Initial O2 Insertion Step of the Tryptophan Dioxygenase Reaction Proposed by a Heme-Modification Study.

L-Tryptophan 2,3-dioxygenase (TDO) is a protoheme-containing enzyme that catalyzes the production of N-formylkynurenine by inserting O2 into the pyrrole ring of L-tryptophan. Although a ferrous-oxy form (Fe²âº-O2) has been established to be an obligate intermediate in the reaction, details of the ring opening reaction remain elusive. In this study, the O2 insertion reaction catalyzed by Pseudomonas TDO (PaTDO) was examined using a heme-modification approach, which allowed us to draw a quantitative correlation between the inductive electronic effects of the heme substituents and the substituent-induced changes in the functional behaviors of the ferrous-oxy form. We succeeded in preparing reconstituted PaTDO with synthetic hemes, which were different with respect to the inductive electron-withdrawing nature of the heme substituents at positions 2 and 4. An increase in the electron-withdrawing power of the heme substituents elevated the redox potential of reconstituted PaTDO, showing that the stronger the electron-withdrawing ability of the heme substituents, the lower the electron density on the heme iron. The decrease in the electron density of the heme iron resulted in a higher frequency shift of the C-O stretch of the heme-bound CO and enhanced the dissociation of O2 from the ferrous-oxy intermediate. This result was interpreted as being due to weaker π back-donation from the heme iron to the bound CO or O2. More importantly, the reaction rates of the ferrous-oxy intermediate to oxidize L-Trp were increased with the electron-withdrawing ability of the heme substituents, implying that the more electron-deficient ferrous-oxy heme is favored for the PaTDO-catalyzed oxygenation. On the basis of these results, we propose that the initial step of the dioxygen activation by PaTDO is a direct electrophilic addition of the heme-bound O2 to the indole ring of L-Trp.

Acute stress-induced antinociception is cGMP-dependent but heme oxygenase-independent

Endogenous carbon monoxide (CO), which is produced by the enzyme heme oxygenase (HO), participates as a neuromodulator in physiological processes such as thermoregulation and nociception by stimulating the formation of 3′,5′-cyclic guanosine monophosphate (cGMP). In particular, the acute physical restraint-induced fever of rats can be blocked by inhibiting the enzyme HO. A previous study reported that the HO-CO-cGMP pathway plays a key phasic antinociceptive role in modulating noninflammatory acute pain. Thus, this study evaluated the involvement of the HO-CO-cGMP pathway in antinociception induced by acute stress in male Wistar rats (250-300 g; n=8/group) using the analgesia index (AI) in the tail flick test. The results showed that antinociception induced by acute stress was not dependent on the HO-CO-cGMP pathway, as neither treatment with the HO inhibitor ZnDBPG nor heme-lysinate altered the AI. However, antinociception was dependent on cGMP activity because pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) blocked the increase in the AI induced by acute stress.

Acute stress-induced antinociception is cGMP-dependent but heme oxygenase-independent.

Endogenous carbon monoxide (CO), which is produced by the enzyme heme oxygenase (HO), participates as a neuromodulator in physiological processes such as thermoregulation and nociception by stimulating the formation of 3',5'-cyclic guanosine monophosphate (cGMP). In particular, the acute physical restraint-induced fever of rats can be blocked by inhibiting the enzyme HO. A previous study reported that the HO-CO-cGMP pathway plays a key phasic antinociceptive role in modulating noninflammatory acute pain. Thus, this study evaluated the involvement of the HO-CO-cGMP pathway in antinociception induced by acute stress in male Wistar rats (250-300 g; n=8/group) using the analgesia index (AI) in the tail flick test. The results showed that antinociception induced by acute stress was not dependent on the HO-CO-cGMP pathway, as neither treatment with the HO inhibitor ZnDBPG nor heme-lysinate altered the AI. However, antinociception was dependent on cGMP activity because pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) blocked the increase in the AI induced by acute stress.

The allosteric role of the AAA+ domain of ChlD protein from the magnesium chelatase of synechocystis species PCC 6803.

Magnesium chelatase is an AAA(+) ATPase that catalyzes the first step in chlorophyll biosynthesis, the energetically unfavorable insertion of a magnesium ion into a porphyrin ring. This enzyme contains two AAA(+) domains, one active in the ChlI protein and one inactive in the ChlD protein. Using a series of mutants in the AAA(+) domain of ChlD, we show that this site is essential for magnesium chelation and allosterically regulates Mg(2+) and MgATP(2-) binding.

Investigation of conditions involved in the susceptibility of the dermatophyte Trichophyton rubrum to photodynamic treatment.

BACKGROUND: Photodynamic treatment (PDT) refers to a treatment with light-activated agents (photosensitizers) in combination with visible light and molecular oxygen. Recently, we have demonstrated that the porphyrins, 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) and deuteroporphyrin monomethylester (DP mme) are excellent photosensitizers to be used against Trichophyton rubrum both in vitro and ex vivo. OBJECTIVES AND METHODS: The objective of this study was to investigate the key factors involved in PDT efficacy of both photosensitizers in an ex vivo situation during different fungal growth stages using a recently developed ex vivo model. The study focused on the influence of pH and ion strength of incubation media, photochemical properties of the photosensitizers (spectra and singlet oxygen production), and the effect of several scavengers of reactive oxygen species (sodium azide, histidine, mannitol) and phenylmethylsulphonylfluoride (keratinase inhibitor) on the PDT efficacy. RESULTS AND CONCLUSIONS: The results show that an optimal pH and low concentrations of calcium are crucial for a selective binding of Sylsens B to the fungus, leading to an increased PDT efficacy. This selective binding to T. rubrum cannot be accomplished for DP mme. It can be concluded that the prerequisite for successful treatment is a use of a low molarity solution of pH 5, supplemented with a chelating agent and a keratinase activity-repressing agent. Under these conditions, PDT with Sylsens B inactivates, initially via singlet oxygen, effectively the fungus in different fungal growth stages.

Tetrapyrrole-photosensitizers vectorization and plasma LDL: a physico-chemical approach.

A photosensitizer is defined as a chemical entity able to induce, under light-irradiation effect, a chemical or physical alteration of another chemical entity. Thanks to their preferential retention in proliferating tissues, some photosensitizers are therapeutically used such as in photodynamic therapy (PDT). Besides, this method has already been approved for several indications. The selectivity of photosenzitizers for cells in proliferation involves both their association with low density lipoproteins (LDLs) and their ability to cross membranes under various pH conditions. The photosensitizers used are in most cases based on the porphyrin structure, but other compounds, of which far-red-light absorption properties are most compatible with biological tissues irradiation, have been developed, such as phthalocyanines. This paper presents physico-chemical studies of the interaction of a disulfonated aluminium phthalocyanine (AlPcS2) with human LDLs. The data obtained are compared with the parameters of the interaction of these lipoproteins with deuteroporphyrin (DP) and chlorin e6 (Ce6). A close attention is paid to the dynamic aspects of these phenomena. The data obtained on these simple systems then allowed us to interpret the sub-cellular localization of the photosensitizers on a human line of fibroblasts, and to evaluate the influence of LDLs on the intracellular distribution of the compounds. This last point is of major importance because the localization of such photosensitizers (in particular AlPcS2) in endocytic vesicles and their subsequent ability to induce a release of the contents of these vesicles - including externally added macromolecules - into the cytosol is the basis for a recent method for macromolecule activation, named photochemical internalization (PCI). PCI has been shown to potentiate the biological activity of a large variety of macromolecules. The comprehension of the mechanisms governing this particular sub-cellular localization could allow the design of better candidates for PCI.

Autocatalytic formation of a covalent link between tryptophan 41 and the heme in ascorbate peroxidase.

Electronic spectroscopy, HPLC analyses, and mass spectrometry (MALDI-TOF and MS/MS) have been used to show that a covalent link from the heme to the distal Trp41 can occur on exposure of ascorbate peroxidase (APX) to H2O2 under noncatalytic conditions. Parallel analyses with the W41A variant and with APX reconstituted with deuteroheme clearly indicate that the covalent link does not form in the absence of either Trp41 or the heme vinyl groups. The presence of substrate also precludes formation of the link. Formation of a protein radical at Trp41 is implicated, in a reaction mechanism that is analogous to that proposed [Ghiladi, R. A., et al. (2005) Biochemistry 44, 15093-15105] for formation of a covalent Trp-Tyr-Met link in the closely related catalase peroxidase (KatG) enzymes. Collectively, the data suggest that radical formation at the distal tryptophan position is not an exclusive feature of the KatG enzymes and may be used more widely across other members of the class I heme peroxidase family.

Heme orientational disorder in human adult hemoglobin reconstituted with a ring fluorinated heme and its functional consequences.

A ring fluorinated heme, 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12,18-trimethyl-porphyrinatoiron(III), has been incorporated into human adult hemoglobin (Hb A). The heme orientational disorder in the individual subunits of the protein has been readily characterized using (19)F NMR and the O(2) binding properties of the protein have been evaluated through the oxygen equilibrium analysis. The equilibrated orientations of hemes in alpha- and beta- subunits of the reconstituted protein were found to be almost completely opposite to each other, and hence were largely different from those of the native and the previously reported reconstituted proteins [T. Jue, G.N. La Mar, Heme orientational heterogeneity in deuterohemin-reconstituted horse and human hemoglobin characterized by proton nuclear magnetic resonance spectroscopy, Biochem. Biophys. Res. Commun. 119 (1984) 640-645]. Despite the large difference in the degree of the heme orientational disorder in the subunits of the proteins, the O(2) affinity and the cooperativity of the protein reconstituted with 2-MF were similar to those of the proteins reconstituted with a series of hemes chemically modified at the heme 3- and 8-positions [K. Kawabe, K. Imaizumi, Z. Yoshida, K. Imai, I. Tyuma, Studies on reconstituted myoglobins and hemoglobins II. Role of the heme side chains in the oxygenation of hemoglobin, J. Biochem. 92 (1982) 1713-1722], whose O(2) affinity and cooperativity were higher and lower, respectively, relative to those of native protein. These results indicated that the heme orientational disorder could exert little effect, if any, on the O(2) affinity properties of Hb A. This finding provides new insights into structure-function relationship of Hb A.

Dynamics of interactions of photosensitizers with lipoproteins and membrane-models: correlation with cellular incorporation and subcellular distribution.

The incorporation and subcellular localization of photosensitizers are critical determinants of their efficiency. Here, we correlate these properties with the interactions of photosensitizers with membrane-models and low density lipoproteins (LDL) in acellular systems. Focus was given on dynamics aspects. Two amphiphilic photosensitizers, deuteroporphyrin (DP) and aluminum phthalocyanine sulfonated on two adjacent isoindole units (AlPcS2a) were selected. The phthalocyanine was bound to LDL with an overall association constant around 5 x 10(7)M(-1). Biphasic association kinetics was indicative of two types of sites. The release of the phthalocyanine into the bulk aqueous medium occurred within less than a second. A similar behavior was found previously for deuteroporphyrin although its affinity was somewhat higher (5.5 x 10(8)M(-1)). Both compounds were previously characterized by high affinity for membrane-models and quick exchange with the bulk solution. However, they strongly differed by their rate of transfer through the lipid bilayer, in the range of seconds for the porphyrin, several hours for the phthalocyanine. In the case of the porphyrin, fluorescence microscopy on human fibroblasts showed diffuse labeling with no significant modification of the distribution upon vectorization by LDL. In contrast, the phthalocyanine was localized in intracellular vesicles. Vectorization by LDL favored lysosomal localization although little effect was found on the overall uptake as shown by extraction experiments. The role of lipoproteins in the cellular localization of photosensitizers is significantly more important for photosensitizers not freely diffusing through bilayers. The dynamics of the interactions of photosensitizers with membranes appears as an important determinant of their subcellular localization.

Magnesium-dependent ATPase activity and cooperativity of magnesium chelatase from Synechocystis sp. PCC6803.

The first committed step in chlorophyll biosynthesis is catalyzed by magnesium chelatase, a complex enzyme with at least three substrates, cooperative Mg(2+) activation, and free energy coupling between ATP hydrolysis and metal-ion chelation. A detailed functional study of the behavior of the intact magnesium chelatase has been performed, including characterization of magnesium cooperativity and the stoichiometry of ATP consumption in relation to the magnesium porphyrin produced. It is demonstrated that, in vitro, this catalyzed reaction requires hydrolysis of approximately 15 MgATP(2-) and that the chelation partial reaction is energetically unfavorable, under our assay conditions, with a DeltaG degrees ' of 25-33 kJ mol(-1). Given the likely metabolite concentrations in vivo, this results in the chelatase reaction operating far from equilibrium. We have also determined the steady-state kinetic behavior of the intact enzyme and have compared the kinetic parameters obtained with those observed for the partial reactions of individual subunits. K(DIX) (where D(IX) represents deuteroporphyrin IX) is estimated to be 3.20 microm, and K(MgATP)(2-) is 0.45 mm. k(cat) for chelation is estimated to be 0.8 min(-1), suggesting that the ATP hydrolysis catalyzed by the isolated ChlI subunit is substantially slower in the intact chelatase. The magnesium-rich form of the chelatase is a more effective catalyst of the chelation reaction; magnesium activation of the chelatase increases V, as well as the specificity constant for the reaction of MgATP(2-) and D(IX), possibly as a result of a magnesium-triggered conformational change.

[Photohemolysis sensitized by deuteroporphyrin-IX derivatives: determination of binding strength of dyes to erythrocytes]. / Fotogemoliz, sensibilizirovannyi proizvodnymi deiteroporfirina-IX: opredelenie prochnosti sviazyvaniia krasitelei s éritrotsitami.

A comparative analysis of the ability of 4-(1-methyl-3-hydroxybutyl)-deuteroporphyrin-IX (I) and 2,4-di-(1-methyl-3-hydroxybutyl)-deuteroporphyrin-IX (II) to photosensitize hemolysis of human erythrocytes was performed. The photohemolytic efficiency of dye I was shown to be about 60 times higher than that of dye II. It was found that a part of each dye tightly binds to erythrocyte membranes and is not removed by washing. A method for estimating the share of the dye tightly bound to the membrane (beta) was proposed, which takes into account the shielding effect produced by the free dye and the photohemolytic efficiency of the bound dye. It was shown that the beta values for dyes I and II are 86 and 61% and correlate with the coefficients of distribution of the dyes in the octanol/water system (20.7 and 17.0, respectively).

GUN4, a regulator of chlorophyll synthesis and intracellular signaling.

Nuclear genes control plastid differentiation in response to developmental signals, environmental signals, and retrograde signals from plastids themselves. In return, plastids emit signals that are essential for proper expression of many nuclear photosynthetic genes. Accumulation of magnesium-protoporphyrin IX (Mg-Proto), an intermediate in chlorophyll biosynthesis, is a plastid signal that represses nuclear transcription through a signaling pathway that, in Arabidopsis, requires the GUN4 gene. GUN4 binds the product and substrate of Mg- chelatase, an enzyme that produces Mg-Proto, and activates Mg-chelatase. Thus, GUN4 participates in plastid-to-nucleus signaling by regulating Mg-Proto synthesis or trafficking.

Characterization of the binding of deuteroporphyrin IX to the magnesium chelatase H subunit and spectroscopic properties of the complex.

Magnesium protoporphyrin chelatase catalyzes the insertion of a Mg(2+) ion into protoporphyrin IX, which can be considered as the first committed step of (bacterio)chlorophyll synthesis. In the present work, the Mg chelatase H subunits from both Synechocystis and Rhodobacter sphaeroides were studied because of the differing requirements of these organisms for modified cyclic tetrapyrroles. Deuteroporphyrin was shown to be a substrate for Mg chelatase. Analytical HPLC gel filtration was used to show that an H-deuteroporphyrin complex can be reconstituted by incubating the magnesium chelatase H subunit with a molar excess of deuteroporphyrin and that these complexes are monomers. The binding process occurs in the absence of Mg(2+) or ATP or the I or D subunits of Mg chelatase. The emission from Trp residues in the H subunit is partly quenched when deuteroporphyrin is bound. Quantitative analysis of Trp fluorescence quenching led to determination of the K(d) values for deuteroporphyrin binding to BchH from Rb. sphaeroides and ChlH from Synechocystis, which are 1.22 +/- 0.42 microM and 0.53 +/- 0.12 microM for ChlH and BchH, respectively. In the case of ChlH, but not BchH, the K(d) increased 4-fold in the presence of MgATP(2-). Red shifts in absorbance and excitation peaks were observed in the B band of the bound porphyrin in comparison with deuteroporphyrin in solution, as well as reduced yield and red shifts of up to 8 nm in fluorescence emission. These alterations are consistent with a slightly deformed nonplanar conformation of the bound porphyrin. Mg deuteroporphyrin, the product of the Mg chelation reaction, was shown to form a complex with either ChlH or BchH; in each case the K(d) for Mg deuteroporphyrin is similar to that for deuteroporphyrin. The implications of the H-Mg protoporphyrin interaction for the next enzyme in the chlorophyll biosynthetic pathway, Mg protoporphyrin methyltransferase, are discussed.

Porphyrin and phthalocyanine antiscrapie compounds.

The transmissible spongiform encephalopathies (TSEs) are fatal, neurodegenerative diseases for which no effective treatments are available. The likelihood that a bovine form of TSE has crossed species barriers and infected humans underscores the urgent need to identify anti-TSE drugs. Certain cyclic tetrapyrroles (porphyrins and phthalocyanines) have recently been shown to inhibit the in vitro formation of PrP-res, a protease-resistant protein critical for TSE pathogenesis. We now report that treatment of TSE-infected animals with three such compounds increased survival time from 50 to 300%. The significant inhibition of TSE disease by structurally dissimilar tetrapyrroles identifies these compounds as anti-TSE drugs.

Horseradish peroxidase-dependent oxidation of deuteroporphyrin IX into chlorins.

Chlorins are cyclic tetrapyrrole derivatives of great interest for use in photodynamic therapy. We have found that horseradish peroxidase (EC 1.11.1.7) (HRP) can convert deuteroporphyrin IX (Deutero) into chlorins. Some characteristics of this enzymatic transformation were investigated. The formation of chlorins was determined spectrophotometrically by monitoring the change in absorbance in the Q-band region (638 nm). The reaction occurred without addition of H2O2 and had a pH optimum of 7.5. The presence of thiol-containing reductants, with a great preference for reduced glutathione, was required and could not be substituted by adding H2O2. Ascorbic acid acted as a potent inhibitor of the reaction, while other organic acids (citric and benzoic) had little to no inhibitory effect. The requirement for O2 was suggested by the inhibitory effect of sodium hydrosulfite and was confirmed by carrying the assay in nitrogen-saturated solutions. Though the reaction occurred without adding H2O2, low amounts of H2O2 (3-30 microM) were stimulatory to the assay. However, concentrations of 300 microM H2O2 or higher were inhibitory. Similarly, light was not required, but was stimulatory at low levels and inhibitory at high levels. Catalase and deferoxamine were inhibitory, but superoxide dismutase and mannitol had no effects. Kinetic analysis and respiratory studies suggest that HRP may initially react with reduced glutathione in a reaction that does not consume much oxygen. The ensuing steps, probably involving an oxygen free radical and porphyrin radical intermediates, consume a large amount of O2 to oxidize Deutero into chlorin.

Ferrochelatase cDNA delivered by adenoviral vector corrects biochemical defect in protoporphyric cells.

Protoporphyria is generally an autosomal dominant disease characterized genetically by mutations in the ferrochelatase gene. The interaction between the wild-type and mutant ferrochelatase protein is unknown. The aim of this study was to evaluate the ability to correct the enzymatic and biochemical defects in cells from patients with protoporphyria, using a replication-defective human adenovirus for gene transfer. Overexpression of ferrochelatase was accomplished by construction of a vector in which expression of the wild-type ferrochelatase cDNA was driven by the constitutive cytomegalovirus (CMV) promoter, introduction and packaging of the cDNA into human adenovirus dl309, and transduction of normal and protoporphyric fibroblasts. Fibroblasts from controls and patients were infected with the ferrochelatase adenovirus or a control adenovirus and assayed for ferrochelatase activity and the accumulation of protoporphyrin upon challenge with the precursor delta-aminolevulinic acid (ALA). At a multiplicity of infection (moi) of 10, greater than 85% of both the wild-type and protoporphyric fibroblasts were infected. The recombinant adenovirus increased the ferrochelatase protein content and activity in the wild-type and protoporphyric fibroblasts with equal efficiency. Therefore, the presence of the mutant ferrochelatase protein did not inhibit the ferrochelatase activity expressed by the transgene.

Interactions of dicarboxylic porphyrins with unilamellar lipidic vesicles: drastic effects of pH and cholesterol on kinetics.

The effect of pH on the transfer of deuteroporphyrin from dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles to human serum albumin is investigated using a stopped-flow with fluorescence detection. The kinetics of this process allows for the determination of the rate constants for the porphyrin exist from the outer vesicle layer to the bulk aqueous medium (koff), and for its movement from the inner to the outer vesicle layer (kto). Both koff and kto are found to strongly depend on the pH. The observed behavior can be described by classical titration curves and is most likely due to protonation equilibria involving the two carboxylic groups of the porphyrin. A pH increase accelerates the exist of the porphyrin. The reverse effect is observed for its movement through the bilayer. The presence of cholesterol in the DMPC bilayer also strongly affects the interactions of the porphyrin with the vesicles. The rate constant kto is dramatically reduced by increasing the cholesterol content. An irregularity is noted around 10-20 mol % cholesterol. The results are discussed in relation to the preferential uptake of porphyrins by tumors, a basis of photodynamic therapy, and to possible pH-mediated relocalization of porphyrins among subcellular structures.

Sites of photodamage by the iminium salt of a copper octaethylbenzochlorin.

Because the benzochlorin derivative copper (II) alpha-meso-N,N'-dimethyloctaethylbenzochlorin iminium chloride (CDS1) is not fluorescent, sites of drug localization in L1210 cells were detected by indirect methods involving using a series of fluorescent probes. The CDS1-mediated cytotoxicity was associated with mitochondrial damage, a decreased membrane potential and an increase in the heterogeneity of membrane sites of binding of a polar analog of diphenylhexatriene. Although CDS1 is a cationic compound, its accumulation was not impaired in a cell line exhibiting the multidrug resistance phenotype.

Evidence that a formyl-substituted iron porphyrin is the prosthetic group of myeloperoxidase: magnetic circular dichroism similarity of the peroxidase to Spirographis heme-reconstituted myoglobin.

To probe the identity of the active site heme-type prosthetic group of myeloperoxidase, whose structure has not been established unambiguously [proposed structures are (i) a chlorin (dihydroporphyrin) or (ii) a formyl-substituted porphyrin such as present in heme a], Spirographis heme (2-formyl-4-vinyldeuteroheme IX) has been incorporated into apo-myoglobin as a possible iron porphyrin model. Comparison of parallel derivatives of these two green proteins with magnetic circular dichroism spectroscopy reveals considerable similarities between several derivatives of these proteins, including the pyridine hemochromogen, the native ferric, ferrous-oxy, and ferrous-CO forms. In contrast, the magnetic circular dichroism spectra of available iron chlorin (octaethylchlorin) model complexes in analogous ligation and oxidation states do not show any significant spectral similarities to myeloperoxidase. This finding provides important evidence in favor of a formyl-substituted porphyrin as the structure of the prosthetic group macrocycle of myeloperoxidase.

In vitro uptake of dicarboxylic porphyrins by human atheroma. Kinetic and analytical studies.

Human atheromatous aorta segments as well as presumably disease-free control aorta were obtained at autopsy. They were incubated with solutions of various purified dicarboxylic porphyrins including hematoporphyrin (HP) and hydroxyethylvinyldeuteroporphyrin (HVD), and with solutions of Photofrin. Selective labelling of the atheroma was shown by macroscopic and microscopic observations of the characteristic porphyrin fluorescence associated with the atheromatous plaques. The time dependence of the uptake, monitored by absorption spectrophotometry or by high performance liquid chromatography, was inferred from the disappearance of the porphyrins in the incubation medium. Significant binding was observed in the absence of albumin or serum proteins. The uptake of HP was less than that of the more hydrophobic compounds HVD or Photofrin when these porphyrins were used alone. The presence of albumin or serum drastically reduces atheroma labelling. Some competition between HP and HVD for binding sites is also seen. The present results do indicate that hydrophobic porphyrins have an intrinsic affinity for atheroma and that they can be taken up through passive processes. Taking into account previous data on animal models (Photochem. Photobiol. (1989), 731-737), it appears however that, in vivo, interactions with proteins and pharmacokinetics will primarily determine plaque labelling.