Antioxidant enzymes in erythrocytes from old and diet restricted old rats.

Diet restriction, prolonging the lifespan of rodents, represents an interesting model for gerontological studies. We analyzed the activity of antioxidant enzymes, Superoxide Dismutase, Catalase and Glutathione Peroxidase in erythrocytes from young, old and old food restricted Wistar rats. Diet restriction was applied feeding the animals on every-other-day schedule starting from the age of 3.5 months. The age-dependent decrease of Catalase and Glutathione Peroxidase activities was prevented by food restriction, whereas Superoxide Dismutase activity was not influenced either by aging and dietary intervention. Present results support the hypothesis that diet restriction increases the protection of cell structure against the peroxidative damage, preserving the activity of antioxidant enzymes.

Evidence for cooperative hydrogen ligand binding to sperm whale metmyoglobin.

Potentiometric titrations of sperm whale metmyoglobin from pH 10 to 3 shows the well-known exposure of groups between pH 4.5 and 4. However the reverse titration, at low protein concentration, results in the regeneration of its ionic property in the form of a reversible hysteresis, which was obtained by titrating the same solution twice. The Soret intensity band indicates reversibility for about 94%. Apomyoglobin shows an acid-base reversible titration from pH 9.5 to 3. Since the analysis by electrostatic interaction models do not adequately describe the overall ionic equilibria of metmyoglobin, a cooperative model was introduced. The model exactly reproduces the titration curve, indicating that 6 groups ionize with a cooperative coefficient of 9. The cooperative model explains the anomalous behaviour toward hydrogen ions and the incomplete spectral reversibility of sperm whale metmyoglobin as a molecular mechanism with physiological significance.

Calorimetric studies of oxyhemoglobin ligand dissociation. III. Evidence for a hemoglobin-catalase-superoxide dismutase integrated system in the oxygen depletion by dithionite.

Calorimetric studies of the effect of superoxide dismutase and/or catalase on the reduction of dioxygen into water by dithionite in oxyhemoglobin have been carried out and the results compared with those in red cell hemolysates. In the absence of the enzymes the stoichiometry (moles dithionite/mole dioxygen) is less than the value of 2:1 which was found previously in red cell hemolysates [Forlani et al., J. Inorg. Biochem. 20, 147-155 (1984)]. In the presence of either superoxide dismutase or catalase alone the stoichiometry increases but is still less than 2:1. In the presence of both enzymes the stoichiometry and the shape of the thermogram is that previously observed for hemolysates, suggesting the presence of a hemoglobin-catalase-superoxide dismutase integrated system. The absence of a calorimetric signal for hydrogen peroxide in the reduction of oxyhemoglobin in the presence of superoxide dismutase suggests a wider biological role of superoxide dismutase than previously thought.

Calorimetric studies of oxyhemoglobin dissociation. II. Erythrocytic oxygen depletion by sodium dithionite.

Dithionite causes the depletion of dioxygen from suspensions of erythrocytes by reduction of the external dioxygen and not by diffusion into the cell. The molar enthalpy for the reduction shows a small difference with respect to the values found for free hemoglobin; and the normal stoichiometry of 2 moles dithionite/mole dioxygen found there is not observed with erythrocytes. At low hematocrit, the stoichiometry is 2.6:1 and decreases to 1.5:1 at high hematocrit. The change is not due to differences in the hemoglobin saturation or to an inability of dithionite to reduce all dioxygen present at the higher hematocrit. Neither catalase nor peroxidase added to the extracellular volume significantly alters the stoichiometry or the enthalpy of dioxygen reduction by dithionite. Addition of superoxide dismutase, however, restores the normal stoichiometry at high hematocrit and further increases the stoichiometry at low hematocrit. The calorimetrical signal of hydrogen peroxide, clearly seen with free dioxygen, is not present with erythrocytes. In all these cases the total heat evolved is the same.

The effect of pH and D-glycerate 2,3-bisphosphate on the O2 equilibrium of normal and SH(beta 93)-modified human hemoglobin.

The present paper reports data for the effect of pH and D-glycerate 2,3-bisphosphate (D-glycerate-2,3-P2) on the oxygen equilibrium of normal and SH(beta 93)-modified human hemoglobin. At sufficiently high D-glycerate-2,3-P2 concentrations, both oxy and deoxy forms of HbA are saturated with the organic phosphate at all pH values between 6 and 9. Furthermore the difference in the affinity for D-glycerate-2,3-P2 between deoxy and oxy hemoglobin remains constant with pH, implying that the pK values of the Bohr effect groups in deoxy and oxyhemoglobin are not affected by the presence of D-glycerate-2,3-P2 on the hemoglobin molecule. In the hemoglobins modified in position beta 93, the difference in affinity between deoxy and oxy hemoglobin for D-glycerate-2,3-P2 decreases with decrease in pH due mainly to a decrease in the affinity of the deoxy form for D-glycerate-2,3-P2. The effect of the chemical modification appears to be primarily a change in pK of a Bohr group in deoxy hemoglobin which is especially pronounced in the presence of phosphates.

Relationship between the pentose phosphate shunt and methemoglobin reductase activity in human erythrocytes: Effect of aging on methemoglobin reductase activity.

The increase in methemoglobin reductase activity in human erythrocytes upon incubation with inosine, phosphate, pyruvate occurs only in the presence of methylene blue. No difference in activity of the methemoglobin reductases was observed between enzyme extracts of fresh cells and aged cells.

The effect of 2-methoxy-5-nitrotropone on the oxygen affinity of human erythrocytes and hemoglobin.

Human hemoglobin reacted with 2-methoxy-5-nitrotropone at pH 7.4 undergoes modification of the four N-terminal amino groups. The modified protein shows increased oxygen affinity with complete abolition of the effect of K-glycerate 2, 3-bisphosphate. The Bohr effect is abolished in the acid range and drastically reduced at alkaline pH values. Changes in the kinetics of ligand reactions parallel the oxygen equilibrium results. Cooperative effects are still present. Human erythrocytes treated with 2-methoxy-5-nitrotropone show increased oxygen affinity and some decrease in methemoglobin reductase efficiency but no change in resistance to hemolysis.

Changes in rate of methemoglobin reduction and oxygen affinity of erythrocytes incubated with inosine, pyruvate and phosphate.

Incubation of human erythrocytes with inosine, pyruvate and phosphate increases several fold the ferrihemoglobin reductase activity, the values of which, however, depend on the age of blood (by 6 to 2 times with respect to the normal value of fresh blood).