PORPHYRINS AND RELATED COMPOUNDS AS PHOTODYNAMIC SENSITIZERS* John D. Spikes Department of Biology University of Utah Salt Lake City, Utah 841 I 2

It was shown early in this century that certain porphyrins are photosensitizers of the photodynamic type, i.e., they sensitize the photooxidative damage of biological systems on illumination in the presence of molecular oxygen.',* An extensive literature has accumulated on porphyrin-sensitized photodynamic effects on multicellular organisms (including humans), cells, biomacromolecules, and small organic molecules of biological importance. The mechanism of the porphyrin-sensitized injury t o multicellular organisms is poorly understood; as will be discussed below, effects a t the cellular level appear t o be mediated by photodynamic damage t o plasma and lysosomal membranes. Damage to proteins involves the photooxidation of certain types of amino acid residues. Efficiencies of porphyrins as photodynamic sensitizers depend critically on the metal chelated by the molecule; porphyrins containing paramagnetic metal atoms are typically nonsensitizers. The sensitizing efficiencies and substrate specificities of metal-free and diamagnetic metalloporphyrins are determined t o some extent by the side chains o n the molecule. In water solution, photosensitizing porphyrins give a good yield of long-lived triplet states on illumination which are quenched by molecular oxygen with high efficiency. Kinetic and product-formation studies with amino acids and proteins suggest that porphyrin-sensitized photooxidations occur by a singlet oxygen mechanism. T h e purpose of this paper is t o review briefly the various kinds of studies that have been made with porphyrins as photosensitizers; the coverage of the literature is extensive rather than intensive in order t o illustrate the great range of systems studied. MECHANISMS O F SENSITIZED PHOTOOXIDATION REACTIONS Formal studies on sensitized photooxidations in biological systems began with the observations of Raab,3 who showed in 1900 that low concentrations of certain dyes, which were without effect in the dark, promoted the rapid killing of paramecia on illumination. A short time later it was found that oxygen was necessary for such phenomena (a few examples of photosensitization reactions in biology, such as the light-promoted interactions of psoralens with nucleic acids, d o not require oxygen4). Considerable progress has been made in recent years in the elucidation of the fundamental mechanisms involved in the sensitized photooxidation of small organic m o l e c ~ l e s . ~ Sensitized photooxidations typically proceed via the triplet state of the sensitizer since it has a much longer lifetime than the singlet

* The preparation of this paper and the original work included were supported by the United States Atomic Energy Commission under Contract No. AT(1 l-l)-875. 49 6

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excited state, which is produced initially o n the absorption of light. Porphyrins such as deuteroporphyrin give a high yield of long-lived triplets on illumination in acidic aqueous solution, as shown in this laboratory.’ Triplet sensitizer molecules react by two primary pathways in photodynamic systems.5They can interact with another molecule ( 1 ) by a hydrogen or electron transfer process, thus producing radicals which in turn react with oxygen, etcetera (“Type I,” radical, or redox reactions); and/or (2) by an energy transfer process (“Type 11” reactions). The most common process in the latter case is energy transfer to ground state molecular oxygen with the production of ground state sensitizer and an excited singlet state of oxygen (‘02, typically the ‘Ag state) which in turn can oxidize susceptible substrate^.^-^ The relative importance of the two pathways depends on the particular photodynamic system (sensitizer, substrate, pH, solvent composition, reactant concentrations, etcetera). It should be stressed that a number of additional reaction pathways are possible, which in some systems become s i g n i f i ~ a n tl.o~ ~ The available evidence suggests that porphyrins sensitize the photooxidation of small organic molecules and of certain amino acids b y way of the singlet oxygen (Type 11) pathway. For example, several investigators’ 2 , 1 have shown that methionine is quantitatively converted into methionine sulfoxide on illumination in the presence of porphyrins; similar results are obtained with “exposed” methionyl residues in small peptides and proteins. Kinetic and chemical studies with a number of different sensitizers suggest that methionine sulfoxide is the Type I1 oxidation product from methionine; other organic sulfides have also been shown t o be photooxidized via the singlet oxygen pathway.’ We have shown by steady-state kinetic studies and flash photolysis measurements that the hematoporphyrin-sensitized photooxidation of methionine t o the sulfoxide occurs by a singlet oxygen pathway.’ Deuteroporphyrin, on illumination, also reacts with ground state oxygen t o give singlet oxygen.’ PORPHYRIN PHOTOSENSITIZATION IN MAMMALS Mammals treated with photosensitizers show a characteristic pattern of symptoms o n illumination, including scratching and hyperactivity; skin damage, involving erythema, edema, cell degeneration, ulceration, and necrosis; and generalized responses, including decreased blood pressure, intestinal hemorrhage, and circulatory collapse.’ The latter symptoms perhaps result from compounds produced photodynamically in the skin, which are then circulated throughout the body. Photosensitivity in mammals involves t w o different mechanisms, usually termed phototoxicity and photoallergy. Phototoxic reactions are a n immediate response t o illumination; with efficient photosensitizers and sufficient illumination all treated organisms will show a response. In photoallergic responses, new haptenes are produced photochemically that combine with protein t o give functional ‘photoantigens’; these ultimately produce delayed, immunological types of responses.’ Present evidence suggests that porphyrins are phototoxic in their behavior rather than photoallergic.’

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*

Porphyrin Photosensitization in Laboratory and Grazing Animals There is a large literature on studies of porphyrin photosensitization in laboratory animals. The first work in this area was probably that of

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H a u ~ m a n n , ’ ~ who ~ ’ ~ in 1908 described the acute and chronic effects of hematoporphyrin photosensitization in the mouse. Blum’s book’ provides an excellent review and interpretation of the earlier work on porphyrin photosensitization in animals and suggests the possible involvement of a histaminerelease process. This possibility has been investigated in greater detail and confirmed to some extent in mice’ and rats*’ photosensitized with hematoporphyrin. Illumination of the mesentery of rats sensitized with hematoporphyrin had rapid and profound effects on the microcirculation with stoppage of the blood flow in capillaries foAlowed by intravascular h e m ~ l y s i s . ’ ~If mice are exposed t o heat (up to 96.8 F) after hematoporphyrin-sensitized photodynamic treatment, lethality is markedly increased; exposure t o heat 12 hours after the photodynamic treatment no longer has any effect.24 Hematoporphyrin-sensitized phototoxicity in mammals appears to be a true photodynamic phenomenon in that oxygen is required. For example, in the rabbit, if the circulation to hematoporphyrin-treated tissue is blocked during illumination, damage is markedly reduced.’ In mice, hematoporphyrin-sensitized phototoxicity was decreased under hypobaric conditions (400 torr).’ A rather large amount of research has been carried out on porphyrin photosensitization in grazing animals, probably because of the economic considerations.’ 7*27 A common form of photosensitivity in ruminants occurs as a result of disturbances in liver function which lead to the accumulation of a photosensitizing chlorophyll derivative, phylloerythrin, in the animal. Hepatic pigmentation associated with photosensitivity has been observed in one flock of Corriedale sheep; the syndrome, which resembles the Dubin-Johnson syndrome in man, is probably inherited.29 Congenital porphyria, inherited in a simple Mendelian fashion, has been repeatedly observed in cattle; clinical manifestations include reddish-brown pigmentation of bones, teeth, and urine, and photosensitivity in unpigmented or lightly pigmented skin areas.’ v 2

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9’

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Porphyrin Photosensitization in Man

Several examples of photosensitivity in humans associated with the appearance of abnormal porphyrin in the urine were reported in the last ~ e n t u r y . ~ ’ ? ~ ~ Experimental porphyrin photosensitization in man was first established in 19 13 by the dramatic experiments of Meyer-Betz, who injected himself with 200 mg of hematoporphyrin and then exposed himself t o light in various ways; he remained photosensitive for approximately two months.34 Cases of hematoporphyrin photosensitization have been reported as a result of using this compound as a therapeutic agent in the treatment of mental d e p r e s ~ i o n The .~~ earlier work on the photosensitizing action of porphyrins in man has been reviewed in detail by Blum.’ The condition known as porphyria results from abnormalities in porphyrin metabolism; the condition can be genetic, or it can be produced by various chemicals. A number of clinical types of porphyria have been recognized. It is beyond the scope of this paper t o discuss the porphyrias in any detail; however, since certain porphyrias result in photosensitization, they should be considered here. One type, which has received considerable study, is the drug-induced porphyria termed porphyria cutanea tarda, sometimes referred t o as acquired hepatic porphyria. Patients with this condition excrete large amounts of uroporphyrin (a known photodynamic sensitizer) and show marked photosensitivity1*; the action spectrum for the skin photoresponse peaks at

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approximately 400 nm, corresponding to the Soret band of p o r p h y r i n ~ . ~ ~ Patients with the rare hereditary disorder, erythropoietic protoporphyria, have elevated levels of protoporphyrin (also a known photodynamic sensitizer) in the red blood cells, the extracellular fluids, and the skin.j7 The action spectrum of skin photosensitivity in this case also shows a peak a t approximately 400 nm, although the sensitivity t o light is lower than in porphyria cutanea tarda.36

Protection against Porphyrin Photosensitization Some years ago Langhof2' made a survey of possible photoprotective compounds using rabbits injected subcutaneously with hematoporphyrin as test organisms. Of those compounds studied, only Ca,Na-ethylene-diaminetetraacetic acid (injected intraarterially) protected the sensitized animals against subsequent illumination. More recently it has been shown that intraperitoneal injections of dithiothreitol give significant protection t o mice sensitized with hematoporphyrin and then i l l ~ m i n a t e d . In ~ ~ 1964 mat hew^^^ showed that p-carotene protected hematoporphyrin-sensitized mice (carotenoid pigments are known t o protect bacteria against photodynamic injury sensitized both by endogenous pigments such as hemes and chlorophylls and by added photosensitizers). She suggested that carotenoids might be useful in the therapy of light-sensitive conditions in humans. Recently, several studies have been reported on the use of /%carotene as a photoprotective agent in patients with erythropoietic protoporphyria; oral administration of this compound produced a remarkable decrease in the light-sensitivity of most of the patients studied.40 - 4 2 The mechanism of protection is not known. However, since p-carotene is an efficient quencher of free radicals>3 singlet ~ x y g e n , ~ - 'and ~ * the ~ ~ triplet states of p h o t o ~ e n s i t i z e r s , it ~ ~ is tempting to speculate that one or more of these mechanisms is operating.

Light, Porphyrins, and Cancer Several workers have demonstrated that photodynamic treatment can induce skin cancers in experimental animals (see the review by S a n t a m a ~ i a ~Actually ~). the first demonstration of this phenomenon involved the use of a porphyrin as the photosensitizer. In 1937 Bungeler showed that if mice were injected subcutaneously with a solution of hematoporphyrin and then illuminated for an extended period, hypertrophy of the epithelium took place followed by the appearance of tumors.47 Neither the porphyrin treatment nor the illumination alone gave this effect. It is not known whether this phenomenon occurs in humans. There is, of course, good evidence that the shorter wavelengths of sunlight (300-320 nm) are directly carcinogenic in humans. I t has been suggested, however, that accumulation of porphyrins in the skin with aging might lead t o photodynamic c a r c i n o g e n e ~ i s . ~ ~ It has been known for a long time that a variety of neoplasms preferentially take u p hematoporphyrin and hematoporphyrin derivatives, as compared t o normal tissues49; in fact, the fluorescence of the incorporated porphyrin has often been used t o localize tumors. Since hematoporphyrin is a powerful photodynamic sensitizer, Diamond and coworkers suggested that the accumulation of this porphyrin in malignant tumors should sensitize them t o selective destruction by visible light.50 Their studies showed that administration of

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hematoporphyrin followed by illumination rapidly killed glioma cells in culture and produced extensive destruction of gliomas transplanted subcutaneously into rats. Thus porphyrins plus light represent a two-edged sword with respect t o neoplasia. Photo therapy of Hyperb ilirubinem ia Jaundice, resulting from the accumulation of bilirubin (an open chain tetrapyrrole derived from the breakdown of heme) in the serum and tissues, is very common in newborn infants. In premature infants, in particular, the bilirubin concentration can rise t o levels that result in brain damage.5' It was observed a number of years ago that exposure t o sunlight or artificial blue light dramatically decreased the serum bilirubin levels in hyperbilirubinemic inf a n t ~ Since . ~ ~ this technique is so simple and convenient compared to the only other effective therapy (complete blood replacement), it has come into widespread use in recent years.53 Relatively little is known about the in vivo photochemistry of bilirubin disappearance, however, bilirubin is known t o sensitize the photodynamic hemolysis of red blood cells,s4 and, in vitro, bilirubin apparently sensitizes its own destruction by a singlet oxygen mechanism.55 The rate constant for bilirubin oxidation b y singlet oxygen is considerably greater than for any other known singlet oxygen a ~ c e p t o r . ~ Although bilirubin is relatively inefficient as a photodynamic sensitizer, considerable concern has been expressed over the use of phototherapy for hyperbilirubinemia because of the lack of fundamental knowledge concerning the in vivo s i t ~ a t i o n . ~ ~ PORPHYRIN PHOTOSENSITIZATION OF CELLS AND SUBCELLULAR STRUCTURES The first experimental studies on porphyrins as photosensitizers were probably those of Hausmann, who showed in 1908 that rabbit erythrocytes were hemolyzed on illumination in the presence of chlorophyll or hernatoporphyrin.58 Mammalian red blood cells have been used extensively ever since in cellular level studies of photodynamic action; the earlier work is reviewed in detail by Blum,' who also made notable contributions t o the field. There are perhaps two major reasons for the use of mammalian erythrocytes in photodynamic studies. Because of the specialized nature of these cells, photodynamic damage appears t o be a pure membrane effect culminating in rupture of the membrane. Thus the red blood cell appears t o provide a much simpler system for study compared t o most other cells. Another reason is that the red blood cells of humans with certain kinds of porphyrias contain elevated levels of free porphyrin and as a result are much more sensitive to hemolysis o n illumination in vitro. F o r example, in vitro illumination of red blood cells with elevated protoporphyrin levels from patients with erythropoietic protoporphyria gave complete hemolysis; normal red blood cells were not appreciably affected by a similar exposure t o light.s9 Essentially all of the cellular potassium was released from the porphyric cells before significant hemolysis occurred, suggesting early damage t o the red blood cell The action spectrum for the photohemolysis of such porphyric red blood cells corresponds to the Soret peak of p ~ r p h y r i n s ~ photohemolysis ~; requires the presence of

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molecular oxygen during the illumination period, as is typical of photodynamic processes.6 ,63 Finally, normal red blood cells undergo photohemolysis if illuminated following treatment with protoporphyrin-IX dimethyl ester6 or with p r ~ t o p o r p h y r i n .Amino ~~ acid residues in the cell membrane are destroyed during protoporphyrin-sensitized p h o t o h e m ~ l y s i s . ~ ~ A variety of reducing agents and other compounds decrease the in vitro sensitivity of red blood cells from patients with erythropoietic porphyria toward photohemolysis.6' Of particular interest, in terms of mechanism, is the observation that 0-carotene is an effective protective agent,6 since, as described above, this compound is an effective quenching agent for singlet oxygen, free radicals, and triplet state sensitizers. Normal human red blood cells are also protected from protoporphyrin-sensitized photohemolysis b y p ~ a r o t e n e . ~ Illumination of ghosts from normal red blood cells and of cholesterolcontaining liposomes, both loaded with hematoporphyrin, led t o the conversion of cholesterol t o 3~-hydroxy-Sa-hydroperoxy-A6-cholestene6(photooxidation of cholesterol in solution by hematoporphyrin also gives this product6 7). Incorporation of the hydroperoxide into normal red blood cells increased their osmotic fragility, leading to hemolysis.66 These studies thus suggest a mechanism for the photohemolysis of erythrocytes from patients with erythropoietic protoporphyria. Illumination of human blood platelets in the presence of hematoporphyrin led t o swelling and a loss of cytoplasm, serotonin, potassium, and acid phosphatase; treated platelets were not aggregated by thrombin plus calcium ~hloride.~~?~~ A number of cellular-level photodynamic studies have been done with cells isolated from animals, or with animal and human cells in tissue culture. F o r example, the photodynamic treatment of rat mast cells from peritoneal fluid with hematoporphyrin caused them t o lose their basophilia and t o become refractory t o the action of histamine-release agents, presumably by an effect o n the permeability of the cell membrane.2 Illumination of human transformed fibroblasts and malignant mouse fibroblasts in culture in the presence of hematoporphyrin resulted in extensive cell degeneration and death within a short time70 (see Reference 7 1 for some of the techniques used in tissue culture studies). The mechanism(s) of cell injury and killing by photodynamic treatment is only poorly understood, although it might be expected that certain subcellular structures, by virtue of their chemical makeup, would be more susceptible t o photooxidation or would concentrate a given photosensitizer t o high levels and thus have a greater probability of damage. It has been shown, for example, that the lysosomes of mammalian cells in culture take u p and concentrate a wide range of basic and neutral substances, as determined b y fluorescence microscopy; the lysosomes of cells cultured in the presence of uroporphyrin I fluoresce red, and in some cases, yellow, due t o uptake of t h e p ~ r p h y r i n A .~~ variety of mammalian cell types were shown t o become sensitive to light in the presence of oxygen following the uptake of uroporphyrin I into their lysosomes. Shortly after illumination, increases in the permeability of the lysosomal membranes were observed (as determined by acid phosphatase staining in unfixed cells); after 24 hours of incubation, cells receiving high light doses were largely destroyed and many had been shed from the coverslips into the m e d i ~ m . ~Phylloerythrin sensitized the photodynamic rupture of lysosomes in cells of rat liver homogenates and in sections of rat skin; skin cell lysosomes were more sensitive than those in liver cells. Both vitamin E and hydrocortisone

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protected rat skin cell lysosomes against photodynamic damage; these results were regarded as supporting the authors’ suggestion that the phylloerythrinsensitized photodynamic damage t o lysosomes involved a free radical mechanism.74 Finally, there is one report on the porphyrin-sensitized photodynamic treatment of isolated cell organelles. Illumination markedly increased the inhibition of the oxidative phosphorylation of isolated rat liver mitochondria in the presence of Cu- and Na-chlorophyllin o r chlorine e6.75 PORPHYRIN PHOTOSENSITIZATION O F PURINES, PYRIMIDINES, AND NUCLEIC ACIDS Gaffron observed in 1926 that the purine, uric acid, was rapidly photooxidized on illumination in the presence of hematoporphyrin; caffeine was oxidized more slowly and alloxan not a t all.76 Uric acid in 60% ethanol is rapidly degraded on illumination in the presence of chlorophylls; adenine, hypoxanthine, and xanthine were not photodegraded under the same conditions. The photodegradation products of uric acid include allantoin, cyanuric acid, parabanic acid, and urea; allantoin and cyanuric acid are not further degraded on illumination in the presence of ~ h l o r o p h y l l . ~K-chlorophyllin, although it does not bind t o DNA at pH 7.0, has a small photosensitizing effect on Micrococcus leisodeikticus DNA and on T4r+ phage (DNA) at this pH, as evidenced by decreases in the melting temperatures of the nucleic acids on illumination in the presence of the p ~ r p h y r i n .In ~ ~contrast, chlorophyll did not sensitize the photoinactivation of an actinophage (#MSP2, a DNA phage).79 The photoconductivity of RNA-chlorophyll systems has been studied.80 PORPHYRIN PHOTOSENSITIZATION OF AMINO ACIDS AND PROTEINS

Photooxidation of A m i n o Acids It was shown in 1926 that tyrosine and tryptophan are photooxidized with hematoporphyrin and chlorophyll as sensitizer^.^^^^ Turacin, the nonfluorescent copper salt of uroporphyrin, did not sensitize the photooxidation of tyrosine, whereas uroporphyrin was an efficient sensitizer. More recent studiesI2 show that the efficiency of porphyrins as sensitizers for the photooxidation of free amino acids in water solution at pH 6.1 depends on the porphyrin side chains as well as on the complexed metal ion. Magnesiumchlorophyll a sensitized the rapid photooxidation of methionine, histidine, tyrosine, and tryptophan. Hematoporphyrin sensitized the photooxidation of methionine and tryptophan, while neither hemin (with a paramagnetic iron ion) nor protoporphyrin IX dimethyl ester sensitized significantly. Histidine and tyrosine were not photooxidized with the chlorophyll derivative in phosphate buffer at pH values below 5 or in acetic acid at concentrations greater than 5%; however, methionine and tryptophan were photooxidized in phosphate buffer at pH 2.5, and especially rapidly in 99--100% acetic acid. Kinetic studies of the porphyrin-sensitized photooxidation of methionine in 90% acetic acid as a function of oxygen concentration suggest that photooxidation occurs via triplet sensitizer and with the involvement of singlet

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*

oxygen. Porphyrins with different side chains (as in the series deutero-, mesoand protoporphyrin and pheophytin a ) have different sensitizing efficiencies. Chelation of the metal ions Mg++ and Zn++ decreases the efficiency of photooxidation with hematoporphyrin, while incorporation of paramagnetic ions such as Cu++ and Fe++ reduces the efficiency essentially to zero.

Photooxidation o f Amino Acid Residues in Small Peptides It might be expected that in some cases amino acid derivatives or amino acids incorporated into simple peptides would have an altered susceptibility to sensitized photooxidation. Further, the chemistry of photooxidation might be different due to the blockage of amino, carboxyl, or other functional groups. Jori, Galiazzo, and Scoffone' examined the hematoporphyrin-sensitized photooxidation of methionine and tryptophan in model peptides. In water at pH 6.1, the methionine residue in N-carbobenzyloxy-methionylaspartic acid was photooxidized by a first-order process to the sulfoxide at the same rate as free methionine. In contrast, although free tryptophan is photooxidized with hematoporphyrin, tryptophan in model peptides (N-carbobenzyloxytryptophylglycine ethyl ester, N-carbobenzyloxy-alanyltryptophyl methyl ester and N-carbobenzyloxytryptophylmethionyl methyl ester) was not photooxidized a t all in water or in acetic acid solution.

Photooxidation of Nonheme Proteins HarrissL showed in 1926 that serum proteins were photooxidized o n illumination in the presence of hematoporphyrin. In the same year, Gaffron7 showed that serum proteins were photooxidized both with hematoporphyrin and with chlorophyll; hematoporphyrin also sensitized the photooxidation of casein. The following free porphyrins also sensitized the photooxidation of serum proteins with efficiencies in the order: uroporphyrin mesoporphyrin coproporphyrin; zinc-hematoporphyrin and metal-free hematoporphyrin sensitized the photooxidation, while hemin, copper-hematoporphyrin, and silver-hematoporphyrin did not sensitize at all. If serum is photooxidized with hematoporphyrin, complement and natural cytotoxic compounds are dest r ~ y e d and , ~ ~albumin and certain globulins are decreased, giving a change in the serum electrophoretic pattern.85 Photooxidized fibrinogen shows an increased clotting time, and firm clots are not formed; such fibrinogen inhibits the clotting of native fibrinogen.s6 The optical rotation of solutions of bovine serum albumin at first increases and then decreases on illumination in the presence of h e m a t o p ~ r p h y r i n .Photodynamic ~~ treatment of myosin solutions with hematoporphyrin decreases the viscosity of dilute solutions and increases the viscosity of more concentrated solutions.s8 Castellani and Lippes9 showed that lysozyme was inactivated on illumination with visible light in the presence of hematoporphyrin; they suggested that inactivation was dependent on a binding of the porphyrin to the protein. Recently, Jori and coworkersL have carried out a more extensive examination of the photodynamic inactivation of lysozyme by porphyrins. The enzymatic activity of lysozyme illuminated in water at pti 6.1 in the presence of hematoporphyrin decreased rapidly by a first-order process to approximately 50% of the initial activity; prolonged illumination caused n o further decrease. Chromatographic analysis showed that only one species of enzyme was present

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Annals New York Academy of Sciences

after illumination; this form differed from native enzyme in the conversion of Met-12 to the sulfoxide. In contrast, if the illumination was carried out in 84% acetic acid, both methionine residues were photooxidized t o the sulfoxide; again, there was n o destruction of other susceptible residues (His, Trp, Tyr). Thus the hematoporphyrin-sensitized photooxidation of lysozyme is a very specific process. The monosulfoxide derivative produced by photodynamic treatment in water showed only slight conformational changes from the native enzyme, while the disulfoxide derivative, which had very little enzymatic activity, showed extensive d e n a t u r a t i ~ n . Adding ~~ lysozyme to hematouorphyrin in water solution at pH 5.9 gives a red shift in the Soret peak of the porphyrin due to the formation of a 2 : 1 hematoporphyrin: lysozyme complex; such binding does not occur in acetic acid solutions where the lysozyme is unfolded. Illumination of the system in water a t 367 nm, where only free hematoporphyrin absorbs, has n o effect on the lysozyme, while illumination at 435 nm, where only the bound porphyrin absorbs, resulted in the photooxidation of Met-12. Thus the specificity of hematoporphyrin for the photooxidation of lysozyme by hematoporphyrin must result from a specific binding of one molecule of the sensitizer t o a site near Met-12 on the enzyme.” Photodynamic treatment with hematoporphyrin inactivated yeast alcohol dehydrogenase; there was a decrease in the number of free -SH groups during inactivation, b u t apparently n o destruction of histidine, tryptophan, or tyro~ine.~ The ~ hematoporphyrin-sensitized photooxidation of methionine residues in ribonuclease A , which had been unfolded t o different extents in water-acetic acid mixtures, indicates that Met-29 is partially exposed in the native protein while Met-13 is partially “buried” and Met-30 and Met-79 are deeply b ~ r i e d ; ~ thus porphyrin-sensitized photodynamic procedures are useful in determining the degree of burial of photosensitive amino acid residues in proteins. Photooxidation of Hemoproteins

Like other types of proteins, most hemoproteins are photooxidized on illumination in the presence of added sensitizers. F o r example, the illumination of horse heart cytochrome c in the presence of hematoporphyrin results in the destruction of all of t h e photooxidizable amino acid residues (His, Met, Trp, Tyr) in the molecule. Hemoproteins, however, permit some unique, selective studies of photodynamic action because of their heme moiety and the presence of specific binding sites for porphyrins. If a photosensitizing molecule is bound or covalently linked t o a protein, only those susceptible amino acid residues within a limited radius (a few Angstrom units) will be photooxidized on illumination.13i94t95 In most hemoproteins the heme moiety is not a photodynamic sensitizer since the iron is in a high spin state (paramagnetic) and is thus ar, effective quencher of the excited states involved in photodynamic reactions; in fact, heme is a good “protective agent” against added photosensitizers. A few hemoproteins, such as cytochrome c , have their iron naturally in a low spin state; thus the prosthetic group would be expected t o b e a photosensitizer. In fact, Jon and c o ~ o r k e r sshowed ~ ~ ~ that ~ ~ illumination of both ferro- and ferricytochrome c resulted in a highly specific destruction of amino acid residues. In both forms, His-18 and Met-80 were photooxidized (these are both ligands for the heme iron); in addition, Trp-59 and Tyr-48 were destroyed in

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ferrocytochrome. Illumination of cells of the alga Prototheca zopfii with blue light resulted in the destruction of cytochrome 0 3 ; cytochromes in yeast and in isolated beef heart mitochondria were also destroyed on i l l ~ m i n a t i o n These .~~ results may also reflect a heme-sensitized photooxidation. Under some conditions, normally high-spin hemoproteins can be converted to low-spin forms, which should then be photosensitive. This strategy has been used t o determine the susceptible amino acid residues adjacent t o the heme in myoglobins. Native sperm-whale and horse myoglobins are insensitive t o visible light. If these hemoproteins are converted to low-spin forms b y ligation with cyanide, illumination results in the destruction of His-93 in both proteins; ligation with carbon monoxide followed by illumination results in the photooxidation of His-93 and His-64 in sperm-whale myoglobin and of His-93, His-64 and Met-131 in horse m y o g l ~ b i n . ~ ~ . ~ ~ A final strategy for obtaining localized porphyrin sensitization in hemoproteins is t o remove the heme, and then replace it with a metal-free porphyrin or with a porphyrin containing a nonparamagnetic metal. Breslow, Koehler, and Girotti' O 0 * l O 1 removed the heme from sperm-whale myoglobin and replaced i t with protoporphyrin IX; illumination of this complex resulted in the photooxidation of histidine residues in the globin. More recent studies on a 1: 1 complex of protoporphyrin IX with sperm-whale m y o obin showed that His-93 is preferentially photooxidized on illumination' o$ this agrees with the observation that His-93 is located very close t o the heme ring in crystalline myoglobin. Thus, porphyrins used as photosensitizers have valuable applications as probes of the three-dimensional structures of hemoproteins in solution.

REFERENCES 1. SPIKES, J. D. 1968. In Photophysiology. A. C. Giese, Ed. Vol. 111: 33-64. Academic Press, New York, N.Y. 2. SPIKES, J. D. & R. LIVINGSTON. 1969. Advan. Radiat. Biol. 3: 29-121. 3. RAAB, 0. 1900. Z. Biol. 39: 524-546. 4. MUSAJO, L. & G. RODIGHIERO. 1972. In Photophysiology. A. C. Giese, Ed. Vol. VII: 115-147. Academic Press. New York, N.Y. 5. FOOTE, C. S. 1968. Science 162: 963-970. 6. FOOTE, C. S. 1968. Acc. Chem. Res. 1: 104-110. 7. FOOTE, C. S. 1971. Pure Appl. Chem. 27: 635-645. 8. WILSON, T. & J. W. HASTINGS. 1970. In Photophysiology. A. C. Giese, Ed. Vol. V: 50-95. Academic Press. New York, N.Y. 9. POLITZER, 1. R., G. W. GRIFFIN & J. LASETER. 1971. Chem. Biol. Interact. 3: 73-93. 10. SPIKES, J. D. & F. RIZZUTO. In Proceedings of the Sixth International Congress on Photobiology, Bochum, Germany. In press. 11. COKER, G. Unpublished Experiments. 12. JORI, G., G. GALIAZZO & E. SCOFFONE. 1969. Biochemistry 8: 2868-2875. 13. SPIKES, J. D. & M. L. MACKNIGHT. 1970. Ann. N.Y. Acad. Sci. 171: 149-162. 14. FOOTE, C. S. & J. W. PETERS. 1971. XXIIIrd International Congress on Pure and Applied Chemistry, Special Lectures. Vol. 4 : 129. Butterworth & Co. London, England. 15. GENNARI, G., G. JORI & J. D. SPIKES. Unpublished work. 16. DALTON, J., C. A. McAULIFFE & D. H. SLATER. 1972. Nature 235: 388-389. 17. BLUM, H. F. 1941. Photodynamic Action and Diseases Caused by Light. Reinhold. New York, N.Y. (Reprinted in 1964 with an updated appendix by Hafner, New York, N.Y.).

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DISCUSSION DR. G. KENNEDY: Boyd in Canada has uv-irradiated solutions of egg albumin containing porphyrin sensitizers and found that the protein breaks down not only to amino acids, but even to ammonia. This might explain the shock produced after injections of photosensitizing porphyrins as t h e tissue protein breaks down releasing histamine, as well as producing a blister. A t Sheffield we’ve found that injection of hematoporphyrin into the tissues a week before injection of a carcinogen produces tumors in half the latent period of the controls. For sarcomas, manganese hematoporphyrin produces tumors even faster. We don’t know the mechanism involved here. DR. SPIKES: If you irradiate proteins only with Soret irradiation and not shorter wave lengths, you don’t rupture peptide bonds o r disulfide bonds generally, but simply oxidize amino acid side chains. Transition type metal porphyrins don’t give appreciable concentrations of triplet states and are generally not good photosensitizers. So I doubt if your tumor mechanism involves a photosensitization, a t least in the manganese case. DR. A. LAMOLA (Bell Laboratory, Murray Hill, N.J.): You just mentioned the fact that having a metal ion in the porphyrin makes a tremendous difference in the excited state lifetime and therefore its photosensitizing ability. Considering the side chain effects in nonmetal ion-containing porphyrins, there are some really intriguing things. For example, red blood cells from lead poisoned people are loaded with coproporphyrin b u t they d o n o t undergo photohemolysis, yet cells from erythropoietic protoporphyria patients are very sensitive t o light. The reason for this difference is presently unknown. Could it be a difference in the excited state properties or a difference in the localization o r a desensitizer? DR. SPIKES: I don’t really know. Certainly incorporating even metals like zinc and magnesium into a porphyrin does alter its photochemical properties t o some degree, such as t h e efficiency of intersystem crossing and the lifetime of the triplet state, Also, different metal-free porphyrins can behave differently as photosensitizers. For example, Dr. Yeou-Jan .Kang of our laboratory has complexed protoporphyrin IX, hematoporphyrin IX,and deuteroporphyrin IX t o apo-horseradish peroxidase. O n illumination of the first t w o complexes he observes the destruction of two histidine residues and one methionine residue in the protein; only a single histidine and no methionine residues are photooxidized in the deuteroporphyrin complex.