BChle behaves differently: its amplitude falls, under reducing conditions, by more than a factor of two during the first 0.5 microsecond (a period during which the Car signal suffers negligible diminution), and is much smaller under nonreducing conditions. The BChle signal is also attributed to transient environment-induced perturbation, but in this case the perturber is a BChle photoproduct (probably a triplet or a radical ion). The absence of long-lived BChle triplets in all three systems, and of long-lived BChla triplets in chlorosomes, indicates that BChle in densely packed assemblies is less vulnerable to photodamage than monomeric BChle and that, in chlorosome, BChla rather than BChle needs, and receives, photoprotection from an adjacent Car.     

HETEROGENEITY IN THE THERMALLY-INDUCED QUENCHING OF THE PHOSPHORESCENCE OF MULTI-TRYPTOPHAN PROTEINS

Abstract— The steady-state intensity and lifetime of the tryptophan phosphorescence from a number of globular proteins in 2:1 (v/v) glycerol buffer were monitored as a function of temperature. The phosphorescence lifetimes are essentially independent of the tryptophan local environment in rigid solution at temperatures < 170K, but vary markedly between proteins at temperatures at which the solutions become fluid. The ratio of steady-state intensity to lifetime P/τ was found to be temperature independent despite the quenching for free tryptophan and the lone residue in myelin basic protein. Heterogeneity in the triplet quenching of the tryptophans in liver alcohol dehydrogenase and alkaline phosphatase were revealed as step-like decreases in the ratio of P/T followed by plateau regions characterizing the homogeneous behavior of the more resistent tryptophans in the proteins. This heterogeneity exists not only between solvent-exposed and buried residues, but reflects variations in the flexibility of the structure surrounding distinct buried tryptophans in the globular proteins.