RESUMO
A new method for the purification of human erythrocyte phosphoglycerate-kinase involving affinity chromatography on dye-ligand media (Red A), in the presence of 3-phosphoglycerate and ATP, is described. The method is rapid and technically simple. The purity of the enzyme was verified by electrophoresis in polyacrylamide gel in the presence of sodium dodecylsulphate, by amino acid analysis and by immunoprecipitation in Ouchterlony plates. Peptide mapping of tryptic digests of the purified enzyme was performed and the immunoneutralization of the enzyme activity evaluated with rabbit antibodies.
Assuntos
Eritrócitos/enzimologia , Fosfoglicerato Quinase/sangue , Cromatografia de Afinidade , Corantes , Eletroforese em Gel de Poliacrilamida , Hemoglobinas , Humanos , Imunoquímica , Ligantes , Fosfoglicerato Quinase/isolamento & purificação , Dodecilsulfato de SódioRESUMO
Isoelectric focusing on thin layer of acrylamide gel has been used for the characterization of 79 different variants of hemoglobin A. This method has replaced the cellulose acetate electrophoresis in clinical laboratories in Martinique, Guadeloupe and Creteil for the detection of abnormal hemoglobins in populations at risk. Up to now 15,000 samples from adults have been evaluated. In addition this method has been used for the screening of 7,000 cord blood samples and for the prenatal diagnosis of severe hemoglobinopathies.
Assuntos
Sangue Fetal/análise , Hemoglobina Fetal/análise , Hemoglobinas Anormais/análise , Adulto , Eletroforese em Acetato de Celulose/métodos , Feminino , Hemoglobina A/análise , Humanos , Recém-Nascido , Focalização Isoelétrica/métodos , Programas de Rastreamento , Gravidez , Relação Estrutura-AtividadeRESUMO
Delayed type hypersensitivity (DTH) to bovine serum albumin (BSA) and to lipid-conjugated BSA were studied comparatively. Unlike the case of BSA with which no DTH can be detected with native antigen, injection of butyric-conjugated BSA (Bu-BSA) in sensitized mice provokes a typical DTH for an early and limited period. Alum-precipitated Bu-BSA (Al-Bu-BSA) provokes from the beginning a stronger DTH which persists a much longer period.
Assuntos
Hipersensibilidade Tardia/imunologia , Lipídeos/farmacologia , Soroalbumina Bovina/imunologia , Compostos de Alúmen/farmacologia , Animais , Butiratos/farmacologia , Bovinos , Feminino , Adjuvante de Freund/farmacologia , CamundongosRESUMO
Hemoglobin Bougardirey-Mali was detected by isoelectrofocusing during a screening in a 32 years old African, a native of Mali. This abnormal Hb, representing 35% of the total, exhibited the same pI as that of Hb F. In contrast, it was indistinguishable from Hb A in all the electrophoretic systems tested, and equally by its resistance to alkaline denaturation. Structural studies have shown that the abnormality was localized on the beta chain. A fingerprint of the tryptic digest of the aminoethylated beta chain indicated the absence of the beta T12 b. The presence of an abnormal beta T12 b was suspected in the T14-15 spot, as indicated by the intensity of staining and its amino acid composition. beta T12 b was isolated by chromatography on PA 35. Its sequential analysis by manual Edman-dansyl degradation showed that glycine 119 was replaced by a valine residue. This mutation is localized in a alpha 1 beta 1 contact, which makes the molecules slightly unstable. The clinical consequences of this mutation seem to be minor; similar observations have been reported for the other Hb mutated at the same locus, i.e. Hb Fannin-Lubbock beta 119 Gly leads to Asp.
