Chemical Modifications of Globular Proteins Phototriggered by an Endogenous Photosensitizer.

The main goal of the present work was to investigate the damages photoinduced by pterin (Ptr), an endogenous photosensitizer present in human skin under pathological conditions, on a globular protein such as ubiquitin (Ub). Particular attention has been paid on the formation of covalent adducts between Ptr and the protein that can behave as photoantigen and provoke an immune system response. Here, a multifaceted approach including UV-visible spectrophotometry, fluorescence spectroscopy, electrophoresis, size exclusion chromatography, and mass spectrometry is used to establish the Ub changes triggered by UV-A irradiation in the presence of Ptr. Under anaerobic conditions, the only reaction corresponds to the formation of a covalently bound Ptr-Ub adduct that retains the spectroscopic properties of the free photosensitizer. A more complex scheme is observed in air-equilibrated solutions with the occurrence of three different processes, that is, formation of a Ptr-Ub adduct, dimerization, and fragmentation of the protein.

Photooxidation of Tryptophan and Tyrosine Residues in Human Serum Albumin Sensitized by Pterin: A Model for Globular Protein Photodamage in Skin.

Human serum albumin (HSA) is the most abundant protein in the circulatory system. Oxidized albumin was identified in the skin of patients suffering from vitiligo, a depigmentation disorder in which the protection against ultraviolet (UV) radiation fails because of the lack of melanin. Oxidized pterins, efficient photosensitizers under UV-A irradiation, accumulate in the skin affected by vitiligo. In this work, we have investigated the ability of pterin (Ptr), the parent compound of oxidized pterins, to induce structural and chemical changes in HSA under UV-A irradiation. Our results showed that Ptr is able to photoinduce oxidation of the protein in at least two amino acid residues: tryptophan (Trp) and tyrosine (Tyr). HSA undergoes oligomerization, yielding protein structures whose molecular weight increases with irradiation time. The protein cross-linking, due to the formation of dimers of Tyr, does not significantly affect the secondary and tertiary structures of HSA. Trp is consumed in the photosensitized process, and N-formylkynurenine was identified as one of its oxidation products. The photosensitization of HSA takes place via a purely dynamic process, which involves the triplet excited state of Ptr. The results presented in this work suggest that protein photodamage mediated by endogenous photosensitizers can significantly contribute to the harmful effects of UV-A radiation on the human skin.

Mechanism of photooxidation of folic acid sensitized by unconjugated pterins.

Folic acid, or pteroyl-l-glutamic acid (PteGlu), is a precursor of coenzymes involved in the metabolism of nucleotides and amino acids. PteGlu is composed of three moieties: a 6-methylpterin (Mep) residue, a p-aminobenzoic acid (PABA) residue, and a glutamic acid (Glu) residue. Accumulated evidence indicates that photolysis of PteGlu leads to increased risk of several pathologies. Thus, a study of PteGlu photodegradation can have significant ramifications. When an air-equilibrated aqueous solution of PteGlu is exposed to UV-A radiation, the rate of the degradation increases with irradiation time. The mechanism involved in this "auto-photo-catalytic" effect was investigated in aqueous solutions using a variety of tools. Whereas PteGlu is photostable under anaerobic conditions, it is converted into 6-formylpterin (Fop) and p-aminobenzoyl-l-glutamic acid (PABA-Glu) in the presence of oxygen. As the reaction proceeds and enough Fop accumulates in the solution, a photosensitized electron-transfer process starts, where Fop photoinduces the oxidation of PteGlu to Fop, and H(2)O(2) is formed. This process also takes place with other pterins as photosensitizers. The results are discussed with the context of previous mechanisms for processes photosensitized by pterins, and their biological implications are evaluated.

Oxidation of 2'-deoxyguanosine 5'-monophosphate photoinduced by pterin: type I versus type II mechanism.

UV-A radiation (320-400 nm) induces damage to the DNA molecule and its components through different photosensitized reactions. Among these processes, photosensitized oxidations may occur through electron transfer or hydrogen abstraction (type I) and/or the production of singlet molecular oxygen ((1)O2) (type II). Pterins, heterocyclic compounds widespread in biological systems, participate in relevant biological processes and are able to act as photosensitizers. We have investigated the photosensitized oxidation of 2'-deoxyguanosine 5'-monophosphate (dGMP) by pterin (PT) in aqueous solution under UV-A irrradiation. Kinetic analysis was employed to evaluate the participation of both types of mechanism under different pH conditions. The rate constant of (1)O2 total quenching (k(t)) by dGMP was determined by steady-state analysis of the (1)O2 NIR luminescence, whereas the rate constant of the chemical reaction between (1)O2 and dGMP (k(r)) was evaluated from kinetic analysis of concentration profiles obtained by HPLC. The results show that the oxidation of dGMP photosensitized by PT occurs through two competing mechanisms that contribute in different proportions depending on the pH. The dominant mechanism in alkaline media involves the reaction of dGMP with (1)O2 produced by energy transfer from the PT triplet state to molecular oxygen (type II). In contrast, under acidic pH conditions, where PT and the guanine moiety of dGMP are not ionized, the main pathway for dGMP oxidation involves an initial electron transfer between dGMP and the PT triplet state (type I mechanism). The biological implications of the results obtained are also discussed.

Pterin pigment granules are responsible for both broadband light scattering and wavelength selective absorption in the wing scales of pierid butterflies.

A small but growing literature indicates that many animal colours are produced by combinations of structural and pigmentary mechanisms. We investigated one such complex colour phenotype: the highly chromatic wing colours of pierid butterflies including oranges, yellows and patterns which appear white to the human eye, but strongly absorb the ultraviolet (UV) wavelengths visible to butterflies. Pierids produce these bright colours using wing scales that contain collections of minute granules. However, to date, no work has directly characterized the molecular composition or optical properties of these granules. We present results that indicate these granules contain pterin pigments. We also find that pterin granules increase light reflection from single wing scales, such that wing scales containing denser granule arrays reflect more light than those with less dense granule collections. As male wing scales contain more pterin granules than those of females, the sexual dichromatism found in many pierid species can be explained by differences in wing scale pterin deposition. Additionally, the colour pattern elements produced by these pterins are known to be important during mating interactions in a number of pierid species. Therefore, we discuss the potential relevance of our results within the framework of sexual selection and colour signal evolution.

Photooxidation of pterin in aqueous solutions: biological and biomedical implications.

Studies of the photochemical reactivity of pterin (= 2-aminopteridin-4(3H)-one; PT) in acidic (pH 5.0-6.0) and alkaline (pH 10.2-10.8) aqueous solutions have been performed. The photochemical reactions were followed by UV/VIS spectrophotometry, thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), and an enzymatic method for H2O2 determination. PT is not light-sensitive in the absence of molecular oxygen, but it undergoes photooxidation in the presence of O2, yielding several nonpteridinic products. The quantum yields for PT disappearance were found to be 8.2 (+/-0.6) x 10(-4) and 1.2 (+/-0.2) x 10(-3) in acidic and alkaline media, respectively. H2O2 was detected and quantified in irradiated solutions of PT; and its importance from a biomedical point of view is discussed. The rate constant of the chemical reaction between singlet oxygen ((1)O2) and PT was determined to be 2.5 (+/-0.2) x 10(5) l mol(-1) s(-1) in alkaline medium, and the role of (1)O2 in the photooxidation of pterin was evaluated.

Oxidative stress in vitiligo: photo-oxidation of pterins produces H(2)O(2) and pterin-6-carboxylic acid.

Patients with vitiligo accumulate millimolar levels of H(2)O(2) in their epidermis. The recycling process of (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin in these patients is disrupted due to deactivation of 4a-OH-BH(4) dehydratase by H(2)O(2). The H(2)O(2) oxidation products 6- and 7-biopterin lead to the characteristic fluorescence of the affected skin upon Wood's light examination (UVA 351 nm). Here we report for the first time the presence and accumulation of pterin-6-carboxylic acid (P-6-COOH) in the epidermis of these patients. Exploring potential sources for P-6-COOH revealed that sepiapterin and 6-biopterin are readily photo-oxidised to P-6-COOH by UVA/UVB irradiation. Photolysis of sepiapterin and 6-biopterin produces stoichiometric H(2)O(2) under aerobic conditions, where O(2) is the electron acceptor, thus identifying an additional source for H(2)O(2) generation in vitiligo. A detailed analysis utilising UV/visible spectrophotometry, HPLC, TLC, and mass spectroscopy showed for sepiapterin direct oxidation to P-6-COOH, whereas 6-biopterin formed the intermediate 6-formylpterin (P-6-CHO) which is then further photo-oxidised to P-6-COOH.

Influence of light on activity of adenylate kinase and pterin-protein complexes from pea chloroplasts.

The activity of adenylate kinase (AK) and of pterin-protein complexes (PPC), whose proteins have adenylate kinase activity comparable to that of the enzyme was studied. It was established that light inhibits adenylate kinase activity and that this effect is partially eliminated by phosphate ions. The forward and reverse reactions catalyzed by AK and PPC were studied and it was found that the activity of native protein complexes is different in the forward and reverse reactions. The thermostable protein both of adenylate kinase and of the pterin-protein complexes had identical activity in the ADP dismutation and the reverse reaction.