KINETICS OF 4-THIOURIDYLATE-SENSITIZED PHOTOINACTIVATION OF RIBONUCLEASE A

Abstract —An action spectrum for the inactivation of ribonuclease A photosensitized with 4-thiouridylic acid follows the shape of the absorption spectrum of an RNase-thiouridylate complex or of the unbound nucleotide. The quantum yield is 3.3 times 10^-3. Statistical analysis of the data suggests that only the bound nucleotide participates in the photoinactivation. An Arrhenius plot for the first-order rate constants of inactivation gives a straight line in the range 0–40C and an apparent activation energy of photoinactivation of 5.7 kcal/mol. The 4-thiouridylate-sensitized photoinactivation of ribonuclease A is regarded as a kind of photodynamic action in which the substrate analogue plays the role of photosensitizer.     

LIMITED CELL-CYCLE DEPENDENCE OF THE MEROCYANINE 540-SENSITIZED PHOTOINACTIVATION OF L1210 LEUKEMIA CELLS

L1210 leukemia cells were synchronized by a double thymidine block technique and then characterized with regard to their susceptibility to merocyanine 540 (MC540)-sensitized photoinactivation. Cells harvested 5 (G2/M phase) h after release from the second thymidine block were most susceptible to MC540-sensitized photoinactivation followed, in order of decreasing sensitivity, by cells harvested 2 (S phase) h and by cells harvested 7 (G1 phase) h after release from the second block. The expression of dye-binding sites changed very little during the cell cycle.     

DIFFERENTIAL INACTIVATION OF SURROGATE VIRUSES WITH MEROCYANINE 540

Bacteriophages may be useful as surrogates for animal viruses when the virucidal properties of different photosensitizing compounds are initially investigated. We studied photoinactivation of four bacteriophages, phi X174, T7, PRD1, and phi 6, by the dye merocyanine 540 (MC540) (15 micrograms/mL). Merocyanine 540 (MC540) should be most effective with lipid-containing viruses, since it is primarily lipophilic (but also binds to proteins). Two of the phages, PRD1 and phi 6 contain lipid, with only phi 6 having an external lipoprotein envelope. Filtered radiation (450-600 nm) from a 750 W projector was used at 16-100 W/m2. The survival curves of the different viruses clearly demonstrated different levels of sensitivity to photoinactivation by MC540, with phi 6 (Do = 1.5 kJ/m2) being the most sensitive, followed by T7 (21-fold less sensitive). While both PRD1 and phi 6 have lipid components, only phi 6 was photoinactivated by MC540. Thus the internal lipid components of PRD1 were not sufficient to allow photoinactivation by this dye, at fluences up to 300 kJ/m2. For comparison, we also photoinactivated Herpes simplex virus (Do = 0.053 kJ/m2) and found it to be 28-fold more sensitive than phi 6 to photoinactivation by the same concentration of MC540. Thus phi 6 may be used as a surrogate for enveloped human viruses for photoinactivation by lipophilic dyes, but the results may only be useful qualitatively.     

PHOTOINACTIVATION OF ENZYMES BY LINEAR AND ANGULAR FUROCOUMARINS

Abstract Furocoumarins with linear (psoralen, 8-methylpsoralen, 8-methoxypsoralen and 3-carbethox-ypsoralen) and angular molecular structures (angelicin and 4,5'-dimethylangelicin) were found to inactivate enzymes to different extents through UV-A irradiation. Moreover, enzymes with different structures (glutamate dehydrogenase, lysozyme, 6-phosphogluconate dehydrogenase, enolase, thermoly-sine and ribonuclease) are inactivated to different extents by the same furocoumarin. UV-A irradiation produces both covalent incorporation of the furocoumarins into the protein molecule and photodegra-dation of amino acid residues; the latter phenomenon seems to be mainly responsible for the photoinactivation process. A close correlation was found between the capacity of the furocoumarins to photoinactivate enzymes and their capacity to modify free amino acids.
A study of the effects of quenchers of various forms of activated oxygen on the photoinactivation of glutamate dehydrogenase, used as a model enzyme, and psoralen and 8-methylpsoralen as a reference for furocoumarins, showed that singlet oxygen is the species most involved in the photoinactivation process.     

SENSITIVITY OF THE ELECTRON TRANSPORT CHAIN OF PIGMENTED AND NON-PIGMENTED SARCINA MEMBRANES TO PHOTODYNAMIC ACTION

Abstract— With malate as substrate, the respiratory system of Sarcina lutea is subject to photodynamic action when toluidine blue is added as a photosensitiser. Both whole cells and isolated cell membranes show similar photoinactivation. Cell and membrane suspensions of a carotenoid-containing strain are significantly less sensitive than those from a non-pigmented mutant prepared from the pigmented strain. Addition of histidine also provides photoprotection. Attempts to locate the site of photoinactivation within the respiratory system lead to the conclusion that several sites are affected. Short periods of illumination do not result in permanent damage since activity is recovered in these suspensions when incubated in the dark subsequent to illumination. The significance of these results and the possible mechanisms involved are discussed.     

THE MECHANISM OF THE FLAVIN SENSITIZED PHOTODESTRUCTION OF INDOLEACETIC ACID*

Abstract— A detailed in vitro study was made of the flavin sensitized photoinactivation of indoleacetic acid, using primarily riboflavin as sensitizer. The dependence of the quantum yield on reactant concentrations, pH, presence of oxygen, viscosity, temperature, KI concentration, and solvent was determined. The involvement of a limiting dark reaction was demonstrated, using an intermittent light technique. The results are consistent with a mechanism involving a metastable state of riboflavin as the photochemically reactive species. The calculated rate constant for intersystem crossing to this state was found to be 2.5 times 10⁸/sec. Riboflavin, in the metastable state, is believed to oxidize indoleacetic acid to indolealdehyde, with subsequent recovery of riboflavin by autoxidation. The maximum quantum yield of the photoinactivation of IAA is 0.71, indicating a highly efficient process, approaching 100% when energy loss due to riboflavin fluorescence is taken into account. Both carotenoids and pure chlorophyll- a were found to be inactive as sensitizers.     

COMPARATIVE KINETICS OF HEMATOPORPHYRIN DERIVATIVE UPTAKE AND SUSCEPTIBILITY OF Bacillus subtilis AND Streptococcus faecalis TO PHOTODYNAMIC ACTION

Hematoporphyrin derivative (HpD) is widely used in photoradiation therapy of tumors and other diseases, and has been shown to affect the viability of gram positive bacteria. This investigation assessed the efficiency of binding of HpD to Bacillus subtilis and Streptococcus faecalis when HpD-treated organisms were exposed to red light. Kinetic studies indicated that the amount of HpD bound increased with increasing external concentration of HpD until saturation of binding sites was reached. S. faecalis had a higher affinity for HpD and was more susceptible to photoinactivation than B. subtilis. The data from this study suggest that differences in susceptibility of microorganisms to photoinactivation are directly related to the affinity of each strain for HpD.     

PHOTODYNAMIC EFFECTS OF PROFLAVINE ON BACTERIOPHAGE φ times 174 AND ITS ISOLATED DNA

Abstract. Proflavine-mediated photoinactivation of φ times 174 phage and its isolated DNA was studied under identical irradiation conditions. The inactivations followed single-hit kinetics and a linear relationship was obtained in reciprocal plots of the inactivation rates vs the proflavine concentrations for both phage and isolated DNA. The phage photoinactivation rate was increased with an increase in the amount of proflavine bound to the phage DNA in a strong binding range (0.01-0.04 proflavine/ nucleotide) as the total proflavine concentration was increased or the ionic strength decreased. Further, a phage-specific factor was also found to affect the inactivation rate. The photodynamic treatment induced mutations in three phage strains from "amber" to "wild type" at a mutation rate per lethal hit of 0.3 times 10^-5 to 2.6 times 10^-5. In contrast to phage infectivity, the φ times 174 DNA infectivity was measurable only at a high multiplicity of infection, and its photoinactivation occurred only at high proflavine concentrations. The photoinactivation rate was enhanced either with a decrease in the multiplicity of infection or with the use of spheroplasts of recA mutants strains. The results are discussed in terms of the nature of and possible repair mechanisms of photodynamically induced lesions in φ times 174 phage DNA.     

PHOTODYNAMIC INACTIVATION OF YEAST CELLS SENSITIZED BY HEMATOPORPHYRIN

Abstract— Yeast cells are inactivated by treatment with hematoporphyrin and light. The inactivation is mainly mediated by singlet oxygen. The quantum yield of singlet oxygen increases with increasing pH, while the efficiency of cellular inactivation decreases with increasing pH. Cells in the stationary phase are much more resistant to the treatment than cells in exponential growth. Membrane damage seems to be the main determining step in the photoinactivation.     

MEROCYANINE 540-SENSITIZED PHOTOINACTIVATION OF LEUKEMIA CELLS: EFFECTS OF DOSE FRACTIONATION

The differential sensitivity to merocyanine 540 (MC540)-sensitized photoirradiation of leukemia cells, selected solid tumor cells, and normal pluripotent hematopoietic stem cells has been successfully exploited for the extracorporeal purging of simulated autologous remission bone marrow grafts. In this communication, we compare the effects of fractionated vs continuous irradiation upon the MC540-sensitized photoinactivation of L1210 and K562 leukemia cells. Exposure to MC540 (15 micrograms/mL) and fractionated doses of white light inactivated fewer in vitro clonogenic cells than exposure to an equivalent dose of continuous irradiation, provided the irradiation doses were small (8.1-16.2 kJ/m2) and spaced 1-2 h apart. The dye-sensitized photoinactivation of leukemia cells was enhanced when cells were stored at 4 degrees C instead of 37 degrees C between irradiation periods, most likely in part because the cells were unable to repair sublethal photodynamic damages at the lower temperature. These data suggest that cells can recover from sublethal damage inflicted by the plasma membrane-active photosensitizer, MC540.     

SENSITIZED PHOTOINACTIVATION OF BACTERIOPHAGE T4

Abstract— The photoinactivation of an osmotic shock resistant mutant of the bacteriophage T 4 can be sensitized by two cationic derivatives of acetophenone. At least part of the sensitized inactivation appears to be due to the formation of thymine dimers in phage DNA by a mechanism which involves triplet excitation transfer from the sensitizer to thymine in the DNA. The photoreactivable sector of the phage inactivated by sensitized irradiation is about 0.6, almost twice as large as the sector for u.v. irradiated phage.     

KINETICS OF SIMIAN VIRUS 40 AND LAMBDA INACTIVATION BY PHOTOADDITION OF PSORALEN DERIVATIVES

Abstract The kinetics of psora/en photoinactivation of two distinct DNA viruses, bacteriophage λ and the papovavirus SV40 were investigated. When λ is treated with near ultraviolet light (UVA, 320-400 nm) and 4,5',8-trimethylpsoralen (TMP) at 1 μg/m/, the phage is rapidly inactivated. The survival curve exhibits a distinct shoulder indicating second or higher-order kinetics. SV40, on the other hand, is much more resistant to psoralen photoinactivation and the survival curve is linear, reflecting first order or'pseudo-first order'kinetics. Two TMP derivatives with increased solubility in aqueous solutions, 4'-aminomethyl-TMP and 4'-hydroxymethyl-TMP, were similarly tested. In both virus systems, TMP was much more effective. In experiments designed to examine the role of psoralen cross-link formation in virus inactivation, treated samples were irradiated a second time in the absence of drug. Since reirradiation causes a decline in λ infectivity as great as that observed in continuously irradiated samples, cross-links are implicated as the primary lethal event. In the case of SV40, the results of such a protocol suggest that both monoadducts and cross-links may be lethal or that monoadduct formation may be rate-limiting.     

Inactivation of iron responsive element-binding capacity and aconitase function of iron regulatory protein-1 of skin cells by ultraviolet A

The ultraviolet-A (UVA) component of sunlight produces in cutaneous cells a highly toxic oxidative stress mediated by redox cycling reactions of Fe ions. A tight regulation of cell iron uptake and storage by iron regulatory proteins (IRP) of keratinocytes and fibroblasts avoids these damaging reactions. We report here that about 40 J/cm2 of UVA are required to inactivate half of the binding capacity of apo-IRP-1 to iron responsive elements (IRE) of RNA whereas 15 J/cm2 already inhibit half of the holo-IRP-1 aconitase activity. No increase in the holo-IRP-1 activity is observed during the apo-IRP-1 photoinactivation suggesting that UVA does not trigger a shift between these two forms. As opposed to holo-IRP-1, which contains a 4Fe-4S cluster, apo-IRP-1 has no UVA chromophore. Thus it should be inactivated indirectly by reactive oxygen species generated by the UVA-induced endogenous photo-oxidative stress. The apo-IRP-1 photoinactivation is weakly prevented by the lipophilic oxyradical scavenger vitamin E but not by the hydrophilic azide anion, a singlet oxygen quencher or by diethyldithiocarbamate, a superoxide dismutase inhibitor. However, full protection against photoinactivation of the apo form is observed after incubation with N-acetylcysteine but the latter only partially protects the aconitase function of the holo-IRP-1 from photoinactivation. The marked difference in the kinetics of photoinactivation of the apo and holo forms, the light dose-independent effect of the sulfhydril group reagent, 2-mercaptoethanol and the partial protection brought by the ferric ion complexing agent desferrioxamine suggest that the photochemistry of the 4Fe-4S cluster of the holo form plays little, if any, role in the photoinactivation of the apo-IRP-1/IRE interaction. It is concluded that the apo/holo equilibrium is irreversibly destroyed by UVA irradiation.     

COMPARISON OF THE EFFECTS OF ULTRAVIOLET RADIATION ON THE INTRACELLULAR CATALASE OF AEROBIC AND ANAEROBIC YEAST

Abstract— Suspensioris of aerobic and anaerobic yeast were subjected to ultraviolet radiation (principally 254 mµ ) under closely comparable experimental conditions, and changes in the level and in the temperature dependence of their catalase activity were determined. Qualita tively, the effects of U.V. on the enzyme of the anaerobic cells were similar to those on that of the aerobic cells. The effect of U.V. on the anaerobic catalase differed from that on the aerobic enzyme in the following respects: I, a considerably greater dose of U.V. was necessary in order to attain the maximum activity and the minimum activation energy of the enzyme-substrate system; 2, a far greater dose was required before appreciable photoinactivation of the maxi mally active enzyme occurred; 3, photoinactivation proceeded at less than one-half the rate; 4, the u.v.-induced increase in the catalase activity of the suspension was virtually complete before appreciable reduction in activation energy occured. The first three of these differences were interpreted in terms of a model, which pictures the anaerohic catalase as being tightly bound to an intracellular chromophore group.     

Action spectrum and quantum yield for the photoinactivation of mnemiopsin, a bioluminescent photoprotein from the Ctenophore mnemiopsis SP.

Abstract— Ctenophores are bioluminescent marine invertebrates closely related to the coelenterates. The isolated bioluminescent systems of the ctenophores Mnemiopsis and Beroë and the hydrozoan jellyfish Aequorea are protein-luciferin complexes (photoproteins) which flash upon the addition of Ca²⁺ ions. The photoprotein mnemiopsin has an oxygen-independent quantum yield for photoinactivation of bioluminescence as high as 0.5, placing it among the most light-sensitive proteins known. We have measured the action spectrum for this photoinactivation at 107 narrow (3.4 nm) wavelength bands between 230 nm and 570 nm, covering a range of four decade units in the action. The action spectrum in the visible region is identical with the absorption spectrum of native photoprotein, implicating bound luciferin. The UV action spectrum implies that absorption by aromatic amino acid residues also leads to extremely efficient photoinactivation. Although photoinactivation is a rapid first-order reaction, destruction of the luciferin is a slower, multiple-order process. Therefore, protein-bound luciferin is not the ultimate target of the photoinactivation. Absorption of light results in the dissociation of “active oxygen” from the photoprotein. Therefore, the ctenophore photoprotein is a precharged enzyme already containing bound luciferin and oxygen.     

COMPARISON OF THE PHOTOTOXICITY OF α-TERTHIENYL WITH THAT OF A SELENIUM AND OF AN OXYGEN ANALOGUE

Abstract— Two analogues of α-terthienyl, namely 2, 5di(2'-thienyl) selenophene and 2, 5di(2'-thienyl) furan have been prepared and their phototoxicities toward several microorganisms have been compared. Dose response studies with Escherichia coli indicate that α-terthienyl is more active than these analogues. α-Terthienyl was shown to be the most effective of the three compounds in the photoinactivation of yeast alcohol dehydrogenase. Diagnostic tests showed the participation of singlet oxygen in the photosensitization to different extents by these three thiophenes.     

PHOTOINACTIVATION OF CHINESE HAMSTER CELLS BY HEMATOPORPHYRIN DERIVATIVE AND RED LIGHT

Abstract— The use of hematoporphyrin derivative (HpD) has previously been demonstrated to be beneficial in clinical cancer therapy. This paper describes cell culture studies used to examine HpD phototherapy in Chinese hamster ovary cells (line CHO). Survival curves have been obtained for both direct HpD toxicity and HpD induced photoinactivation. Examination of HpD induced photoinactivation as a function of stage in the cell growth cycle has also been performed, as has the quantitative measurement of HpD uptake in cells (using ³H-HpD) as a function of cellular incubation time, serum concentration in the incubation medium, and cell cycle position. In the absence of light, no toxicity was observed for HpD incubation levels of up to 400 μg/m/ when incubations times were 3 h or less. Exposure of cells to light alone (> 590 nm, 4.0 mW/cm²) for 9 min was also found to be completely nontoxic. Survival curves obtained for exponentially growing cells labeled with various concentrations of HpD and subsequently illuminated with red light exhibited a threshold or shoulder region at short exposure times followed by exponential killing at longer exposure times. The cell cycle response curves for HpD induced photoinactivation of synchronized CHO cells was nearly flat, indicating no variation in sensitivity for cells treated at time periods from 6 to 15 h after mitosis. Additon of serum to the incubation medium resulted in improved plating efficiency and reproducible survival curves but decreased cellular uptake of HpD.     

CYTOPROTECTION AGAINST MEROCYANINE 540-SENSITIZED PHOTOINACTIVATION OF THE Na+,K+-ADENOSINE TRIPHOSPHATASE IN LEUKEMIA CELLS: GLUTATHIONE AND SELENOPEROXIDASE INVOLVEMENT

When irradiated with broad-band visible light in the presence of merocyanine 540 (MC540), murine leukemia L1210 cells grown under selenium-deficient conditions (Se(-) cells) accumulated lipid hydroperoxides and lost viability more rapidly than selenium-satisfied controls (Se(+) cells). These findings suggest that cytoprotection against photoperoxidation and photokilling is mediated at least in part by selenoperoxidase (SePX) action. Similar protection against photoinactivation of an intrinsic membrane enzyme, the Na⁺,K⁺-ATPase, has been observed. Thus, irradiation of MC540-sensitized Se(-) cells resulted in an immediate and progressive inactivation of ouabain-sensitive Na⁺, K⁺-ATPase; by contrast, activity loss in Se(+) cells was preceded by a prominent lag. Enzyme photoinactivation in Se(-) cells was inhibited by ebselen, an SePX mimetic, confirming that SePX(s) is (are) involved in natural protection. Desferrioxamine treatment (iron sequestration/inactivation) resulted in higher hydroperoxide levels and slower Na⁺,K⁺-ATPase inactivation during MC540/light exposure, whereas ferric-8-hydroxyquinoline treatment (iron supplementation) had the opposite effect. Thus, iron appears to play an important role in both of these processes. In contrast, photoinactivation of another intrinsic enzyme in L1210 cells, acetylcholinesterase (AChE), was unaffected by selenium or iron manipulation. On the basis of these findings, we propose that lipid peroxidation plays an important role in the photoinactivation of Na⁺,K⁺-ATPase, but not AChE. This is consistent with the fact that Na⁺, K⁺-ATPase's active site lies within the membrane bilayer, whereas AChE's active site lies outside the bilayer.     

PHOTODYNAMIC TREATMENT OF YEAST WITH CHLOROALUMINUM-PHTHALOCYANINE: ROLE OF THE MONOMERIC FORM OF THE DYE

The effect of photodynamic treatment on the yeast Kluyveromyces marxianus with aluminum-phthalocyanines has been studied. It was found that the nonsulfonated sensitizer caused light-dependent loss of colony-forming capacity, whereas the mono- and tetrasulfonated forms did not induce loss of clonogenicity. The effect of the nonsulfonated sensitizer increased with longer preincubation periods of cells with the dye. Formation of cellattached, mostly intracellularly localized monomelic sensitizer also increased with time. The amount of cell-bound multimeric nonsulfonated phthalocyanine did not vary with time. Experiments designed to specifically increase the amount of cell-attached monomers led also to an increased photoinactivation of the cells. It is therefore concluded that the photodynamic effect of the nonsulfonated Al-phthalocyanine is mediated by the monomeric form of the dye.     

PHOTOINACTIVATION OF SPINACH NITRATE REDUCTASE SENSITIZED BY FLAVIN MONONUCLEOTIDE. EVIDENCE FOR THE INVOLVEMENT OF SINGLET OXYGEN

Abstract All the activities of the nitrate reductase complex from spinach are irreversibly inactivated by irradiation of the enzyme with blue light in the presence of flavin mononucleotide. The photoinactivation requires oxygen and is prevented by ethylenediaminetetraacetic acid and by reduced nicotinamide adenine dinucleotide, but not by superoxide dismutase plus catalase. On the other hand, the inactivation is markedly enhanced in 77% deuterated water and it is suppressed by the singlet oxygen quenchers azide, histidine and tryptophan. All these results suggest that singlet oxygen generated by light absorption by flavin mononucleotide, rather than excited flavin mononucleotide or other oxygen species, is the primary agent involved in the photooxidative inactivation of the enzyme.