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| Author(s): |
Marilene Silva Oliveira
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Carlos. |
| Institution: | Universidade de São Paulo (USP). Instituto de Química de São Carlos (IQSC/BT) |
| Defense date: | 2008-04-10 |
| Examining board members: |
Marcel Tabak;
Luis Eduardo Almeida;
Daniel Rodrigues Cardoso;
Marilza Castilho;
Sayuri Miyamoto
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| Advisor: | Marcel Tabak |
| Abstract | |
Dipyridamole (DIP), 2,6-bis(dietanolamina)-4,8-dipiperidinapirimida-[5,4-d] pirimidina) is known for its vasodilating and antiplatelet aggregation activity, exhibiting also a potent antioxidant effect, strongly inhibiting lipid peroxidation. In the present work, the quenching of singlet molecular oxygen, O2(1Δg), by dipyridamole, RA47 and RA25 was analyzed in acetonitrile and aqueous acid solutions. Bimolecular quenching constants in acetonitrile (ACN) are consistent with an efficient physical quenching, presenting values slightly lower than the diffusion limit (kt = 3,4-6,8x108 M-1s-1). The quenching process occurs probably, via reversible charge transfer with the formation of an exciplex. Calculation of ΔGet associated to O2(1Δg) quenching corroborates with uncomplete electron transfer. In aqueous acid solutions (pH=3,0) the kt values for DIP and derivatives are 20-fold smaller as compared to ACN. The electrochemical properties of DIP in ACN are characterized by two consecutive one-electron processes with halfwave oxidation potentials of 0,30V and 0,67V vs SCE. However, in aqueous acid medium a single oxidation wave is observed involving a two-electron process (0,80 V vs SCE). Therefore, O2(1Δg) quenching is consistent with electrochemical data. The eletrooxidation of DIP leads to nonfluorescent products and a considerable change in the absorption spectra occurs. The mass spectrum of eletrooxidation products of DIP showed peaks with m/z 472, 269 and 519. In this work the photolysis of DIP in acetronitrile (ACN) and aqueous solutions at pH 3,0 was examined. In aqueous acid solutions DIP was oxidized by photosensitization with methylene blue and by a reaction with a clean chemical source of O2(1Δg), N,N\'-di(2,3-dihydroxypropyl)-1,4 naphtalenedipropanamide (DHPNO2). Photooxidation occurs predominantly by type II mechanism, where DIP is oxidized by O2(1Δg). Some products with low intensity and m/z of 472 were observed, in agreement with eletrooxidation. On the other hand, in ACN, DIP is photooxidated by type I mechanism via radicals generation or by excited triplet state species, as observed by EPR. However, the oxidation produces the same species in both solvents. A dioxetane can be formed as an intermediate of the reaction of the photo-oxidation. The observed product, with m/z 269, can correspond to half of the DIP molecule with an attached oxygen atom or the DIP molecule with two oxygen atom with two protons. The conclusion at this stage is that DIP is photooxidated involving either O2(1Δg) in aqueous solution or molecular oxygen in ACN. Another work involving the giant extracelular hemoglobin of Glossoscolex paulistus (HbGp) was performed. MALDI-TOF-MS analysis of the different subunits from the HbGp in the oxy- form was made. The results are compared to those reported for the homologous hemoglobin of Lumbricus terrestris (HbLt) and some tentative assignments are made for the observed polypeptides. The monomer d is found to exist in, at least, two major forms of identical proportions with masses of 16.355+-25 and 16.428+-24 Da, respectively. Two minor forms were also observed around 16 kDa for the monomers. Upon disulfide bonds reduction the peak associated to the trimer is absent in the mass spectrum, and new peaks assigned tentatively to the monomers a, b and c are observed. Their molecular masses were 18.258+-30 Da, 16.492+-24 Da and 17.363+-17 Da, respectively. Two Linker chains for HbGp were also observed at 25.817+-50 and 26.761+-16 Da. Finally, trimers (abc) were observed at 51-52 kDa. This partial characterization, performed for the first time, is an important step in the characterization of subunits of this giant hemoglobin. The effects of anionic sodium dodecyl sulfate (SDS) and cationic cethyltrimethylammonium chloride (CTAC) on the oligomeric structure of HbGp were also studied by MALDI-TOFMS. The data obtained with this technique show an effective interaction of cationic surfactant CTAC with the two isoforms of monomer d, d1 and d2, both in the whole protein as well as in the pure isolated monomer. The results show that up to 10 molecules of CTAC are bound to each isoform of the monomer. Differently, the mass spectra obtained for SDS-HbGp system showed that the interaction is, probably, significantly less effective as compared to CTAC-HbGp one. The acid pI of the protein around 5,5 is, probably, responsible for this behavior. (AU) | |