Luciano Forlani

Verification of the Ionic Constants of Proteins by Calorimetry

Dissociation curves of proteins have been extensively used as an analytical tool to provide useful information on the nature and number of the ionic groups in a protein. Changes in the ionic behavior of these groups have been related to structural and functional features of a large number of biologically important molecules. An exact count of the number of groups in any region of a Potentiometrie titration is difficult because of the overlapping ionizations of the ionizing groups. The residues which ionize in proteins can be grouped into three major sets corresponding to their ionization constants. (In this paper, the ionization constant is referred to by its negative logarithm, i.e., pK’. The concentrations are expressed as N which is μmoles ionized/μmoles present.) These sets include the acid region which is composed of exterminai carboxyl groups and the γ and δ carboxyls of aspartic and glutamic, the mid-region which includes the imidazoyl and α-amino group ionizations and the alkaline region comprising the ionizations of the sulphy-dryl, phenolic and e-amino groups from cysteine, tyrosine and lysine (1), respectively.

Calorimetric studies of oxyhemoglobin dissociation. I. Reaction of sodium dithionite with oxygen

Abstract Calorimetric studies of the reduction of free oxygen in solution by sodium dithionite are in agreement with a stoichiometry of 2 moles Na2S2O4 per mole of oxygen. The reaction is biphasic with ΔH t - 118±7 kcal mol−1 (−494 ± 29 kJ mol−1). The initial phase of the reaction proceeds with an enthalpy change of ca −20 kcal (−84 kJ) and occurs when 0.5 moles of dithionite have been added per mole dioxygen present. This could be interpreted as the enthalpy change for the addition of a single electron to form the superoxide anion. Further reduction of the oxygen to water by one or more additional steps is accompanied by an enthalpy change of ca −100 kcal (−418. 5 kJ). Neither of these reductive phases is consistent with the formation of hydrogen peroxide as an intermediate. The reduction of hydrogen peroxide by dithionite in 0.1 M phosphate buffer, pH 7.15, is a much slower process and with an enthalpy change of ca − 74 kcal mol−1 (−314 kJ mol−1). Dissociation of oxyhemoglobin induced by the reduction of free oxygen tension with dithionite also shows a stoichiometry of 2 moles dithionite per mole oxygen present and an enthalpy change of ca. −101 ±9 kcal mol−1 (−423± 38 kJ mol−1). The difference in the observed enthalpies (reduction of dioxygen vs. oxyhemoglobin) has been attributed to the dissociation of oxyhemoglobin, which is 17 kcal mol−1 (71 kJ mol−1).

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.

A calorimetric study of the recombination of the isolated α and β chains of human hemoglobin

Abstract The enthalpy change accompanying recombination of αSHO2 and βSHO2 chains of human hemoglobin has been measured calorimetrically. The overall process is exothermal, with a minimal ΔH in the neighborhood of −7000 cal/mole (of dimer). This value appears to be somewhat dependent on the molar ratio of αSHO2 and βSHO2 chains.

A 35Cl−-NMR study of the singular anion-binding properties of dromedary hemoglobin

35Cl(-)-NMR measurements of chloride binding to carbonmonoxy- and deoxy-dromedary hemoglobin reveal the existence of two classes of chloride-binding sites, one of high and the other of low affinity. Although this situation resembles that described for human hemoglobin, it was found that the number of binding sites as well as the association equilibrium constant for chloride binding are significantly higher in the dromedary protein. This difference may be due to the greater number of basic residues exposed to solvent and to the higher flexibility of dromedary hemoglobin. The two oxygen-linked polyanion-binding sites characteristic of this hemoglobin show competition for some of the high-affinity chloride-binding sites in keeping with their location in the cleft enclosed by the beta chains and between the alpha chains termini. It is suggested that the observed anion-binding properties of dromedary hemoglobin may contribute to the control of the physiological osmotic shock after rehydration.

Hybrid formation in mixtures of hemoglobins and isolated hemoglobin chains from different species

Miscele di emoglobina canina e catene isolate di emoglobina umana, tenute a pH neutro e a bassa forza ionica, mostrano la formazione di molecole ibride. La formazione di ibrido in tampone fosfato diluito è minore che in assenza di sali.

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

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.