RESUMO
In order to get catalytic antibodies modelling peroxidases BALB/c mice have been immunized with iron(III)-alpha,alpha,alpha,beta-mesotetrakis-orthocarboxypheny l-porphyrin (Fe-(ToCPP))-KLH conjugates. Monoclonal antibodies have been produced by the hybridoma technology. Three antibodies, 2 IgG1 and 1 IgG2a, were found to bind both Fe(ToCPP) and the free base ToCPPH2 with similar binding constants. None of those antibodies was found to bind tetraphenylporphyrin. Those results suggest that the recognition of Fe(ToCPP) by the antibodies was mainly due to the binding of the carboxylate groups to some amino acid residues of the protein. True Kd values of 2.9 x 10(-9) M and 5.5 x 10(-9) M have been determined for the two IgG1-Fe(ToCPP) complexes. Those values are the best ones ever reported for iron-porphyrin-antibody complexes. UV-vis. studies have shown that the two IgG1-Fe(ToCPP) complexes were high-spin hexacoordinate iron(III) complexes, with no amino acid residue binding the iron, whereas the IgG2a-Fe(ToCPP) complex was a low-spin hexacoordinate iron(III) complex with two strong ligands binding the iron atom. Both IgG1-Fe(ToCPP) complexes were found to catalyze the oxidation of 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) 5-fold more efficiently than Fe(ToCPP) alone whereas the binding of IgG2a to this iron-porphyrin had no effect on its catalytic activity. kcat values of 100 min(-1) and 63 min(-1) and kcat/Km values of 105 M(-1) s(-1) and 119 M(-1) s(-1) have been found respectively for the two IgG1-Fe(ToCPP) complexes.
Assuntos
Anticorpos Catalíticos/metabolismo , Heme/metabolismo , Hemeproteínas/metabolismo , Metaloporfirinas/metabolismo , Peroxidases/metabolismo , Animais , Anticorpos Monoclonais , Benzotiazóis , Feminino , Haptenos/imunologia , Hemeproteínas/síntese química , Hemeproteínas/imunologia , Concentração de Íons de Hidrogênio , Imunoglobulina G , Indicadores e Reagentes , Cinética , Metaloporfirinas/síntese química , Metaloporfirinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Químicos , Oxirredução , Ácidos Sulfônicos/metabolismoRESUMO
Iron deficiency changed markedly the shape of the leaf chlorophyll fluorescence induction kinetics during a dark-light transition, the so-called Kautsky effect. Changes in chlorophyll fluorescence lifetime and yield were observed, increasing largely the minimal and the intermediate chlorophyll fluorescence levels, with a marked dip between the intermediate and the maximum levels and loss of the secondary peak after the maximum. During the slow changes, the lifetime-yield relationship was found to be linear and curvilinear (towards positive lifetime values) in control and Fe-deficient leaves, respectively. These results suggested that part of the Photosystem II antenna in Fe-deficient leaves emits fluorescence with a long lifetime. In dark-adapted Fe-deficient leaves, measurements in the picosecond-nanosecond time domain confirmed the presence of a 3.3-ns component, contributing to 15% of the total fluorescence. Computer simulations revealed that upon illumination such contribution is also present and remains constant, indicating that energy transfer is partially interrupted in Fe-deficient leaves. Photosystem II-enriched membrane fractions containing different pigment-protein complexes were isolated from control and Fe-deficient leaves and characterized spectrophotometrically. The photosynthetic pigment composition of the fractions was also determined. Data revealed the presence of a novel pigment-protein complex induced by Fe deficiency and an enrichment of internal relative to peripheral antenna complexes. The data suggest a partial disconnection between internal Photosystem II antenna complexes and the reaction center, which could lead to an underestimation of the Photosystem II efficiency in dark-adapted, low chlorophyll Fe-deficient leaves, using chlorophyll fluorescence.
RESUMO
Besides existing models of chemical or biotechnological origin for hemoproteins like peroxidases and cytochromes P450, catalytic antibodies (Abs) with a metalloporphyrin cofactor represent a promising alternative route to catalysts tailored for selective oxidation reactions. A brief overview of the literature shows that, until now, the first strategy for obtaining such artificial hemoproteins has been to produce antiporphyrin Abs, raised against various free-base, N-substituted, Sn-, Pd-, or Fe-porphyrins. Four of them exhibited, in the presence of the corresponding Fe-porphyrin cofactor, a significant peroxidase activity, with kcat/K(m) values of 10(2) to 5 x 10(3)/M/s. This value remained low when compared to that of peroxidases, probably because neither a proximal ligand of the Fe, nor amino acid residues participating in the catalysis of the heterolytic cleavage of the O-O bond of H2O2, have been induced in those Abs. This strategy has been shown to be insufficient for the elaboration of effective models of cytochromes P450, because only one set of Abs, raised against meso-tetrakis(para-carboxyvinylphenyl)porphyrin, was reported to catalyze the nonstereoselective oxidation of styrene by iodosyl benzene using a Mn-porphyrin cofactor, and attempts to generate Abs having binding sites for both the substrate and the metalloporphyrin cofactor, using as a hapten a porphyrin covalently linked to the substrate, were not successful. A second strategy is then proposed, which involves the chemical labeling of antisubstrate Abs with a metalloporphyrin. As an example, preliminary results are presented on the covalent linkage of an Fe-porphyrin to an antiestradiol Ab, in order to obtain semisynthetic catalytic Abs able to catalyze the selective oxidation of steroids.
Assuntos
Anticorpos Catalíticos/sangue , Proteínas Sanguíneas/imunologia , Animais , Sistema Enzimático do Citocromo P-450/imunologia , Sistema Enzimático do Citocromo P-450/metabolismo , Haptenos/imunologia , Humanos , Modelos Químicos , Porfirinas/imunologiaRESUMO
UNLABELLED: Neuroendocrine tumours (NETs) are a heterogeneous group of neoplasms whose management can be problematic. In many cases, multiple tumours may occur in the same patient or his or her family, and some of these have now been defined genetically, although in other cases the underlying gene or genes involved remain unclear. We describe a patient, a 63-year-old female, who was diagnosed with a medullary thyroid carcinoma (MTC), which was confirmed pathologically after thyroidectomy, but whose circulating calcitonin levels remained elevated after thyroidectomy with no evidence of metastatic disease. Subsequently, an entirely separate and discrete duodenal NET was identified; this was 2.8âcm in diameter and was removed at partial duodenectomy. The tumour stained immunohistochemically for calcitonin, and its removal led to persistent normalisation of the circulating calcitonin levels. There was no germline mutation of the RET oncogene. This is the first identification of a duodenal NET secreting calcitonin and also the first demonstration of a second tumour secreting calcitonin in a patient with MTC. We suggest that where calcitonin levels remain high after removal of a MTC a search for other NETs should be conducted. LEARNING POINTS: NETs are a complex and heterogeneous group of related neoplasms, and multiple tumours may occur in the same patient.Calcitonin can be produced ectopically by several tumours outside the thyroid.Persistently elevated calcitonin levels after removal of a MTC may not necessarily indicate persisting or metastatic disease from the tumour.The real prevalence of calcitonin-producing NETs may be underestimated, as serum determination is only recommended in the diagnosis of pancreatic NETs.
RESUMO
The topology of the binding site has been studied for two monoclonal antibodies 13G10 and 14H7, elicited against iron(III)-alpha,alpha,alpha,beta-meso-tetrakis(ortho-carboxyphenyl)porph yrin [alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin]], and which exhibit in the presence of this alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] cofactor a peroxidase activity. A comparison of the dissociation constants of the complexes of 13G10 and 14H7 with various tetra-aryl-substituted porphyrin has shown that: (a) the central iron(III) atom of alpha,alpha,alpha,beta-Fe[(o-COOHPh)4-porphyrin] is not recognized by either of the two antibodies; and (b) the ortho-carboxylate substituents of the meso-phenyl rings of alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] are essential for the recognition of the porphyrin by 13G10 and 14H7. Measurement of the dissociation constants for the complexes of 13G10 and 14H7 with the four atropoisomers of (o-COOHPh)4-porphyrinH2 as well as mono- and di-ortho-carboxyphenyl-substituted porphyrins suggests that the three carboxylates in the alpha, alpha, beta position are recognized by both 13G10 and 14H7 with the two in the alpha, beta positions more strongly bound to the antibody protein. Accordingly, the topology of the active site of 13G10 and 14H7 has roughly two-thirds of the alpha,alpha,alpha,beta-Fe[(o-COOHPh)4-porphyrin] cofactor inserted into the binding site of the antibodies, with one of the aryl ring remaining outside. Three of the carboxylates are bound to the protein but no amino acid residue acts as an axial ligand to the iron atom. Chemical modification of lysine, histidine, tryptophan and arginine residues has shown that only modification of arginine residues causes a decrease in both the binding of alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] and the peroxidase activity of both antibodies. Consequently, at least one of the carboxylates of the hapten is bound to an arginine residue and no amino acids such as lysine, histidine or tryptophan participate in the catalysis of the heterolytic cleavage of the O-O bond of H2O2. In addition, the amino acid sequence of both antibodies not only reveals the presence of arginine residues, which could be those involved in the binding of the carboxylates of the hapten, but also the presence of several amino acids in the complementary determining regions which could bind other carboxylates through a network of H bonds.