ABSTRACT
Inactivation of glutathione peroxidase correlates with the rate of hemoglobin chain oxidation. The enzyme inactivation is mainly present in those conditions where the autoxidation of the oxygenated chains is followed by transformation of the oxidized molecule into a hemichrome. Free hemoglobin chains have been encapsulated in human red blood cells by a dialysis technique that involves transient hypotonic hemolysis followed by isotonic resealing. Chain-loaded erythrocytes represent a good in vitro model of thalassemia. The presence of free human chains in the cell alters the intraerythrocytic glutathione peroxidase activity (alpha chains are more effective in the inactivation of the enzyme with respect to the beta chains).
Subject(s)
Erythrocytes/metabolism , Glutathione Peroxidase/blood , Hemoglobins/metabolism , Humans , In Vitro Techniques , Reactive Oxygen Species/metabolismABSTRACT
1. A discontinuous gradient in the region of 45-65% "Percoll" has been utilized for the separation of trout erythrocytes. Three different fractions were obtained. 2. We have evaluated antioxidant enzyme activities and membrane fluidity. The results indicated that catalase and glutathione peroxidase activities increased with the density of the fraction while the membrane fluidity was unchanged. 3. The observed results show a marked difference between nucleated (fish) and unnucleated (human) separated erythrocytes.
Subject(s)
Erythrocytes/chemistry , Trout/blood , Animals , Antioxidants , Cell Separation , Erythrocyte Aging , Erythrocytes/enzymology , Humans , Membrane Fluidity , Superoxide Dismutase/metabolismABSTRACT
Inactivation of erythrocyte GSH-peroxidase correlates with the rate of hemoglobin oxidation. The presence of superoxide dismutase and catalase only marginally reduces the rate of inactivation of the enzyme indicating that the loss of activity is not due to oxygen radicals produced during oxidation of hemoglobin. The inactivation of glutathione peroxidase is due by-and-large to the formation of hemichromes.
Subject(s)
Glutathione Peroxidase/antagonists & inhibitors , Hemeproteins/pharmacology , Hemoglobins/metabolism , Animals , Catalase/pharmacology , Enzyme Activation/drug effects , Erythrocytes/enzymology , Oxidation-Reduction , Superoxide Dismutase/pharmacology , Trout/bloodABSTRACT
Mini-myoglobin, obtained by limited proteolysis of horse heart myoglobin (residues 32 to 139), represents a good model for testing the correlation between an exon and a protein domain. We have shown that ligand binding kinetics, spectral and folding features of mini-myoglobin are very similar to those of native myoglobin. In order to develop further the analysis of the structure-function relationship in this mini-protein, mini-globin was reconstituted with the heme moiety in which iron is replaced by cobalt. The Soret absorption spectra of oxy and deoxy cobaltous mini-myoglobin are very similar to those of cobaltous myoglobin derivatives; in addition. Co-mini-myoglobin binds oxygen reversibly with an n value approximately 1 and a p50 value of 45 to 50 mm Hg (the same as Co-myoglobin). Oxy Co-mini-myoglobin shows a well-resolved electron paramagnetic resonance (e.p.r.) spectrum typical of an oxygenated hemoprotein, while the spectrum of the deoxy derivative, although similar to that of deoxy Co-myoglobin, displays a lower resolution of the complex hyperfine structure. Moreover, photodissociation experiments on oxy Co-mini-myoglobin allow e.p.r. detection of an intermediate state, already observed in most hemoproteins and diagnostic for the interaction of bound oxygen with the distal histidine residue. Thus, reconstitution of mini-globin with cobalt protoprophyrin IX has provided, for the first time, a stable oxygenated complex that reflects a correct folding of the protein surrounding the heme pocket and possesses the functional behaviour typical of a hemoprotein.
Subject(s)
Cobalt/chemistry , Electron Spin Resonance Spectroscopy/methods , Myoglobin/chemistry , Oxygen/metabolism , Peptide Fragments/chemistry , Animals , Horses , Kinetics , Ligands , Myocardium/chemistry , Photolysis , Protoporphyrins/chemistry , SpectrophotometryABSTRACT
Hemoglobin Volga is a rare unstable hemoglobin in which there is a replacement of an internal alanine residue, beta 27 (B9), by an aspartate. From a clinical point of view it is characterized by a moderately severe Heinz body hemolytic anemia. A possible clue to the cause of the hemolysis is the increased vulnerability to oxidation of Hb Volga, with increased free radical turnover and consequent damage to the red cell membrane. Splenectomy performed on carriers of Hb Volga may have a positive outcome leading to improvements in both the clinical condition of the patient and hematological variables such as hemoglobin concentration and bilirubin concentration. With the aim of defining a more complete biochemical picture of the beneficial effect of splenectomy in this disease, we have evaluated some enzymic activities of the red cells of a young patient with Hb Volga disease, before and after splenectomy. In particular, we have investigated the activity of superoxide dismutase (superoxide: superoxide oxidoreductase), glutathione peroxidase (GSH peroxidase, glutathione: hydrogen-peroxidase oxidoreductase) and catalase (hydrogen-peroxide: hydrogen-peroxide oxidoreductase) that catalyze reactions relevant to the steady-state concentration of potentially toxic oxygen derivatives such as O2- and H2O2. Besides, we have carried out experiments on the erythrocytic membrane to evaluate eventual changes on the chemical (i.e. peroxidation) and physico-chemical (i.e. fluidity) properties following surgery.
