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We described the clinical, laboratory and molecular characteristics of individuals with Hb S (HBB: c.20A>T)/β-thalassemia (Hb S/β-thal) participating in the Recipient Epidemiology and Donor Evaluation Study (REDS-III) Brazil Sickle Cell Disease cohort. HBB gene sequencing was performed to genotype each β-thal mutation. Patients were classified as Hb S/β0-thal, Hb S/β+-thal-severe or Hb S/β+-thal based on prior literature and databases of hemoglobin (Hb) variants. Characteristics of patients with each β-thal mutation were described and the clinical profile of patients grouped into Hb S/β0-thal, Hb S/β+-thal and Hb S/β+-thal-severe were compared. Of the 2793 patients enrolled, 84 (3.0%) had Hb S/β0-thal and 83 (3.0%) had Hb S/β+-thal; 40/83 (48.2%) patients with Hb S/β+-thal had mutations defined as severe. We identified 19 different β-thal mutations, eight Hb S/β0-thal, three Hb S/β+-thal-severe and eight Hb S/β+-thal. The most frequent β0 and β+ mutations were codon 39 (HBB: c.118C>T) and IVS-I-6 (T>C) (HBB: c.92+6T>C), respectively. Individuals with Hb S/β0-thal had a similar clinical and laboratory phenotype when compared to those with Hb S/β+-thal-severe. Individuals with Hb S/β+-thal-severe had significantly lower total Hb and Hb A levels and higher Hb S, white blood cell (WBC) count, platelets and hemolysis markers when compared to those with Hb S/β+-thal. Likewise, individuals with Hb S/β+-thal-severe showed a significantly higher occurrence of hospitalizations, vaso-occlusive events (VOE), acute chest syndrome (ACS), splenic sequestration, blood utilization, and hydroxyurea (HU) therapy.

Filing Date: 2006-07-07.-- Priority Data: PT 103303 (2005-07-07), The present invention refers to an acceleration process for macromolecular fragmentation in liquid medium, in gel or in a solid support, by means of joint application of enzymes and ultrasounds, as well as isotopes. The macromolecules, in any of these liquids, are mixed with enzymes and inserted in a recipient (2) and exposed to an ultrasonic radiation field (1). The combined effect of the enzymes and of the ultrasounds allows accomplishing, in time ranges between 10 s and 600 s, the fragmentation of macromolecules or whole proteomes, with the correspondent formation of their constituent lower molecular units.

Filing Date: 2006-07-07.--Priority Data: PT 103303 (2005-07-07), The present invention refers to an acceleration process for macromolecular fragmentation in liquid medium, in gel or in a solid support, by means of joint application of enzymes and ultrasounds, as well as isotopes. The macromolecules, in any of these liquids, are mixed with enzymes and inserted in a recipient (2) and exposed to an ultrasonic radiation field (1). The combined effect of the enzymes and of the ultrasounds allows accomplishing, in time ranges between 10 s and 600 s, the fragmentation of macromolecules or whole proteomes, with the correspondent formation of their constituent lower molecular units.

The gene encoding cytochrome c nitrite reductase (NrfA) from Desulfovibrio desulfuricans ATCC 27774 was sequenced and the crystal structure of the enzyme was determined to 2.3-Å resolution. In comparison with homologous structures, it presents structural differences mainly located at the regions surrounding the putative substrate inlet and product outlet, and includes a well defined second calcium site with octahedral geometry, coordinated to propionates of hemes 3 and 4, and caged by a loop non-existent in the previous structures. The highly negative electrostatic potential in the environment around hemes 3 and 4 suggests that the main role of this calcium ion may not be electrostatic but structural, namely in the stabilization of the conformation of the additional loop that cages it and influences the solvent accessibility of heme 4. The NrfA active site is similar to that of peroxidases with a nearby calcium site at the heme distal side nearly in the same location as occurs in the class II and class III peroxidases. This fact suggests that the calcium ion at the distal side of the active site in the NrfA enzymes may have a similar physiological role to that reported for the peroxidases. publishersversion published

NIGMS NIH HHS (GM 39803) NIGMS NIH HHS (GM 47295) The dissimilatory nitrite reductase from Desulfovibrio desulfuricans ATCC 27774 catalyzes the reduction of nitrite to ammonia. Previous spectroscopic investigation revealed that it is a hexaheme cytochrome containing one high spin ferric heme and five low spin ferric hemes in the oxidized enzyme. The current study uses the high resolution of Mossbauer spectroscopy to obtain redox properties of the six heme groups. Correlating the Mossbauer findings with the EPR data reveals the pair-wise spin-spin coupling among four of the heme groups. The other two hemes are found to be magnetically isolated. Reduction with dithionite and reaction with CO further indicate that only the high spin heme is capable of binding small exogenous ligands. These results confirm our previous finding that Desulfovibrio desulfuricans nitrite reductase contains six heme groups and that the high spin ferric heme is the substrate and inhibitor binding site. publishersversion published

Wellcome Trust NIGMS NIH HHS (GM 47295) Mössbauer and electron paramagnetic resonance (EPR) spectroscopies were used to characterize the diheme cytochrome c peroxidase from Paracoccus denitrificans (L.M.D. 52.44). The spectra of the oxidized enzyme show two distinct spectral components characteristic of low spin ferric hemes (S = 1/2), revealing different heme environments for the two heme groups. The Paracoccus peroxidase can be non-physiologically reduced by ascorbate. Mössbauer investigation of the ascorbate-reduced peroxidase shows that only one heme (the high potential heme) is reduced and that the reduced heme is diamagnetic (S = 0). The other heme (the low potential heme) remains oxidized, indicating that the enzyme is in a mixed valence, half-reduced state. The EPR spectrum of the half-reduced peroxidase, however, shows two low spin ferric species with gmax = 2.89 (species I) and gmax = 2.78 (species II). This EPR observation, together with the Mössbauer result, suggests that both species are arising from the low potential heme. More interestingly, the spectroscopic properties of these two species are distinct from that of the low potential heme in the oxidized enzyme, providing evidence for heme-heme interaction induced by the reduction of the high potential heme. Addition of calcium ions to the half-reduced enzyme converts species II to species I. Since calcium has been found to promote peroxidase activity, species I may represent the active form of the peroxidatic heme. publishersversion published

Trabalho de projecto de mestrado, Ciências da Educação (Administração Educacional), 2009, Universidade de Lisboa, Faculdade de Psicologia e de Ciências da Educação Nível de preservação: critical, Criado em: 2009-08-31 15:33:46, Criado por: creator:MARTA, Alterado em: 2009-11-09 17:10:32, Modificado por: viewer:internal Made available in DSpace on 2010-07-21T10:52:50Z (GMT). No. of bitstreams: 36 18863_ulsd_dep.17604_Texto_do_relatorio_final.pdf: 3813591 bytes, checksum: c0b3a81f626af0be3451f7c912847cea (MD5) 18864_ulsd_dep.17604_ANEXO_1.pdf: 51815 bytes, checksum: 9572db1bf112b2b4ff690c2166b0f38e (MD5) 18865_ulsd_dep.17604_ANEXO_2.pdf: 48002 bytes, checksum: 621013d2831695708d7259003c5e5c6c (MD5) 18866_ulsd_dep.17604_ANEXO_3.pdf: 26107 bytes, checksum: 2eba8d494fbf7e7aeb5a8ac4c416acec (MD5) 18867_ulsd_dep.17604_ANEXO_4.pdf: 77861 bytes, checksum: 75427b1da1311e506c10e621b0fc9d5f (MD5) 18868_ulsd_dep.17604_ANEXO_5.pdf: 68162 bytes, checksum: 24fd6ec853f513cbab3a06d1545396d3 (MD5) 18869_ulsd_dep.17604_ANEXO_6.pdf: 73179 bytes, checksum: 31112db598a87868d98c78d1040d281d (MD5) 18870_ulsd_dep.17604_ANEXO_7.pdf: 47355 bytes, checksum: e87858e764a4d44226d31cf80b00a889 (MD5) 18871_ulsd_dep.17604_ANEXO_8.pdf: 66119 bytes, checksum: 42da74415a6a2dc371fa8f28d7720409 (MD5) 18872_ulsd_dep.17604_ANEXO_9.pdf: 352919 bytes, checksum: 34feb4c4020ec6e9f83c0c6d21e4fa3d (MD5) 18875_ulsd_dep.17604_Anexo1_Analise_descritiva.pdf: 93212 bytes, checksum: 8bef0368a0e5268174e5b9af05682794 (MD5) 18876_ulsd_dep.17604_Anexo1_Objectivos da entrevista.pdf: 48419 bytes, checksum: bc430c0e92e73ef5a60a40bd45629672 (MD5) 18877_ulsd_dep.17604_Anexo1_Protocolo.pdf: 165305 bytes, checksum: f1ffed981be8d61dc4973be6f9d9804e (MD5) 18878_ulsd_dep.17604_Anexo1_Sintese.pdf: 64037 bytes, checksum: fac4d06dfd426d1898f055d009195ca5 (MD5) 18879_ulsd_dep.17604_Anexo2_1_Observ.pdf: 49385 bytes, checksum: 0c570467e4291dcf832fe2878ef9b75c (MD5) 18880_ulsd_dep.17604_Anexo2_2_Observ.pdf: 42545 bytes, checksum: 4dc8f2cbd74d30e2de620361f60b1207 (MD5) 18881_ulsd_dep.17604_Anexo2_3_Observ.pdf: 48130 bytes, checksum: ed6ce0af3bfca900715d88589b356f53 (MD5) 18882_ulsd_dep.17604_Anexo2_4_Observ.pdf: 47202 bytes, checksum: 09742dfd9a880fba0bf9fb7255c8c992 (MD5) 18883_ulsd_dep.17604_Anexo2_5_Observ.pdf: 45668 bytes, checksum: cd03584be0e43fc9488ab79fe66097eb (MD5) 18884_ulsd_dep.17604_Anexo2_6_Observ.pdf: 47882 bytes, checksum: cf0a5d573107ac90f4feb7f561e092be (MD5) 18885_ulsd_dep.17604_Anexo2_Cruzamento.pdf: 54307 bytes, checksum: aa0e5856fd6392ed335aa9e8b451c085 (MD5) 18886_ulsd_dep.17604_Anexo2_Quadro.pdf: 31459 bytes, checksum: 0d422d8bf51cfa9ba8518d8137a7528c (MD5) 18889_ulsd_dep.17604_Anexo3_Guiao_da_entrevista.pdf: 66281 bytes, checksum: 9ef9c19c5d1e3cb1b7a44f0234f88a6c (MD5) 18890_ulsd_dep.17604_Anexo4_Caracterizacao.pdf: 46733 bytes, checksum: d6e63bcbefedcff94a2011cedd9edab2 (MD5) 18891_ulsd_dep.17604_Anexo4_Discurso.pdf: 39938 bytes, checksum: 1e3bb293407eb67674d12c4875625941 (MD5) 18892_ulsd_dep.17604_Anexo4_Texto_escrito.pdf: 53932 bytes, checksum: 5b4b87d2f1258e779fb7e935743feb59 (MD5) 18893_ulsd_dep.17604_Anexo5_1_07.pdf: 27767 bytes, checksum: 6f23c4fda6b20f6c29ce27079e99800a (MD5) 18894_ulsd_dep.17604_Anexo5_6_05.pdf: 34653 bytes, checksum: 18b45cd426dae682b8a4f74c07785c7c (MD5) 18895_ulsd_dep.17604_Anexo5_19_06.pdf: 31506 bytes, checksum: 845121a180697399a9b53b67fa375aae (MD5) 18896_ulsd_dep.17604_Anexo5_20_06.pdf: 33620 bytes, checksum: afc452a7ce8036795e05ef9812292ce0 (MD5) 18897_ulsd_dep.17604_Anexo5_21_04.pdf: 30462 bytes, checksum: a7b774460059744b3cad7f909f6a3ca6 (MD5) 18898_ulsd_dep.17604_Anexo5_21_05.pdf: 41255 bytes, checksum: 8220c1679ce19b36dc72e144905623ef (MD5) 18899_ulsd_dep.17604_Anexo5_23_04.pdf: 27724 bytes, checksum: 72956076f259441191f2bdcf4070fd1e (MD5) 18900_ulsd_dep.17604_Anexo5_27_01.pdf: 30972 bytes, checksum: 99fd7f462c771f4a5c229ef04ae4bd54 (MD5) 18901_ulsd_dep.17604_Anexo5_29_04.pdf: 33423 bytes, checksum: d7d78b8cbaba7dce3f64f7974c358b4c (MD5) 18817.xml: 9611 bytes, checksum: 5ae9ca1f795d9bfcfdab72bacf1efea0 (MD5) Previous issue date: 2009

J Biol Inorg Chem (2008) 13:1321–1333 DOI 10.1007/s00775-008-0416-1 Membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617 can be solubilized in either of two ways that will ultimately determine the presence or absence of the small (Iota) subunit. The enzyme complex (NarGHI) is composed of three subunits with molecular masses of 130, 65, and 20 kDa. This enzyme contains approximately 14 Fe, 0.8 Mo, and 1.3 molybdopterin guanine dinucleotides per enzyme molecule. Curiously, one heme b and 0.4 heme c per enzyme molecule have been detected. These hemes were potentiometrically characterized by optical spectroscopy at pH 7.6 and two noninteracting species were identified with respective midpoint potentials at Em=+197 mV (heme c) and -4.5 mV (heme b). Variable-temperature (4-120 K) X-band electron paramagnetic resonance (EPR) studies performed on both as-isolated and dithionite-reduced nitrate reductase showed, respectively, an EPR signal characteristic of a [3Fe-4S]+ cluster and overlapping signals associated with at least three types of [4Fe-4S]+ centers. EPR of the as-isolated enzyme shows two distinct pH-dependent Mo(V) signals with hyperfine coupling to a solvent-exchangeable proton. These signals, called "low-pH" and "high-pH," changed to a pH-independent Mo(V) signal upon nitrate or nitrite addition. Nitrate addition to dithionite-reduced samples at pH 6 and 7.6 yields some of the EPR signals described above and a new rhombic signal that has no hyperfine structure. The relationship between the distinct EPR-active Mo(V) species and their plausible structures is discussed on the basis of the structural information available to date for closely related membrane-bound nitrate reductases.

Protein Science (2002), 11:2464–2470 Desulfovibrio gigas desulforedoxin (Dx) consists of two identical peptides, each containing one [Fe-4S]center per monomer. Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli. The three forms of the protein, the two homodimers [Fe(III)/Fe(III)]Dx and [Zn(II)/Zn(II)]Dx, and the heterodimer [Fe(III)/Zn(II)]Dx, can be separated by ion exchange chromatography on the basis of their charge differences. Once separated, the desulforedoxins containing iron can be reduced with added dithionite. For NMR studies, different protein samples were prepared labeled with 15N or 15N + 13C. Spectral assignments were determined for [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx from 3D 15N TOCSY-HSQC and NOESY-HSQC data, and compared with those reported previously for [Zn(II)/Zn(II)]Dx. Assignments for the 13C shifts were obtained from an HNCA experiment. Comparison of 1H–15N HSQC spectra of [Zn(II)/Zn(II)]Dx, [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx revealed that the pseudocontact shifts in [Fe(II)/Zn(II)]Dx can be decomposed into inter- and intramonomer components, which, when summed, accurately predict the observed pseudocontact shifts observed for [Fe(II)/Fe(II)]Dx. The degree of linearity observed in the pseudocontact shifts for residues 8.5 Å from the metal center indicates that the replacement of Fe(II) by Zn(II) produces little or no change in the structure of Dx. The results suggest a general strategy for the analysis of NMR spectra of homo-oligomeric proteins in which a paramagnetic center introduced into a single subunit is used to break the magnetic symmetry and make it possible to obtain distance constraints (both pseudocontact and NOE) between subunits.