PORPHYRIN-INDUCED PHOTODAMAGE TO ISOLATED HUMAN NEUTROPHILS

Abstract— Human neutrophils were irradiated with light at 340–380 nm in the presence of low concentrations of protoporphyrin or uroporphyrin. At increasing light doses or increasing concentrations of protoporphyrin, the neutrophils rapidly lost the ability of locomotion. Also, neutrophil chemiluminescence and hexose-monophosphate shunt activity rapidly declined. An early event was leakage of endogenous K⁺ followed by lactate dehydrogenase and at a later stage leakage of particle-bound acid phosphatase. A number of cellular enzymes were inactivated, the susceptibility to inactivation decreased in the order: succinate dehydrogenase > lactate dehydrogenase > glutamate dehydrogenase > acid phosphatase. Uroporphyrin had no effect on neutrophil functions, leakage of K⁺, or cellular enzymes. The results suggest that photodamage to the plasma membrane and the mitochondria are earlier events than photodamage to lysosomes.     

TRYPTOPHAN EMISSION FROM HUMAN HEMOGLOBIN AND ITS ISOLATED SUBUNITS

Abstract— The emission spectra of human adult hemoglobin A₀ and its isolated α and ß subunits were obtained using a highly sensitive photon-counting spectrofluorometer. The quantum yields of the emissions, relative to free tryptophan, were also measured as well as the excitation polarization spectra for hemoglobin A₀ and apohemoglobin. The fluorophore bis-ANS was utilized to probe for the presence of apoproteins in the hemoprotein preparations. The work suggests that tryptophan may be useful as an intrinsic probe to study dynamical processes in hemoglobin.     

SPECTRAL CHARACTERISTICS AND MECHANISM OF CHEMILUMINESCENCE FROM TRYPTOPHAN SOLUTIONS INDUCED BY UV-IRRADIATION

The spectral distribution of the chemiluminescence, fluorescence and phosphorescence of tryptophan aqueous solutions irradiated with high and low pressure mercury lamps has been measured. The blue emission bands in the region of 380–520 nm observed both in the chemi- and photoluminescence, as well as an absorbance increase at 230 and 330 nm, indicate oxidative degradation of tryptophan leading to the formation of derivatives of N-formylkynurenine, xanthurenic and anthranilic acids. Red emission bands at 630 and 705 nm in the spectrum of the chemiluminescence, an enhancement of light intensity by D₂O and its decrease by NaN₃ and DABCO suggest a partial contribution of O₂(¹Δ_g) to the photooxidation and chemiluminescence of tryptophan. The enthalpy of the exergonic reactions, leading to the formation of luminescing products, was calculated to average -270 kJ-mol.     

ISOLATION AND IDENTIFICATION OF TRYPTOPHAN PHOTOPRODUCTS FROM AQUEOUS SOLUTIONS OF TRYPTOPHAN EXPOSED TO NEAR-UV LIGHT*

Abstract—One of the previously unidentified photoproducts isolated from the photolysate of aqueous tryptophan solutions was identified as 2-carboxy-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo(2,3b)-indole by direct comparison with the authentic reference compound synthesized using the established procedure. This pyrroloindole alcohol has been shown to be the reduction product of the 3a-hydroperoxy intermediate (structure 4 in Fig. 1) by Nakagawa et al . (1977). The isolation and identification of this derivative and the detection of the peroxy intermediate 3a-hydroperoxypyrrolidinoindole ( 4 ). from irradiated aqueous tryptophan solutions suggests that the direct photooxidation of l -tryptophan to fromylkynurenine may follow a pathway via a tricyclic intermediate instead of the energetically unfavorable dioxotane intermediate. This scheme is similar to the mechanistic model proposed by Nakagawa et al . (1977) for the rose bengal sensitized photooxidation of tryptophan.     

IRREVERSIBLE BINDING OF CHLORDIAZEPOXIDE TO HUMAN PLASMA PROTEIN INDUCED BY UV-A RADIATION

Abstract— In view of the phototoxicity of chlordiazepoxide (Librium^®) the kinetics of the reaction in the presence of plasma proteins was studied for chlordiazepoxide upon UV-A irradiation and for its oxaziridine in the dark. Two different methods were used to determine the extent of irreversible binding to protein (up to ∼ 50% was found for both situations). Kinetic data support the conclusion that the formation of oxaziridine from photoexcited chlordiazepoxide is the basic event making chlordiazepoxide phototoxic. The half life of oxaziridine is about 30 min even in the presence of a high concentration of plasma proteins, is in agreement with previous in vivo results, showing covalent binding not only to biomolecules of the UV-A irradiated skin but also to those of inner organs, e.g. liver and kidneys.     

RELATIVE CONTRIBUTIONS OF TRYPTOPHAN and TYROSINE TO THE PHOSPHORESCENCE EMISSION OF HUMAN SERUM ALBUMIN AT LOW TEMPERATURES

Abstract— Phosphorescence emission and excitation spectra, as well as decay profiles of human serum albumin, were investigated in the wavelength regions of the tryptophan and tyrosine absorption and emission spectra in potassium phosphate buffer at 77 K. Emission and excitation spectra were found to be linear superpositions of the contributions of the tryptophan and tyrosine residues. It is suggested, therefore, that there is no significant tyrosine to tryptophan energy transfer in this protein at low temperature. The phosphorescence decay is, in general, multiexponential with lifetime components of 5.95, 2.7, and 1.2 s. The longest lifetime is characteristic of tryptophan, whereas the two short components are attributed to two types of tyrosine residues located in different environments within the protein. The latter is confirmed by a detailed analysis of the phosphorescence decay profiles determined at different emission wavelengths, and utilizing different wavelengths of excitation favoring either the tryptophan or tyrosine residues.     

AMIODARONE PHOTOTOXICITY TO HUMAN ERYTHROCYTES AND LYMPHOCYTES

Amiodarone (AD) therapy for cardiac arrhythmia frequently leads to cutaneous phototoxicity. Amiodarone and its metabolite, desethylamiodarone (DEA), photosensitized hemolysis of red blood cells (RBC) and were phototoxic to lymphocytes. Hemolysis photosensitized by 3.3 μ M AD was partially oxygen dependent and was partially quenched 5 m M sodium azide, 50 m M mannitol, superoxide dismutase (251 U/me e ) and catalase (1500 U/m e ), but was unaffected when H₂O was replaced by D₂O. These results suggest that membrane damage may be important in the in vivo phototoxicity to AD, that both oxygen dependent and oxygen independent mechanisms may operate, and that active oxygen species such as O₂ and hydrogen peroxide may be involved. Photohemolysis was more rapid in the presence of DEA than of AD. However, this may be due to the greater fragility of the cell membrane in the presence of DEA. The greater phototoxicity of DEA than AD towards lymphocytes was not due to greater membrane fragility.     

LASER DOPPLER VELOCIMETRY TO QUANTIFY UV-B INDUCED INCREASE IN HUMAN SKIN BLOOD FLOW

Abstract— A laser Doppler velocimeter was used to quantify changes in cutaneous blood flow, in human subjects, after exposure to UV-B radiation. Studies with three subjects showed a peak increase in flow at 6 h post-irradiation. Dose-response studies, on 8 subjects, at 24 h post-irradiation showed that blood flow increased linearly with log UV-B dose over a range of about 0.5 to 5 MED. Comparative studies with pigmented and non-pigmented skins gave a quantitative measure of the photoprotective effect of melanin. The main advantage of the laser Doppler technique is that it allows easy quantification of a vascular response. However, there was no advantage when compared with detection of a visually determined threshold response, viz. a barely perceptible erythema.     

LEVELS OF REDUCED PYRIDINE NUCLEOTIDES AND LENS PHOTODAMAGE

Since most of the known factors that are associated with cataract formation are oxidative in nature, one would expect that a highly reductive environment might arrest or retard the progress of cataract formation. Reduced nucleotides, both NADH and NADPH, are potent reductants with a large negative redox potential of -320 mV. Lenses of certain species contain high levels of these nucleotides, presumably due to the presence of taxon specific crystallins. We have utilized this situation to investigate whether the levels of reduced pyridine nucleotides modulate photo-oxidative damage to the lens. We have monitored the time dependent loss of tryptophan fluorescence upon photodamage for lenses from guinea pig, rabbit and frog (Rana) that contain high levels of pyridine nucleotides and compared with the lenses from rat, Xenopus and a mutant strain of guinea pig that contain significantly lower amounts of these nucleotides. About 75% and 90% of the initial fluorescence intensity is lost in the case of rat and Xenopus lenses, respectively, after a total of 35 min exposure. Rabbit, guinea pig and frog lenses, under identical conditions, show only about 35-40% loss of the initial fluorescence. It appears that the lenses that contain high levels of reduced nucleotides are less susceptible to photodamage. The observed anti-oxidative role of reduced nucleotides in the lenses indicates the possibility of testing reductants (NADPH, NADH and their functional analogues) as potential candidates to therapeutically intervene in the process of cataractogenesis.     

MECHANISMS FOR LUMINOL-AUGMENTED CHEMILUMINESCENCE FROM NEUTROPHILS INDUCED BY LEUKOTRIENE B4 AND N-FORMYL-METHIONYL-LEUCYL-PHENYLALANINE

Neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine (fMLP) or leukotriene B4 (LTB4) generated kinetically distinctive luminol augmented chemiluminescence (LCL). Inhibitors of .O2- [superoxide-dismutase (SOD) or tiron], H2O2 (catalase), myeloperoxidase, MPO, (NaN3), HOCl (taurine) and .OH (mannitol) hampered LCL dose-dependently with similar characteristics for both stimuli. In cell free systems it was found that .O2- (generated in the xanthine/xanthine-oxidase reaction) or H2O2 produced LCL. Superoxide dismutase inhibited .O2- -induced LCL dose dependently. The MPO + H2O2 system, which generated more pronounced LCL than either component alone, was inhibited by catalase and taurine but not by SOD. When neutrophils, treated with luminol, but where extracellular luminol had been removed, were stimulated with fMLP or LTB4, they produced less than 2% of the LCL where luminol was present in the medium. When neutrophil LCL and superoxide formation by the cytochrome C method were assessed in parallel experiments, in all instances the peak LCL response coincided with the linear phase in that response. Thus, LCL, induced by LTB4 and the corresponding fMLP peak, are extracellular events with similar chemical backgrounds, closely related to generation of reactive oxygen species. Consequently, the kinetical differences in LCL between fMLP and LTB4 suggest that LTB4, by yet unknown mechanisms, activates the NADPH oxidase more rapidly than fMLP.     

FLASH PHOTOLYSIS OF AQUEOUS TRYPTOPHAN, ALANYL TRYPTOPHAN AND TRYPTOPHYL ALANINE

Abstract— Tryptophan has been flash photolysed in deoxygenated and air equilibrated solutions. The kinetics of the subsequent reactions of the tryptophyl radical and the hydrated electron have been elucidated. Oxygen has been found to reduce the primary photoionization yield, indicating a triplet state precursor, but no evidence has been found for a biphotonic process. Appreciable differences in the photoionization quantum yields have been found for tryptophan, alanyl tryptophan and tryptophyl alanine.     

TRYPTOPHAN PHOSPHORESCENCE AT ROOM TEMPERATURE AS A TOOL TO STUDY PROTEIN STRUCTURE AND DYNAMICS

Fluorescence and phosphorescence resemble each other and in many ways can give the same type of information. Both originate from a dipolar interaction between light and the molecule. In this regard, both are polarized and subject to the same type of quenching phenomena. In other respects the information which they divulge are complementary. The fluorescence quantum yield is higher for exposed tryptophans and this is expressed in longer lifetime (Grinvald and Steinberg, 1976); in contrast long lifetime of phosphorescence appears to correlate with burial. Phosphorescence, spin-disallowed, is much longer lived than fluorescence. This allows the structural/dynamic characterization of proteins to be studied on a new time regime. A really remarkable finding of studies of protein phosphorescence is that there is such variability both in phosphorescence lifetime and quenchability. We would interpret this to indicate that the tryptophan environment can range from essentially a crystal, almost comparable in rigidity as found at 77 K, to tryptophans in a flexible environment, almost as flexible as free in solution. An interesting task will be to examine the relationship between the yield and lifetime of phosphorescence and details of the tryptophan environment in terms of rigidity and adjacent amino acids among the proteins with known three dimensional structure.     

PHOTOCHEMICAL-LIKE DESTRUCTION OF TRYPTOPHAN IN SERUM ALBUMINS INDUCED BY ENZYME-GENERATED TRIPLET SPECIES

Abstract —Human and bovine serum albumin quench enzyme-generated acetone phosphorescence ( K _sv= ca . 10⁴ M ¹). Concomitantly, these proteins are altered as shown by diminished tryptophan absorption at 280 nm, appearance of products of the formylkynurenine type (_max= ca . 320 nm) and disappearance of tryptophan fluorescence. These alterations—which are similar to those induced photochemically—were also observed with serum albumins exposed to enzyme-generated triplet acetaldehyde. On the other hand, triplet acetone generated by the thermolysis of tetramethyldioxetane failed to induce alterations. Presumably energy transfer occurs from the enzyme-generated triplet species to tryptophan group(s) in the serum albumin associated with the acting enzyme. The detailed mechanism is, however, not yet understood.     

DNA DAMAGE AND REPAIR IN HUMAN CELLS EXPOSED TO SUNLIGHT

Cultured human cells were treated with direct sunlight under conditions which minimised the hypertonic, hyperthermic and fixative effects of solar radiation. Sunlight produced similar levels of DNA strand breaks as equitoxic 254 nm UV in two fibroblast strains and a melanoma cell line, but DNA repair synthesis and inhibition of semiconservative DNA synthesis and of DNA chain elongation were significantly less for sunlight-exposed cells. DNA breaks induced by sunlight were removed more rapidly. Thus, the repair of solar damage differs considerably from 254 nm UV repair. Glass-filtered sunlight (> 320 nm) was not toxic to cells and did not induce repair synthesis but gave a low level of short-lived DNA breaks and some inhibition of DNA chain elongation; thymidine uptake was enhanced. Filtered sunlight slightly enhanced UV-induced repair synthesis and UV toxicity; photoreactivation of UV damage was not found. Attempts to transform human fibroblasts using sunlight, with or without phorbol ester, were unsuccessful.     

PHOTOCHEMICAL REACTIONS BETWEEN TRYPTOPHAN AND POLYNUCLEOTIDES*

Abstract— Anaerobic solutions of ¹⁴C labelled tryptophan and single-stranded synthetic polyribonucleotide form adducts when irradiated with UV light at wavelengths longer than 270 nm. These adducts are stable to cold 10% trichloroacetic acid and are also more resistant to hydrolysis catalyzed by bovine ribonuclease A than non-irradiated controls. All polyribonucleotides reacted with trp to some extent with reactivities in the order poly rU > poly rC = poly rA > poly rG.     

PROFLAVINE-SENSITIZED PHOTOOXIDATION OF TRYPTOPHAN AND RELATED PEPTIDES

Abstract— The photodynamic action of proflavine on the amino acids cystine, rnethionine, tyrosine, histidine, tryptophan and related peptides was tested in anhydrous formic and acetic acids. Only rnethionine and tryptophan were found to be photooxidized: the former, whether free or bound in a polypeptide chain, is quantitatively converted to methioninesulphoxide; the latter, when free, is fragmented to several compounds, which may be divided into two main classes: melanines and kynurenine derivatives. Whereas masking the carboxyl group of tryptophan has no effect on the photooxidation products, N-substituted derivatives of tryptophan are converted in high yields to the corre-sponding kynurenine compounds. The possible applications of the method to biologically active polypeptides are discussed.     

DNA STRAND BREAKS IN MAMMALIAN CELLS EXPOSED TO LIGHT IN THE PRESENCE OF RIBOFLAVIN AND TRYPTOPHAN

Abstract— When mammalian cells were exposed to visible-fluorescent light or near-UV light in the medium containing riboflavin and L-tryptophan, single-strand breaks appeared in their DNA. This did not occur if either riboflavin or tryptophan was omitted from the medium. The same effect was observed when cells were added to the pre-irradiated medium, indicating that a stable photoproduct was responsible. The induced DNA lesions were shown to be equally repairable in both excision proficient and defective (xeroderma pigmentosum) human cell lines. The active photoproduct formed was shown to be hydrogen peroxide. The possible relationship between these results and the near-UV induced killing of mammalian cells is discussed.     

PHOTOCHEMICAL REACTION BETWEEN PROTEIN AND NUCLEIC ACIDS: PHOTOADDITION OF TRYPTOPHAN TO PYRIMIDINE BASES*1

Abstract— The photochemical interactions between tryptophan and nucleic acid bases were studied. When aqueous solutions of tryptophan were irradiated (Λ > 260 nm) at neutral pH in the presence of each of the nucleic acid bases, pyrimidine bases but not purine bases were altered. Air was found to decrease the rate of reaction. Two classes of photoproducts were isolated by thin layer and ion-exchange chromatography and partially characterized. One was the dihydro-pyrimidine forms of the base (see Reeve and Hopkins, 1979) and the other consisted of tryptophan-pyrimidine photoadducts. Four tryptophan-uracil and two tryptophan-thymine adducts each with a 1:1 molecular stochiometry were found. Spectroscopic measurements and a positive reaction with Ehrlich's reagent indicate that the indole nucleus remained intact, but that the pyrimidine base was reduced at the 5, 6 double bond. The absence of a positive ninhydrin reaction and the effect of pH on the quantum yield of the photoadduct formation indicated that the ionized a-amino acid group of tryptophan was involved in photoadduct formation. Indole derivatives lacking an a-amino group were also found to form photoadducts with pyrimidine bases. The experimental results are consistent with a reaction mechanism involving tryptophan excitation to the first excited singlet state as the initial event. Radical scavenging experiments indicate that tryptophan free radicals, formed by electron dissociation from the excited state, react with the ground state pyrimidine.     

ACRYLAMIDE QUENCHING OF TRYPTOPHAN PHOTOCHEMISTRY AND PHOTOPHYSICS

Studies of acrylamide quenching of tryptophan (Trp) fluorescence, photochemistry, and photoionization have been conducted. Quenching of Trp fluorescence in aqueous solution by addition of acrylamide in the concentration range 0.0-0.5 M was measured and resulted in a Stern-Volmer quenching constant of KSV = 21 +/- 3 M-1. Photolysis experiments were performed in which Trp was photolyzed at 295 nm in the presence of varying concentrations of acrylamide. The loss of Trp was monitored using reverse-phase high performance liquid chromatography (RP-HPLC) and was observed to follow first order kinetics. Production of N-formylkynurenine (NFK) was observed by RP-HPLC in irradiated Trp samples both in the presence and absence of added acrylamide. In addition, no new photochemical product was detected. This was taken as evidence that acrylamide did not alter the photochemical pathway but just reduced the reaction rate as expected for a physical quenching mechanism. Plotting the reciprocal of photolysis rate constant versus acrylamide concentration produced a Stern-Volmer constant for quenching of Trp photochemistry of KSV = 6 +/- 2 M-1. The KSV values for both fluorescence quenching and photolysis quenching were thus large, implying efficient quenching of both processes by acrylamide. Assuming an excited singlet state lifetime of 2.8 ns, the calculated second-order quenching rate constants for fluorescence and photolysis were kq = 7.5 x 10(9) and 2.1 x 10(9) M-1 s-1 respectively. The possible involvement of photoionization in the photolysis mechanism was investigated by studies of acrylamide quenching of voltage transients produced by xenon flash lamp excitation of Trp at aqueous/teflon or aqueous/mica interfaces.(ABSTRACT TRUNCATED AT 250 WORDS)