THE IN VITRO PHOTOCHEMISTRY OF BIOLOGICAL MOLECULES-III. ABSORPTION SPECTRA, LIFETIMES AND RATES OF OXYGEN QUENCHING OF THE TRIPLET STATES OF β-CAROTENE, RETINAL AND RELATED POLYENES

Abstract— The triplet-triplet absorption spectra of six polyenes have been characterised using flash photolysis, in the presence of anthracene as sensitizer, and pulse radiolysis, in the absence of a sensitizer. The polyenes include several which contain carbonyl groups whose triplet states, unlike retinal , could not be detected unsensitized by flash photolysis. The triplet lifetimes appear to be a function of the number of conjugated double bonds, and vary between 7 and 14 μ sec. In general, the longer the polyene, the shorter the lifetime. An empirical linear relation was found between the frequencies of the polyene triplet-triplet absorption maxima, and the frequencies of the corresponding ground singlet-singlet maxima. The rate constants for quenching by oxygen of nine polyene triplet states were determined to lie in the range 2–7 × 10⁹ M ^-1 sec^-1. The possible mechanisms for oxygen quenching of triplet states are discussed and analogies between the results for oxygen quenching of polyenes and of polyacenes are drawn. The rate constant for oxygen quenching of all- trans -β-carotene triplet was the same in benzene and hexane.     

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.     

THE IN VITRO PHOTOCHEMISTRY OF BIOLOGICAL MOLECULES—II. THE TRIPLET STATES OF β-CAROTENE AND LYCOPENE EXCITED BY PULSE RADIOLYSIS

Abstract— –Pulse radiolysis has been used to excite the triplet states of β-carotene (τ# 9μ sec) and lycopene (τ= 8μsec) in hexane solution, both in the presence and absence of naphthalene as a triplet sensitiser. The absorption spectra of both triplets have been measured in the range 430–550 nm and have thus been extended into the region of the corresponding singlet absorptions. The overlap of the triplet and singlet spectra is discussed in relation to in vivo studies. Extinction coefficients of 1.3±0.1 × 10⁵ l/mole cm for β-carotene triplet 515 nm and 3.9±0.2 × 10⁵ l/mole cm for lycopene triplet at 525 nm were obtained. Isomerisation of the all- trans polyenes used was detected and preliminary measurements indicate that the yield of isomerisation was greater than the triplet yield. The rate of triplet energy transfer from naphthalene to β-carotene was estimated to be 1.5 × 10¹⁰ l/mole sec. The corresponding value for lycopene was 1.4× 10¹⁰ l/mole sec. The measured efficient quenching of triplet β-carotene by oxygen may occur by an energy transfer mechanism, leading to the formation of singlet oxygen (¹Δ_g. This would suggest that the triplet energy level of β-carotene lies between 121 and 94 kJ mole^-1.     

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.     

PHOTOELECTRON QUANTUM YIELD SPECTRUM AND PHOTOELECTRON MICROSCOPY OF β-CAROTENE

Abstract—The absolute photoelectron quantum yield spectrum for β-carotene in the wavelength range 180–230 nm is reported. β-Carotene is very photoemissive over this wavelength region with photoelectron quantum yields on the order of 2 × 10^--³ electrons per incident photon at 180 nm, 4 × 10^--⁴ at 210 nm, and 3 × 10^--⁵ at 230 nm. At wavelengths longer than 240 nm, β-carotene photoemission dropped off monotonically with increasing wavelength. The photoelectron quantum yield spectrum of β-carotene is very similar to that of chlorophyll a . A photoelectron micrograph of β-carotene deposited on a thin layer of the fatty acid arachidic acid demonstrates the marked photoemission contrast between β-carotene and membrane lipid. Photoelectron micrographs of samples with β-carotene and Chl a in the same field show that prolonged (1 h) illumination in vacuo causes β-carotene to markedly fade while leaving the Chl a photoemissive. This differential bleaching of β-carotene may allow it to be distinguished from Chl in high magnification photoelectron micrographs of photosynthetic membranes.     

SENSITIZED TRIPLET FORMATION OF CHLOROPHYLL-A AND ß-CAROTENE

The naphthalene-sensitized formation of triplet excited chlorophyll-a (Chl-a) and all-transß-carotene has been studied by pulse radiolysis. The rate constants for transfer of triplet energy from naphthalene to Chl-a and all-transß-carotene in benzene at 25°C are (3.6 ± 0.6)·10⁹M^-1 s^-1 and (10.7 ± 1.2)·10⁹M^-1 s^-1, respectively. The decays of the excited triplet states of naphthalene, Chl-a and all-transß-carotene all follow a mixed first-and second-order mechanism. The first-order rate constant for triplet decay is strongly dose dependent for naphthalene but only slightly dependent and independent of dose for Chl-a and all-transß-carotene, respectively. The rate constants for triplet-triplet annihilation are (1.4 ± 0.3)·10⁹M^-1 s^-1 for Chl-a and (3.6 ± 0.4)·10⁹M^-1 s^-1 for all-transß carotene. The nearly constant ratio k(ß-carotene)/k(Chl-a) for the bimolecular triplet energy transfer rate constants is discussed in terms of the molecular shapes of the two molecules. The energetics of the triplet-triplet annihilation of all-transß-carotene are discussed, and it is proposed that production of the excited ¹A_B state may be a major route in the annihilation process.     

ACTION SPECTRA FOR BIOSYNTHESIS OF CHLOROPHYLLS a AND b AND β-CAROTENE

Abstract— Action spectra for the formation of chlorophyll b and β-carotene were determined with etiolated wheat leaves and compared with the action spectrum for the formation of chlorophyll a determined for the same samples. The action spectra were measured with etiolated leaves which had been pre-illuminated for 10 min and incubated in the dark for 4 h to eliminate induction of pigments. The action spectra for chlorophyll b and for β-carotene accorded with the action spectrum for chlorophyll a and with the absorption spectrum of protochlorophyllide in intact etiolated leaves. It is postulated from this result with chlorophyll b that this pigment is formed from protochlorophyllide through chlorophyll a or some intermediates to chlorophyll a. Complexing between chlorophylls and β-carotene and proteins is postulated to interpret the action spectrum for β-carotene. It is assumed that the low concentration of chlorophylls formed photochemically limits the rate of complexing, and that consumption of β-carotene for the complexing induces formation of new β-carotene.     

PHOTOTROPISM IN PHYCOMYCES MUTANTS LACKING β-CAROTENE

Abstract. β-carotene and riboflavin are considered as the major candidates for the photoreceptor for physiological responses to blue light in Phycomyces and a number of other organisms. Mutants of Phycomyces blocked in all six steps of the biosynthesis of β-carotene from phytoene contain no detectable β-carotene (less than 4 times 10^-5 of wild-type amount) but exhibit phototropic responses identical to wild-type. Moreover, wild-type Phycomyces , while abundant in trans -β-carotene, contains no detectable cis -β-carotene, sometimes proposed as a photoreceptor candidate on the basis of the close similarity of the cis -species absorption spectrum in the near UV region to many action spectra for blue-light responses. These results indicate that β-carotene cannot be the photoreceptor for phototropism in Phycomyces.     

PHOTOOXIDATION OF A REFINED PETROLEUM OIL: INHIBITION BY β-CAROTENE AND ROLE OF SINGLET OXYGEN

Abstract— β-Carotene, at concentrations 0.1 m M , inhibits the formation of hydroperoxides and other oxidation products in a refined petroleum oil exposed to Pyrex-filtered UV. The effect appears to be due to ¹O₂ quenching. A mechanism incorporating ¹O₂ and radical processes is proposed as a model for environmental photooxidation of petroleum.     

PHOTOINITIATION OF SPORANGIOPHORES IN PHYCOMYCES MUTANTS DEFICIENT IN PHOTOTROPISM AND IN MUTANTS LACKING β-CAROTENE

Abstract—The formation of sporangiophores from mature Phycomyces mycelium is inhibited in a closed system. Irradiation of the mycelium with blue light reverses the inhibition of spordngiophore formation. Dose response curves for this reaction are established for wild type. β-caroteneless mutants and for mutants that are deficient in phototropism.
Phototropic-negative mutants. altered in genes madA and madB , have a raised threshold in this light reaction. whereas mutants deficient in genes madD to madG are unaffected. β-caroteneless mutants deficient in genes carA, carB , or carR have a threshold raised by a factor of 100–2000 depending on the amount of residual synthesis of β-carotene.     

THE TRIPLET STATE OF 8-METHOXYPSORALEN

Abstract— Transient absorption spectra produced by laser flash photolysis of an aqueous solution of 8-methoxypsoralen (8-MOP) have been studied. The biphotonic production of hydrated electrons and of the radical ions, 8-MOP ⁺ and 8-MOP^- is reported. The hydrated electron was found to react with ground state 8-MOP with k ˜ 3 × 10¹⁰ M ^-1 s^-1. In order to obtain a true triplet-triplet absorption spectrum. contributions from the radical ions were subtracted from the overall transient absorption. In addition, contributions from e^-_aq to the transient spectrum were removed by using N₂O, low laser intensity to minimize photoionization or by measuring the transient O.D. after the electron has decayed. These three methods each produced the same triplet-triplet spectrum which differs in the red region from previously reported spectra.     

ROLE OF THE TRYPTOPHAN FLUORESCENT STATE IN THE ULTRAVIOLET-INDUCED INACTIVATION OF β-TRYPSIN*

Abstract— Stern-Volmer quenching constants for β-trypsin at pH 3 were determined for fluorescence quenching by histidine, acrylamide, and nitrate ion. A modified Stern-Volmer plot (Lehrer, 1971) was employed to show that all of the fluorescent tryptophanyl residues of β-trypsin were equally susceptible to quenching by acrylamide at pH 3 when the enzyme was either in its native conformation or denatured in 6 M guanidine hydrochloride (GuHCl). Fluorescence lifetime measurements indicated that acrylamide quenched β-trypsin fluorescence by a purely collisional mechanism. Solvation of tryptophanyl residues of the protein was maximal at 2.5 M GuHCl, as monitored by fluorescence emission wavelength.
Investigations of the ultraviolet-induced inactivation of β-trypsin at 295 nm were performed in the presence of acrylamide at pH 3. The quantum yields for enzyme inactivation and indole destruction (determined using the PDAB reagent) were unchanged upon depopulation of the fluorescent state by 65 per cent, whether the enzyme was in its native conformation or denatured by 6 M GuHCl. It is concluded that the fluorescent state of tryptophanyl residues of β-trypsin is not involved in enzyme inactivation or tryptophan destruction.     

EFFECT OF DIETARY FAT LEVEL ON UV-B INDUCED SKIN TUMORS, AND ANTI-TUMOR ACTION OF β-CAROTENE

Abstract —Hairless mice (SKH-hr strain) maintained on diets containing either 4.5 or 14.1% fat were exposed to UV-B irradiation for a 16-week period and tumor incidence was evaluated. The animals on the low fat diet developed tumors significantly more slowly and in smaller numbers than those on the high fat diet. In addition, a group of animals on a high fat diet (11.1%) were supplemented with β-carotene in their food at a final level of 3%. At the end of the experiment there was no difference in the prevalence of tumors in the β-carotene group, although these animals developed significantly fewer tumors per mouse than the control animals receiving 14.1% fat. These results would indicate that the fat level of the diet can decrease or eliminate the ability of β-carotene to protect animals against UV-B induced skin tumors.     

PHOTOREDUCTION OF SOME NITROBIPHENYL ETHER HERBICIDES TO NITRO RADICAL ANIONS BY β-CAROTENE AND RELATED COMPOUNDS

Abstract —The nitrobiphenyl ether herbicides; 4-nitrobiphenyl ether, bifenox, nitrofen, acifluorfen, acifluorfen-methyl, acifluorfen-ethyl, and oxyfluorfen were reduced to their corresponding nitro radical anions by visible light in the presence of β-carotene, lycopene, retinol, retinal, retinoic acid and retinyl acetate in anaerobic solutions at high pH. It was more difficult to obtain spectra of bifenox, nitrofen and oxyfluorfen than acifluorfen derivatives, probably due to their poor solubilities. In neutral solutions the steady-state concentration of the nitro radical anions was low due to their faster rate of dispropor-tionation and the poor solubility of β-carotene. In the presence of retinal, the nitro radical anion of acifluorfen was produced at pH 7.4. Compounds containing conjugated double bonds such as crotonaldehyde and 2.4-hexadienal also reduced acifluorfen and its derivatives to their respective nitro radical anions in the presence of light. Ubiquinone-50 which does not contain conjugated double bonds in the side chain did not reduce acifluorfen under similar conditions.     

THE ANAEROBIC PHOTOLYSIS OF LENS α-CRYSTALLIN: EVIDENCE FOR TRIPLET STATE MEDIATED PHOTODAMAGE

Anaerobic solutions of lens alpha-crystallin were subjected to near-UV (greater than 295 nm) irradiation, and the photoproducts were analyzed by fluorescence and room-temperature phosphorescence spectroscopy. The principal photoproduct was excited maximally at 340 nm, fluoresced maximally at 430 nm, and phosphoresced with an emission maximum at 510 nm. The phosphorescence intensity decay of this species was well fit by a sum of two exponentials with lifetimes of 9.2 ms (78%) and 61 ms (22%); this report is the first demonstration of a long-lived triplet state associated with a protein photolysis product. As reported previously, 3trp* is also long-lived in deoxygenated alpha-crystallin solution at room-temperature (Berger and Vanderkooi, 1989, Biochemistry 28, 5501-5508), hence both tryptophan and photoproduct triplet states are good candidates to mediate photodamage. Photolysis experiments in the presence of agents known to alter the tryptophan triplet yield provide evidence for the importance of triplet-state-mediated photodamage of lens crystallins in anaerobic solution. In 30 mM acrylamide where 3trp*, but not 1trp*, is efficiently quenched, anaerobic solutions exhibited marked resistance to protein photodamage, whereas the photoprotection in aerobic solution was minimal. In D2O, where photoionization is suppressed but triplet states are longer-lived, photodamage was accelerated in anaerobic solution but reduced in aerobic solutions. Finally, the anaerobic photodestruction rate was increased in 500 mM Cs+ solution where the triplet yield is increased by a heavy atom effect.     

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.