ABSTRACT
The hemoglobin Porto Alegre (HbPA) disulfide polymer dodecamer and the HbPA, HbA disulfide polymer octamer subunit dissociation by NaCl was studied by measuring the osmotic pressure of CO-hemoglobin solutions at pH = 6.9 and 20 degrees C. The dissociation equilibrium constants were evaluated from the osmotic pressure data. The subunit dissociation of the two types of disulfide polymers follows a reaction of the type A3 in equilibrium 3A. The HbPA disulfide polymer dodecamer appears to be more susceptible to NaCl induced dissociation than is normal HbA and less resistant to dissociation on protein dilution. The HbPA, HbA disulfide polymer octamer appears to be much more susceptible to dissociation on protein dilution than is the HbPA disulfide polymer dodecamer and normal HbA. The standard free energy of dissociation of the HbPA disulfide polymer at 2 M NaCl is delta GOD(25 degrees C) = 12 kcal/mole. The type of dissociation reaction (A3 in equilibrium with 3A) support the conclusion that the HbPA disulfide polymer has a quaternary molecular structure of a closed ring of three tetramers.
Subject(s)
Hemoglobin A/metabolism , Hemoglobins, Abnormal/metabolism , Osmotic Pressure , Polymers/metabolism , Sodium Chloride/pharmacology , Humans , Mathematics , Molecular WeightABSTRACT
The cleavage of HbPA disulfide polymer by GSH and its indirect cleavage by yeast glutathione reductase, via reduced glutathione is obtained. Decreasing the initial proportion of GSH in the hemolysate increases the formation of HbPA disulfide polymer. In the experimental conditions used, yeast glutathione reductase is unable to perform the direct cleavage of the mixed disulfide of HbPA and GSH, using it as substrate. The reduction of HbPA polymer to tetramers by DTE is analyzed by a pseudo-first-order kinetic and two rate constants are obtained. That of 265 X 10(-3) min-1 should be concerned with one disulfide of the closed ring and one of the open ring structure of dodecamer, while that of 38 X 10(-3) min-1 is related to disulfide reduction of the octamer. The enthalpy of activation values of 8.0 kcal.mol-1 an 17.4 kcal.mol-1 obtained, from the Arrhenius plot, for the "fast" and "slow" rate disulfide reduction, respectively, are indicative that a strong conformational strain of S--S bonds in the closed ring structure is maintained. The entropy of activation values of 24 e.u. and 52 e.u. are found for the activation of disulfides from dodecamers and octamers, respectively.
Subject(s)
Glutathione Reductase/metabolism , Hemoglobins, Abnormal/metabolism , Polymers/metabolism , Brazil , Electrophoresis, Starch Gel , Genetic Carrier Screening , Hemoglobins, Abnormal/genetics , Humans , KineticsABSTRACT
Population surveys performed in different Brazilian ethnic groups, and comprising 23,606 subjects, disclosed, besides the common S and D types, hemoglobins G and Por to Alegre, as well as high F, A'2 and an apparently unstable A2 variant. Additional studies on isolated families revealed the occurrence of hemoglobins I, Niterói, D Punjab, M Boston and J Rovigo. Screening for sickle cells was also performed on 17,412 individuals. Detailed clinical studies on series of sickle cell anemics were also performed. Due to their special interest, the physico-chemical properties of Hb porto Alegre are reviewed in some detail.