Andree M. Dozy

Studies on the heterogeneity of hemoglobin : IX. The use of tris(hydroxymethyl)aminomethane—HCl buffers in the anion-exchange chromatography of hemoglobins

Abstract A modified procedure for the separation of various hemoglobin types by anion exchange chromatography has been presented. DEAE-Sephadex, A-50 medium, was preferred over DEAE-cellulose as chromatographic medium. Complete separations of many hemoglobin fractions were obtained by applying a single pH gradient to the columns using TRIS—HCl buffersof reasonably high concentrations (0.05 M). The method was applicable both for analytical and preparative purposes.

Studies on the heterogeneity of hemoglobin : XIII. Chromatography of various human and animal hemoglobin types on deae-sephadex

Abstract A modification of the anion exchange chromatography of various hemoglobin types using DEAE-Sephadex is presented. It has been observed that the replacement of the DEAE-Sephadex A-50, 100 to 270 mesh, by a similar preparation, but particle size 40 to 120 μ, greatly improved the resolution of several human and animal hemoglobin types.

Studies on the heterogeneity of hemoglobin: XIV. Chromatography of normal and abnormal human hemoglobin types on CM-Sephadex

Abstract A new chromatographic procedure for the separation and quantitation of human hemoglobin components is described. The method utilizes columns of carboxymethyl-Sephadex G-50 and 0.05 M Tris-maleic acid buffers as developers; application of a simple pH gradient is preferred over stepwise elution. The procedure allows the separation of Hb-A 2 from the hemoglobins S and C, and of Hb-D 2 from the slow moving hybrid component SDα. Fetal hemoglobin is eluted in front of the normal Hb-A. The minor hemoglobins of normal red cell hemolysates were fractionated into at least four components, well separated from the major hemoglobin component. The procedure has the disadvantage of requiring several days to complete the chromatographic separations.

Studies on the heterogeneity of hemoglobin : IV. Chromatographic behavior of different human hemoglobins on anion-exchange cellulose (DEAE-cellulose)

Abstract 1. A new column chromatographic procedure for the separation of different hemoglobin fractions using DEAE-cellulose as adsorbent is described. Except for fetal hemoglobin the elution rates of the hemoglobin components were found to be in accordance with the differences in electrophoretic points. The elution rates, as measured by their elution volumes, are within certain limits independent of the total amount of Hb chromatographed; the elution rates were greatly influenced by the initial pH values of the adsorbent. 2. Examples of hemoglobin separations are presented for cases with the following Hb-abnormalities: AS, AC, AJ (Georgia), SS, SC, normal cord blood, cord blood samples from babies heterozygous for Hb-S and for Hb-C, cord blood sample with the abnormal Barts Hb, Thalassemia minor and major, Hb-E-thalassemia disease, persistent high Hb-F abnormality, persistent high Hb-F abnormality combined with Hb-S, Hb-B2 trait, Hb-B2 trait associated with thalassemia, the Hb “Lepore” abnormality, and a case with an unknown abnormal minor Hb-component. 3. Since small amounts of Hb-F are generally eluted with the minor Hb-A1 fraction, application of an alkali denaturation method to this isolated fraction offered a new and fairly accurate procedure for the determination of small amounts of Hb-F. Applications to cases with thalassemia minor and the Hb “Lepore” abnormality have been presented. 4. The quantitative data obtained with the procedure were in agreement with the results obtained by starch block electrophoresis and carboxymethylcellulose chromatography. It was emphasized that the method was of particular use for the minor Hb fractions: A2, B2, Lepore and of small amounts of Hb-F. 5. The heterogeneity of the hemoglobins of normal adults, sickle cell anemia patients, and cord blood samples as observed by DEAE-cellulose chromatography was found to be closely related to those found by electrophoretic methods and by CM_cellulose chromatography. 6. The DEAE-cellulose chromatography was found of great use for the preparation of large quantities of pure Hb-fractions: examples for Hb-A2 and Hb-Lepore have been presented.

STUDIES ON ANIMAL HEMOGLOBINS. II. THE INFLUENCE OF INORGANIC PHOSPHATE ON THE PHYSICO-CHEMICAL AND PHYSIOLOGICAL PROPERTIES OF THE HEMOGLOBIN OF THE ADULT CHICKEN.

Abstract The equilibrium of dilute and concentrated adult chicken red blood cell hemolysates with O 2 has been studied using potassium phosphate buffers and NaCl solutions of different molarities. The results indicate an increase in O 2 affinity with increase in ionic strength. The same phenomenon was observed for the isolated minor hemoglobin component; the isolated major hemoglobin component showed a rather high O 2 affinity at low ionic strength, which decreased with increase in molarity. The minor hemoglobin fraction exhibited an ability to form rather stable complexes with phosphate ions; such complexes possessed a high O 2 affinity at low ionic strength. The data also indicated that the two hemoglobin components in a hemolysate interacted with each other. This extra-molecular interaction interfered with the physiologic properties of the hemolysate. No physico-chemical interaction could be detected, as was demonstrated by the determination of the molecular weight and by viscosity measurements.