Luigi Rossi-Bernardi

Isoelectric focusing and electrophoresis at subzero temperatures

Abstract Electrophoretic and isoelectric focusing separations have been achieved at −20 to −30°C, i.e., at temperatures considerably lower than previously reported by using as supporting media gels of acrylamide-methylacrylate copolymers and dimenthylsulfoxide-water mixtures. Hybrids of human and sickle cell hemoglobin and partially oxidized human carboxyhemoglobin have been separated in the temperature range −20 to −30°C, both by a discontinuous buffer gel electrophoresis and by isoelectric focusing.

[12] Detection of hemoglobin hybrid formation at subzero temperature

Publisher Summary This chapter discusses the detection of hemoglobin hybrid formation at subzero temperature. The study of hybrid species (asymmetric hybrids) in mixtures of unlike tetrameric hemoglobins yields significant information on the structure–function relationship of hemoglobin. The formation of hybrid molecules between hemoglobin S and minor hemoglobin components present in the red cells of sickle cell blood—such as hemoglobin A, hemoglobin A2, and hemoglobin F—is recognized as an important factor in the inhibition of hemoglobin S gelling promoted by these minor components. Intermediates in the reactions of hemoglobin with ligands or with oxidants can also be considered hybrid molecules. In this case, the dimers forming the tetrameric hybrid belong to the same hemoglobin species but exist in different liganded or valency states (ligand and valency hybrids). Hybridization reactions that occur via the dimerization of tetramers and subsequent reassociation of dimers are one possible source of instability of the asymmetric hybrid molecules. Standard chromatographic and electrophoretic methods are used for the isolation of stable valency and asymmetric hybrids. The ability to isolate hybrids by these methods depends on a favorable ratio between the rate of tetramer dissociation and the rate of protein separation.

The functional properties of sickle cell blood

It is known that whole blood from patients with sickle cell anemia has a decreased oxygen affinity [ 1,2]. Purified hemoglovin S solutions, however, are normal [3,4]. A systematic study of the effect of 2,3-DPG and of other known allosteric regulators of oxygen affinity of HbS in whole blood seems desirable, indeed essential, to understand the reason for the altered oxygen affinity of the hemoglobin S molecule in the intact red cell environment. A more complete understanding of the functional properties of SSblood would also be important to assess the physiological significance of such an alteration, and to evaluate the therapeutic effect of cyanate, administration.

The interaction between hemoglobin and its "oxygen-linked" ligands.

The primary function of hemoglobin, i.e., its oxygen carrying ability, is controlled, in mammalian blood, by some small molecules or ions.

Hemoglobin-liganded intermediates.

Publisher Summary This chapter discusses the hemoglobin (Hb)-liganded intermediates. The study of liganded intermediates is crucial to the clarification of the mechanisms of Hb cooperativity. Information on the concentrations of the intermediates in solutions of Hb at equilibriumwith oxygen or carbon monoxide (CO) can be obtained by the analysis of the binding isotherms. The determination of the concentrations of the intermediates under dynamic conditions, such as in the course of the association reaction between Hb and CO as studied by classic optical stopped-flow techniques: is subject to similar limitations. The isolation and study of the liganded intermediates are difficult because (1) the ligand can dissociate from the heme site to rebind to a different site and (2) the Hb tetramer dissociates reversibly into dimers by cleavage along the α1β2and α2β1 contacts. An asymmetrical intermediate, or hybrid, in which the ligand is not distributed symmetrically with respect to the dimer interface, dissociates into dimers that can yield on reassociation the original hybrid and its parent tetramers.

[28] Measurement of CO2 equilibria: The chemical-chromatographic method

Publisher Summary This chapter describes the measurement of CO 2 equilibria. The method described in this chapter stems from the exhaustive work of Faurholt on the determination of the carbamino compounds of simple amines. This method was first applied to the determination of carbonylhemoglobin by Ferguson and Roughton. The basic principles of the chemical method herein described were then used by Rossi-Bernardi et al. and by Perrella et al. for the development of the chemical-chromatographic method. The accuracy of this method is limited by the need to estimate various factors correcting for (1) the protective colloidal effect of hemoglobin on BaCO 3 precipitation, (2) the reaction of free CO 2 with the ɛ amino groups of the lysines, both processes leading to an overestimation of carbonylhemoglobin, (3) the coprecipitation of hemoglobin with BaCO 3 , (4) the decomposition of carbonylhemoglobin during centrifugation, both processes leading to carbonylhemoglobin underestimation, and (5) hemoglobin denaturation because of the long exposure to pH 12. The chapter describes the rapid chemical-chromatographic method.

paH gradients in isoelectric focusing experiments at subzero temperatures

Abstract An improved method for the subzero separation of proteins by isoelectric focusing in gels of acrylamide/ethylacrylate copolymers in dimethylsulfoxide-water mixtures is described, together with the measurements of the rate of formation and stability of the p a H gradient in gels under such conditions.