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1.
Biochim Biophys Acta ; 421(1): 87-96, 1976 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-1247590

RESUMO

Two simultaneous reactions take place between ferrihemoglobin and phenyldiazene in the absence of excess ferricyanide or of oxygen, namely the reduction of ferrihemoglobin to ferrohemoglobin and the binding of an exogenous ligand by ferrihemoglobin to form a compound with the optical spectrum of a ferrihemochrome. In the presence of excess ferricyanide, only the formation of a ferrihemochrome is observed. This compound differs from the ferrihemochrome induced by salicylate or by benzoate with respect to optical spectrum, concentration of inducer, and stability. One phenyl group is bound per heme, probably as phenyldiazene. Phenyl groups are also bound to globin, but phenyldiazene does not react anaerobically with thiols. Each ring-substituted isomer of methylphenyldiazene or bromophenyldiazene yields a different ferrihemochrome spectrum with ferrihemoglobin in the presence of ferricyanide. Only reduction of ferrihemoglobin occurs with 2- or 4-diazenylbenzoic acid in the absence of excess ferricyanide, but partial formation of ferrihemochrome occurs with 4-diazenylbenzoic acid in excess ferricyanide. The ability of an aryldiazene to bind quantitatively to ferrihemoglobin parallels the ability of the corresponding arylhydrazine to induce in vivo hemolysis.


Assuntos
Compostos Azo/sangue , Metemoglobina , Sítios de Ligação , Ferricianetos/sangue , Humanos , Iminas , Oxigênio/sangue , Ligação Proteica , Espectrofotometria , Espectrofotometria Ultravioleta
2.
Biochim Biophys Acta ; 672(2): 214-8, 1981 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-7225414

RESUMO

We have compared the rates of reaction of ortho and para substituted halophenylhydrazines with oxygen, and we have found that the reaction rates of these phenylhydrazines are accelerated by metal ions and oxyhemoglobin. Stimulation of the reaction rate by oxyhemoglobin was 20-times that by Fe3+ at the same concentration. In the presence of oxyhemoglobin, the initial decrease in the concentration of oxygen was followed by an increase. We propose that phenyldiazene produced from the oxidation of phenylhydrazine by oxyhemoglobin reduced oxygen to superoxide and caused the initial rapid decrease in oxygen concentration. The partial restoration of oxygen in the reaction mixture could be accounted for by the dismutation of superoxide to oxygen and hydrogen peroxide, and of hydrogen peroxide to oxygen and water.


Assuntos
Compostos Férricos , Ferro , Oxigênio , Oxiemoglobinas , Fenil-Hidrazinas , Adulto , Fenômenos Químicos , Química , Humanos
3.
Biochim Biophys Acta ; 564(3): 488-94, 1979 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-497224

RESUMO

Biosynthesis of the alpha and beta chains of rabbit and human adult hemoglobin is initiated with a methionyl residue, which is removed during elongation of the peptide chain. To study the initiation of biosynthesis of the delta chain of human fetal hemoglobin, fresh placental blood was used for labeling experiments with radioactive amino acids. Labeled nascent peptide chains were purified from the polysomal fraction of placental blood reticulocytes. The number of amino acid residues in nascent gamma chain at the time of removal of its N-terminal methionine was estimated to be 40--60 from the relative yields of labeled tryptic peptides.


Assuntos
Hemoglobina Fetal/biossíntese , Iniciação Traducional da Cadeia Peptídica , Animais , Feminino , Sangue Fetal/metabolismo , Humanos , Isoleucina/metabolismo , Substâncias Macromoleculares , Metionina/metabolismo , Placenta , Gravidez , Coelhos , Reticulócitos/metabolismo
4.
Biochim Biophys Acta ; 955(2): 220-30, 1988 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-3395625

RESUMO

Reactions of nitrosobenzene, phenyl isocyanide and their ring-substituted analogues with hemoglobin, ferrous phthalocyanine and a synthetic model compound of hemoglobin were investigated by optical, 1H-NMR and infrared spectroscopy. Complexes of chelated ferromesoheme, the model compound, with 2-methyl-, 2-ethyl, 2-isopropyl- or 2,6-disubstituted nitrosobenzene were less stable than its complex with nitrosobenzene. Formation of a complex of the model compound with 2-tert-butylnitrosobenzene was incomplete. Previous studies showed that 2,6-disubstituted nitrosobenzenes are not ligands of ferrohemoglobin. In the present work 2,6-dimethylphenyl isocyanide was found to be a ligand of ferrohemoglobin. These results are consistent with binding of the nitrogen of the nitroso group of nitrosobenzene and of the carbon of the isocyanide group of phenyl isocyanide to ferroheme. The same bonding modes of these ligands to ferrous phthalocyanine were inferred from ring-current-induced shifts in the 1H-NMR spectra of the respective complexes.


Assuntos
Cianatos/metabolismo , Hemoglobinas/metabolismo , Indóis/metabolismo , Isocianatos , Mesoporfirinas/metabolismo , Compostos Nitrosos/metabolismo , Compostos Organometálicos/metabolismo , Porfirinas/metabolismo , Cloretos/metabolismo , Espectroscopia de Ressonância Magnética , Espectrofotometria Infravermelho
5.
J Thorac Cardiovasc Surg ; 120(5): 947-56, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11044321

RESUMO

OBJECTIVE: The objective of this study was to examine the feasibility of human interleukin 10 gene transfer into rat lung isografts and to investigate the effect of gene transfer on subsequent ischemia-reperfusion injury. METHODS: Male F344 rats were divided into 4 groups and underwent left lung isotransplantation. Twenty-four hours before harvest, 5 x 10E9 pfu (group I, n = 6) or 1 x 10E10 pfu (group II, n = 7) of AdRSVhIL-10 was intravenously administered to donor rats. In group I-C (n = 6) and group II-C (n = 6), serving as controls, 5 x 10E9 pfu and 1 x 10E10 pfu of AdCMVLacZ were administered, respectively. Grafts were preserved for 18 hours at 4 degrees C before implantation and assessed 24 hours after reperfusion. Transgene expression of human interleukin 10 was assessed by both reverse transcriptase-polymerase chain reaction and immunohistochemistry. Graft inducible nitric oxide synthase, tumor necrosis factor alpha, intercellular adhesion molecule-1, growth-regulated gene product/cytokine-induced neutrophil chemoattractant-1, and monocyte chemotactic protein-1 mRNA expression were assessed by reverse transcriptase-polymerase chain reaction. Isograft gas exchange, exhaled nitric oxide, and myeloperoxidase activity were also analyzed. RESULTS: Dose-dependent transgene expression was detected by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Arterial PO (2) in groups I (164.72 +/- 85.3 mm Hg) and II (153.19 +/- 113 mm Hg) was significantly higher than in groups I-C (82.37 +/- 19.1 mm Hg) and II-C (77.95 +/- 33.4 mm Hg) (P =.022 and P =.031, respectively). Arterial PCO (2) in group I (33.40 +/- 6.80 mm Hg) was significantly lower than in group I-C (51.23 +/- 11.9 mm Hg) (P =.0096). Myeloperoxidase activity in group II (0.083 +/- 0.031 DeltaOD. min(-1). mg(-1)) was significantly lower than in group II-C (0.117 +/- 0.028 DeltaOD. min(-1). mg(-1)) (P =.044). The inducible nitric oxide synthase mRNA expression in group II (0.627 +/- 0.28) was significantly lower than in group II-C (1.125 +/- 0.63) (P =. 039). CONCLUSION: Adenovirus-mediated human interleukin 10 gene transfer in vivo into lung isografts ameliorates subsequent ischemia-reperfusion injury. This results in improved graft gas exchange, reduced neutrophil sequestration, and down-regulation of graft inducible nitric oxide synthase mRNA expression.


Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Interleucina-10/genética , Transplante de Pulmão , Traumatismo por Reperfusão/prevenção & controle , Análise de Variância , Animais , Regulação para Baixo , Estudos de Viabilidade , Expressão Gênica , Vetores Genéticos , Humanos , Imuno-Histoquímica , Interleucina-10/metabolismo , Pulmão/enzimologia , Pulmão/patologia , Transplante de Pulmão/patologia , Masculino , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Peroxidase/genética , Peroxidase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos F344 , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
6.
J Thorac Cardiovasc Surg ; 122(1): 29-38, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11436034

RESUMO

BACKGROUND: Recent studies suggest that viral interleukin 10 suppresses alloimmune response in transplantation and that cationic lipids are one of the most promising nonviral vehicles for gene therapy. The aim of this study was to examine the effect of ex vivo lipid-mediated viral IL10 gene transfer into rat lung allografts on subsequent rejection. METHODS: Male F344 rats (RT1lvl) underwent left lung transplantation with allografts from Brown Norway rats (RT1n). Allografts were transvascularly transfected 15 minutes after harvest with 5 mL of 1:20-diluted (group 1, n = 7) or 1:40-diluted (group 2, n = 6) GL67-pCMVievIL-10 complex. Group 3 (n = 7), serving as the control group, received 1:40-diluted GL67-pCF1-chloramphenicol acetyltransferase complex. All allografts were preserved for 3 hours at 10 degrees C before transplantation. In all groups recipients were killed on postoperative day 5. Transgene expression of viral interleukin 10 was assessed by means of both reverse transcriptase-polymerase chain reaction and immunohistochemistry. Histologic rejection score, allograft gas exchange, exhaled nitric oxide level, and allograft cytokine mRNA expression were also assessed. RESULTS: Dose-dependent transgene expression of viral interleukin 10 was detected by means of both reverse transcriptase-polymerase chain reaction and immunohistochemistry. Allograft gas exchange (PaO2) in groups 1 (114.06 +/- 61.1 mm Hg) and 2 (108.58 +/- 35.7 mm Hg) was significantly better than that in group 3 (66.4 +/- 8.22 mm Hg; P =.020 and P =.023, respectively). The vascular rejection score in group 1 was significantly lower than that in group 3 (P =.032, Kruskal-Wallis test). Exhaled nitric oxide levels in group 2 (5.150 +/- 6.38 ppb) were significantly lower than those in group 3 (13.517 +/- 10.4 ppb; P =.039). Allograft interleukin 2 mRNA expression levels in group 1 (1.123 +/- 0.23 relative units) were significantly lower than those in group 3 (1.753 +/- 0.71 relative units; P =.038 vs group 3). CONCLUSIONS: Lipid-mediated ex vivo viral IL10 gene transfer into rat lung allografts improved graft gas exchange, reduced histologic rejection scores, downregulated graft interleukin 2 mRNA expression, and reduced exhaled nitric oxide levels by postoperative day 5. These results suggest a therapeutic potential of graft viral IL10 gene transfer as an effective immunosuppressive strategy against lung allograft rejection.


Assuntos
Técnicas de Transferência de Genes , Rejeição de Enxerto/imunologia , Terapia de Imunossupressão/métodos , Interleucina-10/uso terapêutico , Transplante de Pulmão/imunologia , Animais , Expressão Gênica , Vetores Genéticos , Rejeição de Enxerto/prevenção & controle , Imuno-Histoquímica , Interleucina-10/imunologia , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transgenes
7.
Ann Thorac Surg ; 67(2): 332-9, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10197650

RESUMO

BACKGROUND: This study was designed to investigate the efficacy of partial liquid ventilation (PLV) on acute allograft dysfunction after lung transplantation. METHODS: The canine left lung allotransplantation model was used, with the graft preserved in 4 degrees C low-potassium dextran glucose solution for 18 hours. The control group (n = 6) had conventional mechanical ventilation, and the PLV group (n = 6) had perfluorooctylbromide instilled into the airway 30 minutes after reperfusion. For 360 minutes, allograft function and hemodynamics were evaluated. After the evaluation, myeloperoxidase activity of the graft tissue was assayed. RESULTS: All dogs survived for 360 minutes. In the PLV group, PaO2, shunt fraction, and alveolar to arterial gradient for O2 were significantly better than those in the control group after 120, 180, and 120 minutes, respectively (p < 0.05). After 240 minutes, peak airway pressure became significantly lower than that in the control group (p < 0.05). The PaO2 at 360 minutes was 102 +/- 55 mm Hg in the control group and 420 +/- 78 mm Hg in the PLV group (p < 0.0001), and the peak airway pressure was 21.4 +/- 4.1 mm Hg in the control group and 14.7 +/- 5.0 mm Hg in the PLV group (p < 0.05). Myeloperoxidase activity in the PLV group was lower than that in the control group. CONCLUSIONS: The study shows that PLV alleviated acute allograft dysfunction after lung transplantation.


Assuntos
Fluorocarbonos , Transplante de Pulmão/fisiologia , Pulmão/irrigação sanguínea , Traumatismo por Reperfusão/terapia , Respiração Artificial , Animais , Cães , Fluorocarbonos/administração & dosagem , Hidrocarbonetos Bromados , Pulmão/patologia , Transplante de Pulmão/patologia , Oxigênio/sangue , Troca Gasosa Pulmonar/fisiologia , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Transplante Homólogo
8.
Ann Thorac Surg ; 71(4): 1126-33, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11308148

RESUMO

BACKGROUND: Recent studies suggest that viral interleukin-10 (vIL-10) suppresses alloimmune response in transplantation. Tissue mRNA expression of inducible nitric oxide synthase (iNOS) and exhaled nitric oxide (NO) levels have been observed to increase in lung allograft rejection. The aims of this study were to examine the feasibility of vIL-10 gene transfer into rat lung allografts and to investigate its effect on subsequent allograft rejection. METHODS: Male Lewis rats (RT1l) underwent left lung transplantation with allografts from Brown Norway rats (RT1n). The donor rats were endobronchially transfected 2 minutes before harvest with 400 microg (group I, n = 5), 600 microg (group II, n = 5), or 800 microg (group III, n = 5) of naked pCMVievIL-10. Group IV (n = 5) animals, serving as control, received 400 microg of naked pCF1-CAT. All recipients were sacrificed on postoperative day 5. Transgene expression of vIL-10 was assessed by both reverse transcriptase-polymerase chain reaction and immunohistochemistry. Allograft gas exchange, exhaled NO level, histologic rejection score, and mRNA expression of graft cyokines were also assessed. RESULTS: Transgene expression of lung graft vIL-10 was detected by both reverse transcriptase-polymerase chain reaction and immunohistochemistry. The iNOS mRNA expression in groups I, II, and III was significantly lower than that of group IV (p < 0.05, analysis of variance). Exhaled NO levels in groups I, II, and III were significantly lower than in group IV (p < 0.01, analysis of variance). There was no significant difference between groups with respect to gas exchange, peak airway pressure, or histologic rejection score. CONCLUSIONS: It appears that endobronchial transfection of naked vIL-10 plasmid in a rat lung allotransplant model is feasible and suppresses lung iNOS mRNA expression and exhaled NO levels. An association between iNOS upregulation and high exhaled NO levels in lung allograft resection was also noted.


Assuntos
Interleucina-10/genética , Transplante de Pulmão/imunologia , Transplante de Pulmão/métodos , Proteínas Virais/genética , Análise de Variância , Animais , Sequência de Bases , Broncoscopia , Regulação da Expressão Gênica , Rejeição de Enxerto , Sobrevivência de Enxerto , Imuno-Histoquímica , Masculino , Modelos Animais , Dados de Sequência Molecular , Óxido Nítrico/análise , Reação em Cadeia da Polimerase , Probabilidade , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Sensibilidade e Especificidade , Estatísticas não Paramétricas , Transfecção , Imunologia de Transplantes , Transplante Homólogo
9.
Acta Med Okayama ; 52(3): 131-7, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9661739

RESUMO

To study the effect of partial liquid ventilation (PLV) with perfluorocarbon on acute respiratory failure, 3 groups of 17 rabbits were examined to compare. After acute respiratory failure was induced by lung lavage with sea water in 12 of the 17 rabbits, 7 of the 12 rabbits were treated with conventional mechanical ventilation (AC group) and 5 of the 12 rabbits were treated with PLV using perfluorocarbon (AP group). The remaining 5 normal rabbits without acute respiratory failure were treated with PLV with perfluorocarbon as a control group (PL group). In the PL group, PaO2, PaCO2, blood pH, pulmonary compliance or pathological findings were not so changed after PLV. In the AC and AP groups, PaCO2 significantly increased, and in contrast, PaO2 and pulmonary compliance significantly decreased after lung lavage. However, these findings improved to almost the same levels as those of a control group within 2 h after the PLV treatment in the AP group, but in the AC group, these gradually deteriorated over time. As for the pathological findings, pulmonary vascular congestion, alveolar hemorrhage and inflammatory infiltration were observed in the AC group. However, these findings were not observed in the specimens of the AP group. From these results, PLV with perfluorocarbon was shown to be useful to improve gas exchange and pulmonary functions without major side effects.


Assuntos
Lavagem Broncoalveolar/métodos , Ventilação Pulmonar , Insuficiência Respiratória/terapia , Água do Mar , Doença Aguda , Animais , Dióxido de Carbono/metabolismo , Concentração de Íons de Hidrogênio , Complacência Pulmonar , Oxigênio/metabolismo , Pressão Parcial , Coelhos , Insuficiência Respiratória/etiologia
15.
Proc Natl Acad Sci U S A ; 67(2): 485-92, 1970 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-5289003

RESUMO

Phenylhydrazine is oxidized stoichiometrically by two equivalents of ferricyanide to produce phenyldiimide and ferrocyanide. This reaction permits accurate determination of the absorption maxima and molar absorption coefficients of phenyldiimide in aqueous solution. These values are identical or extremely similar to those of phenyldiimide produced by decarboxylation of phenylazoformic acid. Knowledge of the stoichiometry of the reaction confirms an earlier suggestion that phenyldiimide is the product of ferricyanide-oxidized phenylhydrazine that reacts with heme proteins. Homologous structures postulated for phenyldiimideferrihemoglobin and nitrosobenzeneferrohemoglobin suggest a common factor in the production of Heinz bodies after exposure of erythrocytes to phenylhydrazine and to arylamines.


Assuntos
Corpos de Heinz/metabolismo , Hemoglobinas/metabolismo , Iminas , Derivados de Benzeno , Eritrócitos/metabolismo , Ferricianetos , Oxirredução , Oxigênio , Fenil-Hidrazinas , Espectrofotometria , Raios Ultravioleta
16.
Biochem J ; 226(3): 767-71, 1985 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-3985944

RESUMO

Coupled oxidation of octaethylhaemin and phenylhydrazine hydrochloride with 16,16O2 and 18,18O2 produced octaethyl[16O]verdohaemochrome and octaethyl[18O]-verdohaemochrome respectively. Reactions of these products with 16,16O2 in the presence of phenylhydrazine hydrochloride yielded octaethyl[16O, 16O]biliverdin and octaethyl[18O, 16O]biliverdin. The same reactions with 18,18O2 yielded octaethyl[16O, 18O]biliverdin and octaethyl[18O, 18O]biliverdin. Accordingly, the two oxygen atoms of biliverdin are incorporated from different O2 molecules in separate reactions, namely the formation of verdohaemochrome and the conversion of verdohaemochrome into biliverdin. These reactions account for a "two-molecule mechanism' of biliverdin formation from haem with verdohaemochrome participating as an intermediate product.


Assuntos
Pigmentos Biliares/biossíntese , Heme/metabolismo , Biliverdina/análogos & derivados , Biliverdina/biossíntese , Peróxido de Hidrogênio/metabolismo , Hidrólise , Modelos Químicos , Oxigênio/metabolismo , Fenil-Hidrazinas/metabolismo
17.
Biochemistry ; 15(16): 3529-35, 1976 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-952876

RESUMO

By means of affinity chromatography on poly(adenylic acid) (poly(A))-fixed Sepharose, protein fractions having strong affinity to poly(A) were prepared from postribosomal supernatants of rabbit reticulocyte and rat liver. These fractions contained several proteins similar by electrophoretic analysis to rabbit globin messenger ribonucleoprotein. Protein fractions from both sources were shown to form ribonucleoprotein complexes with rabbit globin mRNA, and these complexes sedimented at the same rate as native globin messenger ribonucleoprotein. Binding of the proteins to RNA was not highly specific, since not only poly(A) but also other polynucleotides as poly(C) or poly(U) were bound to these proteins. Ribosomal RNAs, tRNA, or DNAs did not bind the proteins. In order to ascertain the function of the poly(A)-Sepharose purified proteins, their effects on translation of globin mRNA was studied in vitro. Addition of rabbit reticulocyte protein to globin mRNA resulted in no more than a slight stimulation of both alpha- and beta-chain synthesis. Poly(A)-Sepharose purified protein from rat liver, however, caused a marked preferential reduction of alpha-chain synthesis. These results showed that at least some proteins in the poly(A)-Sepharose purified proteins affect the translation of globin. This inference suggested a possibility that protein moiety in globin mRNP might be involved in control of globin synthesis.


Assuntos
Proteínas Sanguíneas/fisiologia , Globinas/biossíntese , Fígado/metabolismo , Biossíntese de Proteínas , Reticulócitos/metabolismo , Animais , Proteínas Sanguíneas/isolamento & purificação , Carcinoma Krebs 2/metabolismo , Cromatografia de Afinidade , Feminino , Peso Molecular , Poli A , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Coelhos , Ratos , Sefarose
18.
J Biol Chem ; 253(10): 3477-81, 1978 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-649583

RESUMO

The affinity of a substituted nitrosobenzene for ferrohemoglobin and the visible absorption spectrum of the resulting compound was influenced by the nature, position, and number of substituents on its benzene ring. Alkyl ring substituents inhibited the binding of nitrosobenzene to ferrohemoglobin, and binding was blocked by an ortho tertbutyl group or by a pair of ortho methyl groups. A single halogen atom increased binding affinity except that iodine decreased affinity, more at the ortho than at the para position. Binding occurred with a pair of ortho fluorine atoms but not with a pair of ortho chlorine or bromine atoms. These results favor a model of nitrosoarene ferrohemoglobin in which the iron of ferroheme is bonded to the nitrogen atom of the nitroso group since a bond to the oxygen atom would not be hindered by ortho substituents. The presence of a carboxylate substituent resulted in prevention of binding, which was reversed by esterification of the group. Large neutral para substituents, which cannot directly affect formation of the Fe-NO bond, inhibited binding, although not to the same degree as ortho substituents. It thus appears that the affinity of a substituted nitrosobenzene for ferrohemoglobin depends on interactions of the nonbonding part of the ligand molecule with the heme crevice as well as on the ability of its nitroso group to form a bond with the iron of heme. Nitrosoarenes also exhibited in varying degrees the property of removing an electron from ferrohemoglobin to form ferrihemoglobin.


Assuntos
Hemoglobinas , Compostos Nitrosos , Humanos , Cinética , Ligação Proteica , Espectrofotometria , Relação Estrutura-Atividade
19.
Proc Natl Acad Sci U S A ; 70(7): 2059-63, 1973 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-4516205

RESUMO

Synthetic alphaT1 and betaT1, the N-terminal tryptic peptides of alpha-chain and beta-chain of hemoglobin, and MetalphaT1 and MetbetaT1, peptides in which N-terminal methionyl residues are peptide-bonded to alphaT1 and betaT1, were prepared by the solid-state method of Merrifield. These synthetic peptides were used to establish conditions for chromatographic purification and analysis. When tryptic digests of nascent globin chains from rabbit and sickle-cell anemia reticulocytes incubated with (35)S- and (3)H-labeled amino acids were analyzed, radioactivity not present in tryptic digests of labeled hemoglobin appeared at the elution positions of MetalphaT1 and MetbetaT1. The fraction of nascent chains with N-terminal methionine was higher in sickle-cell anemia reticulocytes than in rabbit reticulocytes. If rate of peptide-chain elongation in polysomes is uniform, nascent human chains must attain a greater length before removal of the initial methionyl residue. Length of nascent chain at time of removal was calculated from two independent sets of data, one obtained from [(35)S]methionine incorporation into MetalphaT1, MetbetaT1, alphaT5, and betaT5, and the other obtained from [(3)H]lysine and [(3)H]valine incorporation into MetbetaT1 and betaT1.


Assuntos
Anemia Falciforme/sangue , Hemoglobinas Anormais/biossíntese , Hemoglobinas/biossíntese , Metionina/metabolismo , Reticulócitos/metabolismo , Animais , Cromatografia , Hemoglobina Falciforme/biossíntese , Humanos , Técnicas In Vitro , Lisina/metabolismo , Biossíntese Peptídica , Elongação Traducional da Cadeia Peptídica , Peptídeos/isolamento & purificação , Coelhos , Isótopos de Enxofre , Trítio , Valina/metabolismo
20.
Proc Natl Acad Sci U S A ; 79(5): 1393-7, 1982 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-6951184

RESUMO

Several studies have shown that both terminal oxygen atoms of biliverdin are derived from molecular oxygen. Since the conversion of verdohemochrome to biliverdin has been assumed to be hydrolytic, these findings seemed to exclude verdohemochrome as an intermediate in the degradation of heme to biliverdin. Coupled oxidation of myoglobin and ascorbate yielded a pure preparation of verdohemochrome IX alpha. The structure and ferrous state of this product were determined from its composition, ligand reactions, 1H NMR spectrum, and conversion to biliverdin IX alpha dimethyl ester. Reaction with ascorbate and 18O2 converted this compound to biliverdin that contained an atom of 18O. Successive treatment of verdohemochrome, first oxidation with H2O2 and then reduction with phenylhydrazine, yielded the iron complex of biliverdin. These results showed that hydrolysis is not an obligatory step in the conversion of verdohemochrome to biliverdin and, moreover, indicated how heme can be converted, with verdohemochrome as an intermediate, into biliverdin in which the two terminal oxygen atoms are derived from different O2 molecules.


Assuntos
Pigmentos Biliares , Ácido Ascórbico , Biliverdina , Hemoglobinas , Mioglobina , Oxirredução , Análise Espectral
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