TRIPLET-TRIPLET ENERGY TRANSFER IN P-TRYPSIN 1 THE ROLE OF THE INDOLE TRIPLET IN THE ULTRAVIOLET-INDUCED PHOTOLYSIS OF β-TRYPSIN

Detection of triplet-triplet energy transfer in an aqueous solution of P-trypsin is reported. This conclusion is based on the observation that a light excited phenolate side chain can sensitize the destruction of an adjacent indole side chain. The role that the indole triplet might play in the UV-induced photolysis of /l-trypsin is also investi-gated. The results suggest that the UV (309 nm)-induced inactivation of P-trypsin is not caused by indole ring destruction but by the disruption of disulfide bonds without thiol formation.     

OPTICALLY DETECTED MAGNETIC RESONANCE OF THE TRYPTOPHAN PHOSPHORESCENT STATE IN NATIVE PROTEINS*

Abstract— The phosphorescent triplet state of tryptophan has been studied by the method of optically detected magnetic resonance (ODMR) at pumped helium temperatures in zero magnetic field. Only one of the triplet sublevels is found to be significantly radiative; the other two decay radiationlessly. Although the phosphorescence and ODMR decay lifetimes are influenced by spin–lattice relaxation processes at T = 1.3°K, the lifetime of the radiative level can be estimated as approximately 2 s, whereas the lifetimes of the non–radiative levels are in excess of 10 s. Comparison of the ODMR signals and the phosphorescence spectra has been made for tryptophans in native proteins with the following results: the ODMR signals of the two types of tryptophan sites in horse liver alcohol dehydrogenase can be resolved due to a shift in the D and E values of the respective triplet states; binding of the substrate tri- N -acetylglucosamine to hen lysozyme leads to a considerable narrowing of the phosphorescence peaks and ODMR signals as well as to a shift in the E value of the triplet state.
The following tentative conclusions can be reached: the tryptophan triplet D and E values are measurably affected by the environment of the chromophore in the protein, as are the linewidths of the magnetic resonance transitions. The | E | value is reduced and the magnetic resonance linewidth is increased with increasing exposure of the tryptophan to hydroxylic solvent. Although a considerable part of the width of the magnetic resonance transition can be ascribed to a heterogeneity of environments in the sample, there appears to exist an intrinsic line–broadening process which at present is not understood.     

KINETICS OF THE ULTRAVIOLET-INDUCED DIMERIZATION OF THYMINE IN FROZEN SOLUTIONS*

Abstract— It is known that thymine forms dimers when aqueous solutions are irradiated with ultraviolet light while in the frozen state, but does not form dimers when solutions are irradiated in the liquid state. The eutectic point of aqueous thymine solutions was found to be. —0.02°C. Since the irradiation of frozen solutions is always carried out at lower temperatures, the dimerization must be occurring in the solid state. Activation energies and quantum yields for dimer formation were determined by irradiating 1–mm layers of thymine solution at —5°C to — 707deg;C for various lengths of time. As expected, the activation energy was zero. After measuring the amount of radiation scattered by samples of ice, the extreme values for the quantum yield were found to be 0.73 and 4.08. The lower limit assumed that all the scattered light was absorbed by thymine; the upper limit assumed that none was absorbed. Since the theoretical maximum quantum yield is 2, the best estimate of the quantum yield is considered to be between 1 and 2.     

PRIMARY PRODUCTS IN THE FLASH PHOTOLYSIS OF TRYPTOPHAN*

There has been considerable interest in the photochemistry of tryptophan in connection with ultraviolet inactivation of enzymes. Earlier flash photolysis work has demonstrated that the hydrated electron (e^-_aq) is an initial product in the irradiation of indole derivatives, accompanied by a longer-lived transient absorption near 500 nm attributed to an aromatic radical species[1–5]. Similar transients were observed in a recent flash photolysis study of lysozyme[6] in which it was proposed that inactivation is a consequence of electron ejection from 1 to 2 essential tryptophan residues in the active center. However, there has been uncertainty concerning the tryptophan radical structure and its relationship to the triplet state and radical spectra reported for tryptophan photolysis in low-temperature rigid media. This note reports a flash photolysis investigation of L-tryptophan (Trp) and 1-Methyl-L-tryptophan (1-MeTrp) undertaken to clarify these points. The flash photolysis apparatus and methods employed are described in Ref. [6].     

THE LIFETIME OF THE EXCITED SINGLET STATE OF β-CAROTENE: CONSEQUENCES TO PHOTOSYNTHETIC LIGHT HARVESTING

Abstract The mechanism of singlet-singlet energy transfer to chlorophyll from carotenoid auxiliary pigments in photosynthetic apparatus is considered. Transmittance studies and resonance-enhanced Raman spectroscopy on a picosecond time scale lead to the conclusion that the de–excitation lifetime of the β-carotene singlet state is not greater than one picosecond. This would require close contiguity on the part of the transferring partners.     

KINETIC STUDIES OF THE TRYPTOPHAN TRIPLET STATE IN SOLID FILM AND IN WOOL KERATIN

Abstract— The triplet state of tryptophan in the solid environments of a polyvinyl alcohol) (PVA) film and the protein wool keratin has been studied by emission and absorption spectroscopy at room temperature. The decay kinetics of the triplet state vary depending on the conditions under which the study is made. The observation of second order kinetics suggests triplet-triplet interactions play a major role in the deactivation of the tryptophan triplet state in PVA films plasticised by the presence of water vapour. For wool keratin in the presence of air the major mechanism appears to be a first order reaction between tryptophan residues and oxygen. The actual effects of moisture and oxygen on the tryptophan triplet state are discussed.     

THE PHOTOCHEMISTRY OF SPECIFIC TRYPTOPHAN RESIDUES IN PROTEINS AS ANALYZED BY THE FLUORESCENT SCANNING OF TRYPTIC PEPTIDE MAPS

Abstract— The fact that most proteins contain several tryptophans hinders the investigation of the photochemistry of a particular indole residue. A method is presented here that can be used to investigate the photochemistry of specific tryptophan residues in proteins. It consists simply of separating the peptides of a proteolytically digested protein by TLC and then scanning the peptides at the fluorescent maximum of tryptophan. The assignment of the resultant peaks to a particular peptide is based on the chromatographic comparison of the scans with peptide maps.
Using this method, the photochemistry of the tryptophan residues in alpha crystallin, a major protein of lenses, was investigated. Under photolytic conditions that mimic the transmission characteristics of the cornea (>293 nm), it was found that there is a differential photolysis of the tryptophan residues in the protein; with Trp-9 in the N-terminal peptide photolyzing at a considerably faster rate than Trp-60. In addition to tryptophan, photolytic losses of tyrosine were assessed by scanning the peptide maps at the tyrosine fluorescent maximum. Only one tyrosine residue photolyzes under these conditions. The differential photolysis of the tryptophan residues is explained in part by the presence of residues in the vicinity of the indole moieties.     

THE PHOTOSENSITIZED OXIDATION OF β-CAROTENE

Abstract— –Products of oxidation of β-carotene, photosensitized by hypericin in acetone, appear to include mutatochrome, aurochrome, and a number of other compounds absorbing in the violet and near u.v. regions of the spectrum. The 5, 6-monoepoxide is not formed in appreciable quantity, though it is the principal product of oxidation of carotene by perphthalate. Mutatochrome, lutein, and zeaxanthin are photooxidized more slowly than β-carotene, and the products do not appear to include 5, 6-epoxides. β-carotene-5, 6-monoepoxide is oxidized to what is probably luteochrome. Quantum yields appear to be low. We found no evidence to support a photochemical contribution to the oxidation of zeaxanthin to violaxanthin, a process known to accompany photosynthesis.     

MULTIPLE EFFECTS OF FLUORESCENT LIGHT ON REPAIR OF ULTRAVIOLET-INDUCED DNA LESIONS IN CULTURED GOLDFISH CELLS

Abstract— It is known that fluorescent light illumination prior to UV irradiation (FL preillumination) of cultured fish cells increases photorepair (PR) ability. In the present study, it was found that FL preillumination also enhanced UV resistance of logarithmically growing cells in the dark. This enhancement of UV resistance differs from induction of PR because it was not suppressed by cycloheximide (CH) and it occurred immediately after FL preillumination. The effects of FL preillumination on repair of UV-induced DNA lesions in the dark were examined by an endonuclease-sensitive site assay to measure the repair of cyclobutyl pyrimidine dimers, and by enzyme-linked immunosorbent assay to quantitate the repair of (6-4) photoproducts. It was found that excision repair ability for (6-4) photoproducts in the genome overall was increased by FL preillumination. Moreover, a decrease in (6-4) photoproducts by FL illumination immediately after UV irradiation of the cells was found, the decrement being enhanced by FL preillumination with or without CH.     

THE CONFORMATIONAL STATUS OF A PROTEIN INFLUENCES THE AEROBIC PHOTOLYSIS OF ITS TRYPTOPHAN RESIDUES: MELITTIN, β- LACTOGLOBULIN and THE CRYSTALLINS

We have studied the aerobic photolysis of the tryptophan residues of the proteins melittin and beta-lactoglobulin when the proteins are in ordered conformations and when they are in randomly coiled states. The results suggest that the conformational status of the protein is a factor that influences the photolysis of the constituent tryptophan residues. This point appears to be of relevance to the photo-oxidation of the tryptophan residues of the eye lens proteins crystallins.     

THE RATES OF PHOTOLYSIS OF THE FOUR INDIVIDUAL TRYPTOPHAN RESIDUES IN UV EXPOSED CALF γ-II CRYSTALLIN

Aqueous buffer solutions of the lens protein bovine gamma-II crystallin were irradiated at 295 nm in the presence of dithiothreitol to determine the individual photolysis susceptibilities of the four tryptophan residues. Reverse-phase high performance liquid chromatography was utilized to compare the tryptic peptide maps before and after irradiation. Sequence analysis of collected tryptic peptides showed that the four tryptophans in calf gamma-II crystallin. TRP-42, TRP-68, TRP-131, and TRP-157 appeared in four distinct tryptic peptides. Fluorescence and absorption (diode array) monitoring of the eluting peptides allowed assessment of the changes in peptide absorbance and fluorescence following irradiation. Tryptophan fluorescence losses of (40 +/- 15)%, (17 +/- 4)%, (35 +/- 5)% and (15 +/- 4)% were observed for the peptides containing TRP-42, TRP-68, TRP-131 and TRP-157, respectively. Thus the four tryptophans in calf gamma-II crystallin did not all photolyze at the same rate. The rate differences are presumably related to the microenvironments of the individual tryptophan residues, and this is discussed in terms of the known crystal structure of calf gamma-II crystallin.     

THE PRESENCE OF PHOTOREACTIVE β-CARBOLINE FLUOROPHORES IN WEATHERED WOOL KERATIN

Abstract Solvent-cleaned Merino wool, which was enzymatically digested with protease K, displayed a fluorescence maximum at 500 nm when excited at 430 nm. The yield of this emission was approximately 15 times greater for a fiber tips digest than for a digest of the mid-length region of the same fibers.
Separation of the components in the wool tip digest by silica gel thin-layer chromatography revealed the presence of several fluorescent species. The chromatographic mobility of these species was similar to the behavior observed in a preparation of authentic 1-methyltetrahydro-P-carboline, 1,3-dicarboxylic acid.
Mass spectroscopy of the fluorescent compounds in the wool tip digest displayed molecular ions, [M + H]⁺ with m/z = 273.0890 and 257.0560, and molecular weights that define the formulae C₁₄H₁₂N₂O₄ and C₁₁H₈O₄N₂, respectively, which correspond to β-carboline 1,3-dicarboxylic acids. Mass spectral evidence also indicates the presence of two other β-carbolines.     

THE ROLE OF PYRIMIDINE DIMERS AND NON-DIMER DAMAGE IN THE INACTIVATION OF ESCHERICHIA COLI BY UV RADIATION

Abstract— We have quantitated the role of pyrimidine dimers and non-dimer damage in the inactivation of Escherichia coli by far-UV radiation, near-UV radiation, and triplet state sensitized near-UV radiation. The extent of photoreactivation in vivo of an excision and postreplication repair-deficient strain of E. coli after the different radiation treatments has been correlated with the relative proportion of pyrimidine dimers and non-dimer lesions produced. Using an excision deficient strain of E. coli, the susceptibility to recA ⁺ -dependent repair of the damage produced by the different radiation treatments has also been quantified.     

PHOTOCHEMICAL INACTIVATION OF SINGLE-STRANDED VIRAL DNA IN THE PRESENCE OF UROCANIC ACID*

Abstract— Urocanic acid (UA) has previously been shown to react photochemically in vitro with N,N-dimethylthymine. In this study, mixtures of UA and phage G4 single-stranded DNA have been irradiated with UV light (λ≥ 254 nm) and the DNA assayed for infectivity. At the concentrations of UA employed (typically 5.4 × 10^-3 M ) there is extensive absorption of the incident light by the UA. The DNA is inactivated at rates greater than that predicted from the calculated shielding by UA, indicating that photosensitization is occurring. Photosensitization is also indicated by the fact that at high UA concentrations the inactivation rate does not decrease to zero but approaches a residual value. Furthermore, the ability to photoreactivate DNA that has been photolyzed in the presence of UA is much reduced relative to that observed upon photolysis of the DNA alone. UA is therefore responsible for the production of UV-induced DNA lesions, which are resistant to photoreactivation.
A general analysis of the effects of photosensitization on the kinetics of UV inactivation is presented in an appendix.     

THE ROLE OF DNA DAMAGE IN PM2 VIRAL INACTIVATION BY METHYLENE BLUE PHOTOSENSITIZATION

Abstract— This study investigates the importance of DNA damage in viral inactivation by phenothiazines and light. Phenothiazines, including methylene blue (MB), toluidine blue and azure B are of particular interest because of their ability to bind to nucleic acids in vitro. Initial studies employing phages T7, MS2 and PM2 indicated that both DNA and RNA phages as well as enveloped and nonenveloped phages can be inactivated by phenothiazine photosensiti-zation. PM2, which contains a lipid-protein bilayer and supercoiled DNA, was used for the mechanistic studies to model blood-borne viruses. Viral DNA damage was assessed following treatment of phage to known levels of viral inactivation by extracting the DNA and analyzing for both direct and piperidine-catalyzed strand cleavage by gel electrophoresis. DNA strand cleavage was found to be both sensitizer concentration and light dose dependent. Both viral inactivation and DNA damage were found to be oxygen-dependent events. In parallel experiments, strand cleavage of isolated PM2 DNA treated with MB and light was also found to be oxygen dependent, in contrast to some previous reports. Transfection studies, which measure the infectivity of the extracted viral DNA, indicated that DNA from MB-treated phage was just as capable of generating progeny virus as the untreated controls. It was therefore concluded that the observed DNA damage is not correlated with loss of phage infectivity.     

A SELF-CONSISTENT-FIELD STUDY OF THE EXCITED STATE π-ELECTRON CHARGES IN PHENOL

Abstract— –The semiempirical self-consistent-field (SCF) method was used to calculate the net π-electron charges for phenol in the first excited singlet and first excited triplet states. These calculations differ from the usual ground state calculations in that (i) recently available static excited state data were used wherever possible as the empirical basis for evaluating SCF parameters and (ii) the theory of density matrices was used to include the effects of all singly-excited configurations in the configuration interaction contributions to the excited state π-electron charge densities.