RESUMO
Hemoglobin (Hb) Esch, is an alpha1 variant, expressed at less than 5%, resulting from the duplication of the 12 nucleotides corresponding to CD65 through 68. The effect of this insertion is the repetition of the sequence Ala-Leu-Thr-Asn, which corresponds to the last turn of helix E. In this variant the presence of a one-turn elongated helix E causes instability and increased ligand affinity. Hb Esch was characterized by DNA sequencing and confirmed by electrospray mass spectrometry. Functional studies were performed by flash photolysis measurements on a fraction isolated by flatbed isoelectric focusing, which was enriched in the abnormal hemoglobin. Similar to other alpha chain variants due to short insertion (or deletion), Hb Esch probably results from a slipped mispairing mechanism. The stability of such modified proteins depends upon the region which is added or deleted and usually is more stable when involving a flexible loop or complete helix turn(s) near by.
Assuntos
Hemoglobinas Anormais/genética , Hemoglobinas/genética , Mutagênese Insercional , Fragmentos de Peptídeos/genética , Adulto , Sequência de Aminoácidos , Duplicação Gênica , Variação Genética , Hemoglobinas Anormais/biossíntese , Humanos , Masculino , Portugal , Talassemia alfa/diagnóstico , Talassemia alfa/genéticaRESUMO
Hemoglobin Lyon-Bron was found in two members of a family of German ascent presenting with a moderate normocytic anemia. In this alpha 2 globin variant, the N-terminal valine of the chain was replaced by an alanine. Electrospray mass spectrometry of the alpha chain showed that, as normally, the initiator methionine was cleaved during globin processing but that the N alpha-terminal group was totally acetylated. This resulted in structural modifications of a region crucial for oxygen binding. As a consequence, hemoglobin Lyon-Bron displayed both a reduced chloride effect and a decreased oxygen affinity, this last point explaining the apparent anemia.
Assuntos
Globinas/genética , Hemoglobinas Anormais/genética , Oxigênio/metabolismo , Acetilação , Adolescente , Substituição de Aminoácidos , Sítios de Ligação/genética , Cloro/metabolismo , Saúde da Família , Feminino , Variação Genética , Globinas/química , Hemoglobinas Anormais/química , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Masculino , Pessoa de Meia-Idade , Oxiemoglobinas , Espectrometria de Massas por Ionização por Electrospray , TitulometriaAssuntos
Hemoglobina Falciforme/genética , Hemoglobinas Anormais/genética , Deleção de Sequência/genética , Talassemia alfa/genética , Adulto , Substituição de Aminoácidos , Camarões , Análise Mutacional de DNA , Feminino , Globinas/análise , Globinas/genética , Heterozigoto , Humanos , Focalização Isoelétrica , Mutação , Mapeamento de Peptídeos , Gravidez , Complicações Hematológicas na Gravidez/diagnósticoRESUMO
Laboratory methods allowing the detection and characterization of hemoglobin variants are reviewed. Protein chemistry techniques such as isoelectrofocusing, electrophoreses under various experimental conditions, cation exchange and reversed phase high performance liquid chromatography, are the most frequently used for the detection of variants. When associated with a few additional data they may lead to a presumptive diagnosis. DNA studies are also developed in many laboratories. Final identification of a variant may be achieved either by molecular biology techniques or by protein sequence analysis in which mass spectrometry now occupies a key position.
Assuntos
Técnicas de Laboratório Clínico , Hemoglobinas Anormais/análise , Técnicas de Química Analítica/métodos , Técnicas de Laboratório Clínico/instrumentação , Variação Genética , Hemoglobinas Anormais/genética , Humanos , Análise de Sequência de DNA , Análise de Sequência de Proteína/métodosAssuntos
Anemia Hemolítica Congênita/sangue , Globinas/genética , Hemoglobinas Anormais/análise , Complicações Hematológicas na Gravidez/sangue , Adulto , Substituição de Aminoácidos , Fenômenos Químicos , Físico-Química , Cromatografia Líquida de Alta Pressão , Etnicidade/genética , Feminino , França , Hemoglobinas Anormais/genética , Humanos , Focalização Isoelétrica , Espectrometria de Massas , Marrocos/etnologia , Mutação de Sentido Incorreto , GravidezRESUMO
Hb Sitia [beta128(H6)Ala-->Val] was found in a Greek female with slightly reduced red blood cell indices. The abnormal hemoglobin was indistinguishable from Hb A by electrophoresis but eluted after Hb A on cation exchange high performance liquid chromatography. DNA sequence analysis revealed a GCT-->GTT mutation at codon 128, which is predicted to encode an Ala-->Val substitution. This was confirmed by mass spectrometry analyses of the beta-globin chain. Since alanine at beta128(H6) interacts with several amino acids of the alpha1beta1 contact, its replacement by a larger residue results in a mild instability of the molecule and slight modifications of the oxygen binding properties.
Assuntos
Substituição de Aminoácidos , Globinas/genética , Hemoglobinopatias/genética , Hemoglobinas Anormais/isolamento & purificação , Mutação de Sentido Incorreto , Adulto , Cromatografia por Troca Iônica , Códon/genética , Análise Mutacional de DNA , Eritropoetina/sangue , Feminino , Globinas/biossíntese , Globinas/isolamento & purificação , Grécia , Corpos de Heinz/ultraestrutura , Hemoglobinopatias/sangue , Hemoglobinas Anormais/química , Hemoglobinas Anormais/genética , Humanos , Cinética , Espectrometria de Massas , Oxigênio/metabolismo , Ligação Proteica , Conformação Proteica , RNA Mensageiro/sangue , Receptores da Transferrina/sangue , Transcrição GênicaRESUMO
Hb Mont Saint-Aignan [beta128(H6)Ala-->Pro] is a mildly unstable variant, associated with hemolytic anemia, marked microcytosis and increased alpha/beta biosynthetic ratio (1.55 versus 1.1 +/- 0.1 in the control). The abnormal chain was isolated by selective precipitation with isopropanol and the structural modification determined by protein chemistry methods (reversed phase high performance liquid chromatography and mass spectrometry). Possible mechanisms underlying the beta(+)-thalassemia-like expression of this variant are discussed.
Assuntos
Substituição de Aminoácidos , Anemia Hemolítica Congênita/genética , Globinas/genética , Hemoglobinopatias/genética , Hemoglobinas Anormais/isolamento & purificação , Mutação de Sentido Incorreto , Adulto , Sequência de Aminoácidos , Anemia Hemolítica Congênita/sangue , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Códon/genética , Feminino , Globinas/biossíntese , Hemoglobinopatias/sangue , Hemoglobinas Anormais/química , Hemoglobinas Anormais/genética , Humanos , Espectrometria de Massas , Pessoa de Meia-Idade , Dados de Sequência Molecular , Oxigênio/metabolismo , Gravidez , Complicações Hematológicas na Gravidez/sangueAssuntos
Substituição de Aminoácidos , Globinas/genética , Hemoglobinopatias/genética , Hemoglobinas Anormais/genética , Hemoglobinas Anormais/isolamento & purificação , Mutação de Sentido Incorreto , Camarões/etnologia , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , França , Hemoglobinopatias/sangue , Hemoglobinas Anormais/química , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A procedure for the separation and identification of small peptides from the water-soluble fraction of a goat cheese was developed. The water-soluble extract was ultrafiltered (1000 Da membrane cutoff), and peptides were isolated by sequential chromatography: size exclusion chromatography (HPLC-grade water), anion exchange chromatography (phosphate buffer gradient), and semipreparative reverse-phase high-performance liquid chromatography (water/acetonitrile gradient). The fractions obtained were analyzed by combined mass spectrometry methods including electrospray ionization, liquid secondary ionization, and tandem mass spectrometry to identify and to confirm the sequences of 28 tri- to octapeptides naturally appearing in goat cheese during ripening. Among these peptides, 26 are produced by degradation of caseins but do not correspond to the known specific cleavages due to chymosin. Only low correlation was found between hydrophobicity of peptides and HPLC elution time with acetonitrile gradient.
Assuntos
Queijo/análise , Cabras , Espectrometria de Massas , Oligopeptídeos/isolamento & purificação , Sequência de Aminoácidos , Animais , Fenômenos Químicos , Físico-Química , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Oligopeptídeos/análise , Oligopeptídeos/química , Solubilidade , ÁguaAssuntos
Hemoglobina Fetal/genética , Hemoglobinas Anormais/genética , Substituição de Aminoácidos , Cromatografia Líquida de Alta Pressão , França/etnologia , Variação Genética , Globinas/genética , Humanos , Recém-Nascido , Peso Molecular , Triagem Neonatal , Portugal/etnologia , Espectrometria de Massas por Ionização por ElectrosprayAssuntos
Hemoglobinas Anormais/genética , Adulto , Substituição de Aminoácidos , Análise Mutacional de DNA , Variação Genética , Hemoglobinas Anormais/química , Hemoglobinas Anormais/metabolismo , Humanos , Focalização Isoelétrica , Masculino , Oxigênio/metabolismo , Estrutura Quaternária de Proteína , Subunidades ProteicasRESUMO
Hb Bushey, found in a Chinese baby and his father, is a new variant with a point mutation leading to the substitution Phe-->Leu at position beta122. Hb Casablanca, found in a family from Morocco, is a further example of a hemoglobin variant that carries two abnormalities in the same chain; the first is identical to that of Hb Bushey and the second to that of Hb J-Antakya [beta65 (E9)Lys-->Met]. Structural abnormalities of both Hbs were determined by protein chemistry methods including electrospray and tandem mass spectrometry. Their stability and oxygen binding properties were found to be identical to those of Hb A. Various mechanisms that may lead to two point mutations in the same chain are reviewed briefly.
Assuntos
Hemoglobinas Anormais/química , Hemoglobinas Anormais/genética , 2,3-Difosfoglicerato/farmacologia , Adulto , Substituição de Aminoácidos , Povo Asiático/genética , Cromatografia Líquida de Alta Pressão , Saúde da Família , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Variação Genética , Globinas/química , Globinas/genética , Hemoglobinas Anormais/efeitos dos fármacos , Humanos , Recém-Nascido , Masculino , Marrocos/etnologia , Oxigênio/metabolismo , Mutação Puntual , Reino UnidoRESUMO
Nod factors are substituted N-acyl chito-oligomers secreted by plant symbiotic bacteria of the Rhizobium family. Substitutions on the oligosaccharide core specify their recognition by host plants. A method using tandem mass spectrometry is proposed to locate the O-acetyl and O-carbamoyl substituents on the nonreducing terminal residue of the chito-oligomers. As model compounds, all the positional isomers of monoacetyl and monocarbamoyl esters of 1-O-methyl-N-acetyl-alpha-D-glucosamine were synthesized. Oxonium ions (MH - CH3OH)+ were generated by liquid secondary ion mass spectrometry (LSIMS) and their decomposition was recorded on a tandem magnetic instrument. Large differences were observed in the relative abundances of ions resulting from elimination of water and of the O-ester substituent from metastable oxonium ions. Deuterium exchange reactions indicated parallel elimination pathways involving either exchangeable or carbon-linked hydrogens. The intensity ratios of some of the ions generated by collisions with helium atoms allowed the isomers to be distinguished. The main dissociation routes were identified. Metastable and collision-induced decomposition of the B1 ions from Nod factors of Sinorhizobium meliloti and Azorhizobium caulinodans resembled that of the 6-O-substituted N-acetylglucosamine models. Decomposition of the B1 ion from Mesorhizobium loti and Rhizobium etli Nod factors, was similar to that of 3-O-carbamoyl N-acetyl-glucosamine and different to that of the 4-O isomer. 6-O- and 3-O-carbamoylation specified by the nodU and nolO genes, respectively, of Rhizobium. sp. NGR234 were confirmed.
Assuntos
Acetilglucosamina/química , Oligossacarídeos/química , Ésteres , Fungos/química , Fungos/enzimologia , OniocompostosRESUMO
Two new fetal hemoglobin variants affecting the Ggamma chain are reported. Hb F-Clamart was found during investigation of a French newborn who presented with a mild microcytemia. The second variant was found during neonatal screening for hemoglobinopathies of 30,000 babies from a population-at-risk living in the Paris region. It was named Hb F-Ouled Rabah because its structural modification and ethnic distribution is similar to that of Hb D-Ouled Rabah [beta19(B1)Asn-->Lys]. Hb F-Ouled Rabah is clinically silent and occurs at a frequency of ca. 0.1% in newborns originating from Maghreb. Structural characterization of both variants was done by protein chemistry methods, including amino acids analysis and mass spectrometry.
Assuntos
Hemoglobina Fetal/química , Hemoglobina Fetal/genética , Globinas/química , Globinas/genética , Hemoglobinas Anormais/química , Hemoglobinas Anormais/genética , África do Norte/etnologia , Argélia/etnologia , Substituição de Aminoácidos , Cromatografia Líquida de Alta Pressão , Saúde da Família , França/epidemiologia , França/etnologia , Variação Genética , Heterozigoto , Humanos , Recém-Nascido , Peso Molecular , Marrocos/etnologia , Triagem Neonatal , Tunísia/etnologia , Talassemia alfa/genéticaRESUMO
Analysis of globin chains by reversed phase high performance liquid chromatography, used as an additional tool for characterizing hemoglobin variants, has led to the discovery of a new class of variants that display only differences in hydrophobicity. Two such variants are here described. Hb Ernz was found in a man of Italian origin who was polycythemic, and in two of his three daughters who were hematologically normal. Hb Renert, a slightly unstable variant, was found in a man from Cape Verde who also carried Hb S and presented with chronic hemolysis. The structural abnormalities were characterized by protein structure methods involving reversed phase high performance liquid chromatographic separations of globins and peptides, followed by mass spectrometry studies (electrospray, ion trap, tandem mass spectrometry).
