Importance of UDP-glucuronosyltransferase 1A1 expression in skin and its induction by UVB in neonatal hyperbilirubinemia.

UDP-glucuronosyltransferase (UGT) 1A1 is the sole enzyme that can metabolize bilirubin. Human infants physiologically develop hyperbilirubinemia as the result of inadequate expression of UGT1A1 in the liver. Although phototherapy using blue light is effective in preventing jaundice, sunlight has also been suggested, but without conclusive evidence, to reduce serum bilirubin levels. We investigated the mRNA expression pattern of human UGT1A1 in human skin, human skin keratinocyte (HaCaT) cells, and skin of humanized UGT1 mice. The effects of UVB irradiation on the expression of UGT1A1 in the HaCaT cells were also examined. Multiple UGT1A isoforms, including UGT1A1, were expressed in human skin and HaCaT cells. When HaCaT cells were treated with UVB-exposed tryptophan, UGT1A1 mRNA and activity were significantly induced. Treatment of the HaCaT cells with 6-formylindolo[3,2-b]carbazole, which is one of the tryptophan derivatives formed by UVB, resulted in an induction of UGT1A1 mRNA and activity. In neonates, the expression of UGT1A1 was greater in the skin; in adults, UGT1A1 was expressed mainly in the liver. Treatment of humanized UGT1 mice with UVB resulted in a reduction of serum bilirubin levels, along with increased UGT1A1 expression and activity in the skin. Our data revealed a protective role of UGT1A1 expressed in the skin against neonatal hyperbilirubinemia. Sunlight, a natural and free source of light, makes it possible to treat neonatal jaundice while allowing mothers to breast-feed neonates.

UV-radiation induced disruption of dry-cavities in human γD-crystallin results in decreased stability and faster unfolding.

Age-onset cataracts are believed to be expedited by the accumulation of UV-damaged human γD-crystallins in the eye lens. Here we show with molecular dynamics simulations that the stability of γD-crystallin is greatly reduced by the conversion of tryptophan to kynurenine due to UV-radiation, consistent with previous experimental evidences. Furthermore, our atomic-detailed results reveal that kynurenine attracts more waters and other polar sidechains due to its additional amino and carbonyl groups on the damaged tryptophan sidechain, thus breaching the integrity of nearby dry center regions formed by the two Greek key motifs in each domain. The damaged tryptophan residues cause large fluctuations in the Tyr-Trp-Tyr sandwich-like hydrophobic clusters, which in turn break crucial hydrogen-bonds bridging two ß-strands in the Greek key motifs at the "tyrosine corner". Our findings may provide new insights for understanding of the molecular mechanism of the initial stages of UV-induced cataractogenesis.

Discovery and biological characterization of 1-(1H-indol-3-yl)-9H-pyrido[3,4-b]indole as an aryl hydrocarbon receptor activator generated by photoactivation of tryptophan by sunlight.

Activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is required for AHR dependent transcriptional activation and TCDD toxicity. We previously reported that aqueous tryptophan exposed to sunlight through window glass (aTRP) contains multiple photoproducts, including the well characterized 6-formylindolo[3,2-b]carbazole (FICZ), capable of activating the AHR and inducing CYP1A and CYP1A-mediated enzyme activities. We report here the isolation from aTRP and chemical characterization and synthesis of 1-(1H-indol-3-yl)-9H-pyrido[3,4-b]indole (IPI), a compound previously identified as a natural product of marine ascidia and now shown to be a TRP photoproduct with AHR-inducing properties. IPI, FICZ and TCDD produced equieffective induction of CYP1A-mediated 7-ethoxyresorufin deethylase (EROD) activity in chick embryo primary hepatocytes and mammalian Hepa1c1c7 cells. EROD induction by IPI was markedly curtailed in AHR-defective c35 cells, supporting the AHR dependence of the IPI response. Although IPI had a higher EC(50) for EROD induction than FICZ, the much larger amount of IPI than FICZ in aTRP makes IPI a prominent contributor to EROD induction in aTRP. IPI was detected in TRP-containing culture medium under ambient laboratory conditions but not in TRP-free medium, consistent with its production from TRP. Cotreatment of hepatocytes with submaximal EROD-inducing doses of IPI and FICZ or TCDD produced additive increases in EROD without synergistic or inhibitory interactions. IPI and FICZ were readily metabolized by cultured hepatocytes. In addition to increasing CYP1A4 mRNA and EROD, IPI and FICZ decreased hepatocyte phosphoenolpyruvate carboxykinase mRNA expression and glucose output, biological effects associated with TCDD metabolic dysregulation. The findings underscore a role for sunlight in generating AHR-activating bioactive molecules.

Flash photolysis of cutinase: identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues.

Aromatic amino acids play an important role in ultraviolet (UV)-induced photochemical reactions in proteins. In this work, we aim at gaining insight into the photochemical reactions induced by near-UV light excitation of aromatic residues that lead to breakage of disulfide bridges in our model enzyme, Fusarium solani pisi cutinase, a lipolytic enzyme. With this purpose, we acquired transient absorption data of cutinase, with supplemental experimental data on tryptophan (Trp) and lysozyme as reference molecules. We here report formation kinetics and lifetimes of transient chemical species created upon UV excitation of aromatic residues in proteins. Two proteins, lysozyme and cutinase, as well as the free amino acid Trp, were studied under acidic, neutral, and alkaline conditions. The shortest-lived species is assigned to solvated electrons (lifetimes of a few microseconds to nanoseconds), whereas the longer-lived species are assigned to aromatic neutral and ionic radicals, Trp triplet states, and radical ionic disulphide bridges. The pH-dependent lifetimes of each species are reported. Solvated electrons ejected from the side chain of free Trp residues and aromatic residues in proteins were observed 12 ns after excitation, reaching a maximum yield after approximately 40 ns. It is interesting to note that the formation kinetics of solvated electrons is not pH-dependent and is similar in the different samples. On the other hand, a clear increase of the solvated electron lifetime is observed with increasing pH. This observation is correlated with H3O+ being an electron scavenger. Prolonged UV illumination of cutinase leads to a larger concentration of solvated electrons and to greater absorption at 410 nm (assigned to disulphide electron adduct RSSR *-), with concomitant faster decay kinetics and near disappearance of the Trp* radical peak at 330 nm, indicating possible additional formation of TyrO* formed upon reaction of Trp* with Tyr residues. Prolonged UV illumination of cutinase also leads to a larger concentration of free thiol groups, known to originate from the dissociation of RSSR *-. Additional mechanisms that may lead to the near disappearance of Trp(*) are discussed. Our study provides insight into one key UV-light-induced reaction in cutinase, i.e., light-induced disruption of disulphide bridges mediated by the excitation of aromatic residues. Knowledge about the nature of the formed species and their lifetimes is important for the understanding of UV-induced reactions in humans that lead to light-induced diseases, e.g., skin cancer and cataract formation.

Formation and spectroscopy of a tryptophan radical containing peptide in the gas phase.

A doubly deprotonated tryptophan containing peptide was electrosprayed and isolated in an ion trap. UV excitation on this peptide leads to electron detachment and to the formation of an indolyl radical. The photogenerated radical was fragmented by a second laser. The visible spectrum of the gas-phase neutral tryptophan radical containing peptide has been recorded and constitutes a benchmark for calculations and optical measurements.

Photodynamic efficacy of chlorin p6: a pH dependent study in aqueous and lipid environment.

Photodynamic efficacy of chlorin p6, a potential candidate of photodynamic therapy (PDT), has been studied at pH 5.0, 6.0 and 7.6 in aqueous and lipid environment. Increased chlorin p6 mediated photodynamic bleaching of N,N-dimethyl-4-nitrosoaniline (RNO), a measure of singlet oxygen yield, was obtained at higher pH. Rate of photodynamic bleaching of RNO was also higher at higher pH and the rate decreased with lowering in pH of irradiated solution. Photodynamic oxidation of tryptophan was also found to be higher at higher pH. Diminished oxidation of RNO was obtained with decrease in pH of irradiated solution. Both, RNO bleaching and tryptophan oxidation was significantly reduced by sodium azide, a known quencher of singlet oxygen. At lower pH, chlorin p6 mediated photodynamic malondialdehyde (MDA) and lipid hydroperoxide formation in egg lecithin liposome was higher. At higher pH chlorin p6 was found to be photodynamically more effective in aqueous environment whereas at lower pH chlorin p6 was photodynamically more effective in hydrophobic environment.

Lanthanide-binding tags with unnatural amino acids: sensitizing Tb3+ and Eu3+ luminescence at longer wavelengths.

Lanthanide-binding tags (LBTs) are small, genetically encoded, versatile protein fusion partners that selectively bind lanthanide ions with high affinity. The LBT motif features a strategically positioned tryptophan residue that sensitizes Tb3+ luminescence upon excitation at 280 nm. Herein, we describe the preparation of new LBT peptides that incorporate unnatural amino acids in place of tryptophan, and which sensitize both Tb3+ and Eu3+ luminescence at lower energies. We also report the semisynthesis of proteins tagged with these new LBTs using native chemical ligation. This expands the scope of LBTs and will enable their wider use in luminescence applications.

Photodynamic properties of dipeptide-modified hypocrellin B derivatives: the role of tyrosine and tryptophan groups.

Three long-wavelength absorbing dipeptide-modified hypocrellin B (HB) derivatives, Gly-HB, Tyr-HB, and Trp-HB, were prepared for application in photodynamic therapy (PDT). Their abilities to produce free radicals and singlet oxygen were compared in detail with EPR technique, and their binding interactions with calf thymus DNA (CT DNA) were studied by absorption spectra and DNA melting temperature measurements. Tyr-HB and Trp-HB distinguish themselves from Gly-HB and HB remarkably by their significantly improved efficiencies to generate semiquinone anion radicals, superoxide anion radicals, and hydroxyl radicals, as well as their affinity to CT DNA, as the result of the electron-donating properties and intercalating abilities of tyrosine and tryptophan groups. Tyr-HB and Trp-HB show remarkably enhanced photodamage capabilities on CT DNA than their parent HB in aerobic conditions. Moreover, they possess moderate photodamage abilities on CT DNA even in anaerobic conditions, indicating the role of Type I mechanism in their photodynamic behaviors.

Effects of tryptophan photoproducts in the circadian timing system: searching for a physiological role for aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) mediates adverse effects of dioxins, but its physiological role remains ambiguous. The similarity between AhR and canonical circadian clock genes suggests potential involvement of AhR in regulation of circadian timing. Photoproducts of tryptophan (TRP), including 6-formylindolo[3,2-b]carbazole (FICZ), have high affinity for AhR and are postulated as endogenous ligands. Although TRP photoproducts activate AhR signaling in vitro, their effects in vivo have not been investigated in mammals. Because TRP photoproducts may act as transducers of light, we examined their effects on the circadian clock. Intraperitoneal injection of TRP photoproducts or FICZ to C57BL/6J mice dose dependently induced AhR downstream targets, cytochrome P4501A1 (CYP1A1) and cytochrome P4501B1 mRNA expression, in liver. c-fos mRNA, a commonly used marker for light responses, was also induced with FICZ, and all responses were AhR dependent. A rat-immortalized suprachiasmatic nucleus (SCN) cell line, SCN 2.2, was used to examine the direct effect of TRP photoproducts on the molecular clock. Both TRP photoproducts and FICZ-increased CYP1A1 expression and prolonged FICZ incubation altered the circadian expression of clock genes (Per1, Cry1, and Cry2) in SCN 2.2 cells. Furthermore, FICZ inhibited glutamate-induced phase shifting of the mouse SCN electrical activity rhythm. Circadian light entrainment is critical for adjustment of the endogenous rhythm to environmental light cycle. Our results reveal a potential for TRP photoproducts to modulate light-dependent regulation of circadian rhythm through triggering of AhR signaling. This may lead to further understanding of toxicity of dioxins and the role of AhR in circadian rhythmicity.

Identification of radiation-induced cross-linking between thymine and tryptophan by electrospray ionization-mass spectrometry.

Upon exposure to ionizing radiation, DNA undergoes a variety of modifications including the production of a covalent bond between the nucleobase thymine and aromatic amino acids. In this work, electrospray ionization-mass spectrometry(ESI-MS) was used to identify the gamma radiation-induced covalent cross-linking of model peptides (sequence YPPW and pYPPW) with the nucleobase thymine. Tandem electrospray ionization-mass spectrometry (ESI-MSn) was employed to investigate the cross-linking sites. The results showed that irrespective of whether tyrosine was phosphorylated or not, the nucleobase thymine was cross-linked with the tryptophan residue. Possible cross-linking mechanisms are proposed by investigating the related mass peaks.

Statistical vs. non-statistical deactivation pathways in the UV photo-fragmentation of protonated tryptophan-leucine dipeptide.

The excited state dynamics of protonated tryptophan-leucine ions WLH+, generated in an electrospray source, is investigated by photo-induced fragmentation in the gas phase, using femtosecond laser pulses. Two main features arise from the experiment. Firstly, the initially excited pipi* state decays very quickly with 2 time constants of 1 and 10 ps. Secondly, the transient signals recorded on different fragments are not the same which indicates two competing primary fragmentation processes. One involves a direct dissociation from the excited state that gives evidence for a non-statistical deactivation path. The other is attributed to a statistical decay following internal conversion to the ground electronic surface.

Radiation abolishes inducer binding to lactose repressor.

The lactose operon functions under the control of the repressor-operator system. Binding of the repressor to the operator prevents the expression of the structural genes. This interaction can be destroyed by the binding of an inducer to the repressor. If ionizing radiations damage the partners, a dramatic dysfunction of the regulation system may be expected. We showed previously that gamma irradiation hinders repressor-operator binding through protein damage. Here we show that irradiation of the repressor abolishes the binding of the gratuitous inducer isopropyl-1-beta-D-thiogalactoside (IPTG) to the repressor. The observed lack of release of the repressor from the complex results from the loss of the ability of the inducer to bind to the repressor due to the destruction of the IPTG binding site. Fluorescence measurements show that both tryptophan residues located in or near the IPTG binding site are damaged. Since tryptophan damage is strongly correlated with the loss of IPTG binding ability, we conclude that it plays a critical role in the effect. A model was built that takes into account the kinetic analysis of damage production and the observed protection of its binding site by IPTG. This model satisfactorily accounts for the experimental results and allows us to understand the radiation-induced effects.

Minimization of photooxidative insult to calf lens protein irradiated with near UV-light in the presence of pigmented glucosides derived from human lens protein.

An aqueous solution of a pigmented glucoside associated with human lens protein, 2-amino-3-hydroxyacetophenone-O-beta-D-glucoside (AHA-Glc), was irradiated with near UV-light. The near UV-irradiated glucoside was shown to generate a much lower level of active species of molecular oxygen as compared to the level of the active species generated from the irradiated aglycon, 2-amino-3-hydroxyacetophenone (AHA). This result suggests that the glycon of the glucoside is functioning as a scavenger for active oxygen generated from the aglycon of the irradiated glucoside. Superoxide dismutase (SOD) was shown to remove a large portion of the active oxygen generated from the irradiated AHA, so the bulk of the active species generated is assumed to be superoxide anion. The small portion of active oxygen remains after removal of superoxide anion may include singlet oxygen. The photooxidation of tryptophan residues of calf alpha-crystallin irradiated with near UV-light in the presence of AHA-Glc or AHA was investigated to confirm the role that the glycon plays in diminution of the active species of oxygen generated through the photosensitized aglycon of the glucoside. A decrease with time in the fluorescence intensity of the tryptophan residues irradiated with AHA-Glc was shown to be much slower as compared to the time-dependent decrease with AHA, indicating that the photooxidation proceeds with an increase in accumulation of active oxygen generated through the aglycon and that the glycon of the glucoside deactivates the active species as it is formed in the photodynamic process. Similar effects have also been observed in calf lens crystallin irradiated with either 3-hydroxykynurenine-O-beta-D-glucoside (HKN-Glc) or 3-hydroxykynurenine (HKN). Furthermore, effects of near UV-irradiation on calf lens soluble protein in the presence of AHA-Glc or AHA were studied by monitoring changes in the SDS-PAGE profile of the irradiated protein. Near UV-irradiation with AHA-Glc was shown to bring about a slight change in cross-linking of the polypeptides, while irradiation with AHA was shown to give rise to a significant increase in cross-linking of the polypeptides. In conclusion, pigmented glucoside associated with human lens protein is not only a photosensitizer for near UV-light but also an anti-photooxidant to deactivate active oxygen formed through the in situ photosensitizer, in order that photooxidative insults to lens proteins may be minimized during aging.

Fluorescence of the single tryptophan of cutinase: temperature and pH effect on protein conformation and dynamics.

The cutinase from Fusarium solani pisi is an enzyme with a single L-tryptophan (Trp) involved in a hydrogen bond with an alanine (Ala) residue and located close to a cystine formed by a disulfide bridge between two cysteine (Cys) residues. The Cys strongly quenches the fluorescence of Trp by both static and dynamic quenching mechanisms. The Trp fluorescence intensity increases by about fourfold on protein melting because of the disruption of the Ala-Trp hydrogen bond that releases the Trp from the vicinity of the cystine residue. The Trp forms charge-transfer complexes with the disulfide bridge, which is disrupted by UV light irradiation of the protein. This results in a 10-fold increase of the Trp fluorescence quantum yield because of the suppression of the static quenching by the cystine residue. The Trp fluorescence anisotropy decays are similar to those in other proteins and were interpreted in terms of the wobbling-in-cone model. The long relaxation time is attributed to the Brownian rotational correlation time of the protein as a whole below the protein-melting temperature and to protein-backbone dynamics above it. The short relaxation time is related to the local motion of the Trp, whose mobility increases on protein denaturation.

Induction of cytochrome p450 1A1 and 1B1 by photooxidized tryptophan in transformed human keratinocytes.

L-Tryptophan was oxidized by UVA, UVB, or UVC irradiation and the effect of oxidized tryptophan (OT) was investigated for its ability to induce CYP1A1 and 1B1 in a human keratinocyte cell line (HaCat). The results obtained demonstrate that tryptophan oxidized by UVA, UVB, or UVC produced photoproducts which caused a significant induction of 7-ethoxyresorufin O-deethylase (EROD) activity in HaCat cells. The induction of EROD activity by UVB-OT was studied in greater detail. The results demonstrate that induction of EROD activity by UVB-OT was dose- and time-dependent. Results with immunoblot analyses showed that administration of UVB-OT, but not unoxidized tryptophan, caused a marked induction of CYP1A1 and 1B1 proteins in HaCat cells.

Suitability of tryptophan radiation products as markers for the detection of gamma-irradiated protein rich food.

During gamma-irradiation (5 kGy) of aqueous tryptophan (Trp) solutions small amounts of 5-, 6-, and 7-hydroxytryptophan (OH-Trp) (0.04-0.08 Mol-%) are formed. Protein rich food like shrimps contain reasonable amounts of non-protein bound Trp (100 mg/kg). In order to detect the treatment of shrimps with gamma-irradiation a method for the determination of OH-Trp in gamma-irradiated shrimps was developed. After homogenization, squeezing of shrimp samples and protein precipitation, a two-step-SPE-clean up was performed using a C18-cartridge and a propylsulfonic acid cation-exchange SPE followed by HPLC analysis with electrochemical detection (750 mV). Results showed that 5-OH-Trp contents in shrimp samples increased with applied doses up to 3 kGy and then decreased with higher doses. Other OH-Trp isomers were not detectable in the irradiated shrimps. Similarly no formation of 4-, 6-, and 7-OH-Trp was detected in model solutions containing the same amino acid composition as in shrimps. This indicates a suppression of the reaction of OH-radicals with Trp by the 300 fold molar excess of other amino acids acting as well as radical scavengers. Therefore, non-physiological OH-Trp isomers formed from free Trp are not suitable as markers for the detection of gamma-irradiated protein-rich foodstuff.

Photodynamic properties of amphiphilic derivatives of aluminum tetrasulfophthalocyanine.

Photodynamic therapy (PDT) is a promising treatment modality that has recently been accepted in clinics as a curative or palliative therapy for cancer and other nonmalignant conditions. Phthalocyanines (Pc) are attractive photosensitizers for PDT because of their enhanced photophysical and photochemical properties. The overall charge and solubility of Pc play a major role in their potential usefulness for PDT. A series of amphiphilic derivatives of tetrasulfonated aluminum Pc (AlPcS4) was prepared by substituting one of the four sulfonate groups with aliphatic side chains of 4, 8, 12 and 16 carbon atoms. The photodynamic properties of the derivatives were compared with those of AlPcS4 and the adjacent disulfonated aluminum Pc. Parameters studied included reversed-phase high-performance liquid chromatography (HPLC) retention times, capacity to generate singlet oxygen (1O2), in vitro cell uptake and phototoxicity, as well as PDT response of transplantable EMT-6 tumors in mice. The monomerized AlPcS4 derivatives showed similar or higher capacities to generate 1O2 as compared with the parent AlPcS4 as measured from relative L-tryptophan photooxidation yields. A549 cell uptake of the AlPcS4 derivatives decreased in the following order: AlPcS4(C16) > AlPcS4(C12) > AlPcS4(C8) > AlPcS4(C4). Human low-density lipoprotein at high concentrations (40 micrograms/mL) completely prevented uptake, whereas at 4 micrograms/mL uptake was decreased for the more lipophilic compounds and yet remained unaffected for the more hydrophilic dyes. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, A549 cell survival was assessed; it showed that photocytotoxic activity varied directly with the HPLC retention times, i.e. more hydrophilic compounds were less phototoxic. As 1O2 yields were similar for the four substituted AlPcS4 derivatives, it was postulated that the increased cytotoxic activity was caused by enhanced subcellular localization as a result of the long aliphatic side chains. These amphiphilic compounds proved to be photodynamically potent against the EMT-6 mouse mammary tumor model implanted in Balb/c mice. At dye doses of 0.2 mumol/kg and a fluence of 400 J/cm2 complete tumor regression was observed with no morbidity. The substitution of AlPcS4 with long aliphatic chains on the macrocycle greatly enhances its photodynamic efficacy both in vitro and in vivo.

Photodynamic activity of 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin on the Hep-2 human carcinoma cell line: effect of light dose and wavelength range.

The photodynamic activity of 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin (TMP) has been investigated in two systems: reverse micelles of n-heptane/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/water-bearing photooxidizable substrates and on a Hep-2 human carcinoma cell line. The effect of variation in the light dose and wavelength range (360-800, 455-800, and 590-800 nm) was compared in both media. The aerobic singlet oxygen-mediated photooxidation of L-tryptophan (Trp) was used as a model of biological substrate in a micellar system. A considerable increase of the observed rate constants of Trp (k(Trp)(obs)) was noted, increasing the irradiated area of the TMP spectrum. In vitro, the survival curves of Hep-2 cells, treated with TMP, were markedly dependent on the light wavelength ranges used for irradiation. A linear behavior between k(Trp)(obs) and the photoinactivation rate of Hep-2 cells was found, indicating that the singlet oxygen (1O2 ) is the main species responsible for cell inactivation. These results contributed to an understanding of the photodynamic process yielded by this porphyrin in vitro and the sensitivity of Hep-2 cells to photodamage.

Study of the peptide prebiotic synthesis in context of exobiological investigations on earth orbit.

Dry films of amino acids mixtures glycine+ tryptophan and tryptophan were exposed on the surface of "Mir" station. Similar films were irradiated by vacuum ultra violet (145 nm) and ultra violet (254 nm) in the laboratory experiments. Gly-Gly, Trp-Gly, Gly-Trp, Tpr-Trp and Trp-Trp-Trp were the main reaction products for the experimental mixture glycine + tryptophan and Tpr-Trp and Trp-Trp-Trp for tryptophan. The presence of Lunar soil both in flight and in laboratory experiments increases the reaction yield by 1.5-2.0 times. Therefore, the hypothesis concerning the possibility of safe delivery of peptides and amino acids required for the emergence of life and associated with mineral have got yet another approval.

Photodynamic therapy: tumor targeting with adenoviral proteins.

A brief summary of the mechanisms involved in photodynamic therapy (PDT) and the role of delivery vehicles for photosensitizer targeting is addressed. Phthalocyanines (Pc) have been coupled to adenovirus type 2 capsid proteins including the hexon, the penton base and the fiber to enhance their target selectivity. Adenovirus penton base proteins contain the arginine-glycine-aspartic acid peptidic sequence (RGD) motif known to bind with great affinity and high specificity to integrin receptors, expressed by several types of cancer. Tetrasulfonated aluminum phthalocyanine (AlPcS4) was covalently coupled to the various capsid proteins via one or two caproic acid spacer chains (A1 or A2) in 7:1 up to 66:1 molar ratios. The capacity of the bioconjugates for singlet oxygen production, as measured by an L-tryptophan oxidation assay, was strongly reduced, likely reflecting scavenging by the carrier. Cell adsorption and in vitro photocytotoxicity assays were carried out using the A549 and HEp2 human cell lines expressing integrin receptors, and one murine, the EMT-6 cell line, which lacks receptors for the RGD sequence. The AlPcS4A2-protein complexes induced greater cytotoxicity as compared to the analogous AlPcS4A1 preparations. The penton base-AlPcS4A2 derivative was the more phototoxic for all cell lines tested. Tumor response studies using Balb/c mice with EMT-6 tumor implants demonstrated that the free AlPcS4A2 induced complete tumor regression at a dose of 1 mumol/kg and 400 J/cm2, which is comparable to the activity of the known AlPcS2adj. A mixture of adenovirus type 2 soluble proteins covalently labeled with AlPcS4A2 required 0.5 mumol/kg to induce the same response with the same light dose, suggesting that the high affinity RGD/receptor complex is able to target Pc for PDT.