M. Stallings

Separation of Human Hemoglobins by Deae-Cellulose Chromatography using Glycine-Kcn-Nacl Developers

This chromatographic procedure uses DEAE-cellulose as ion exchanger and glycine-KCN-NaC1 solutions as developers. Blood samples from several adults and newborn infants with alpha, beta, delta, or gamma chains variants have been analysed. The hemoglobins are eluted as compact and symmetrical zones, and the separation of many hemoglobin types is greatly improved. The procedure is relatively fast, simple, and inexpensive.

An Improved Chromatographic Procedure for Quantitation of Human Fetal Hemoglobin

The DEAE-cellulose chromatographic procedure which uses glycine-KCN-NaCl solutions as Developers and a NaCl gradient to elute the hemoglobin zones was found to be useful for the quantitation of Hb F in samples containing Hb A, provided the level of Hb F exceeds 2 percent. The method has been evaluted through a study of artificial mixtures and of blood samples from patients with various disorders. The data have been compared with results obtained by four other procedures that are used for the quantitation of Hb F.

Demonstration of a minor hemoglobin with modified α chains and additional modified hemoglobins in normal and diabetic adults

We have attempted to separate various hemoglobins in 10 normal and 11 diabetic persons by Bio-Rex 70 chromatography at 4 degrees C with an exponentially increasing sodium phosphate gradient. Minor hemoglobins, namely AIa1, AIa2, AIb1, AIb2, AIb3, AIc, AId1, AId2 and AId3, have been separated and eluted in that order. Most of these minor hemoglobins were newly observed and could not be detected by the previous chromatographic techniques. On the basis of colorimetric assay protein-bound ketoamine was present in all the minor hemoglobins, which was confirmed by chromatographic separation of hemoglobins after reduction with NaB3H4. All the minor hemoglobins, with the exception of Hb AIa1, HbIa2, and Hb AIb1, showed a 2-fold increase in the diabetic patients. Hb AIc (5.0% in normals; 9.0% in diabetics) and Hb AId3 (1.9% in normals; 3.2% in diabetics) were present in the largest amounts. Both Hb AId3 and Hb Ao showed a decrease in oxygen affinity in the presence of 2,3-diphosphoglycerate, whereas Hb AIc showed no effect. Separation of globin chains by cellulose acetate electrophoresis at pH 8.6 showed that the cathodal mobility of the alpha chains of Hb AId3 was slower than those of Hb Ao. Glycosylation of Hb with [14C]glucose followed by separation of hemoglobins by two Bio-Rex 70 chromatographic methods indicated that the minor Hb formed by glycosylation of the alpha-chain amino-terminus was separated from Hb Ao, while the minor hemoglobins formed by the glycosylation of epsilon-NH2 groups chromatographed with Hb Ao.

Minor hemoglobins in sickle-cell heterozygotes and homozygotes with and without diabetes

Abstract Minor hemoglobins in the red cell lysates of Hb S heterozygotes (AS) and homozygotes (SS) with and without juvenile-onset diabetes were separated by Biorex 70 column chromatography. These minor hemoglobins were identified as Hb A Ia+b , Hb A Ic , Hb S Ia+b , and Hb S Ic in AS persons and Hb F I , Hb F 0 , Hb S Ia+b , and Hb S Ic in SS patients. The levels of all four components were elevated 2 to 3 fold in AS persons with diabetes. Likewise, the percentages of Hb S Ia+b and Hb S Ic were significantly increased in a diabetic SS patient. The proportions of the Hb A I and Hb S I components in the AS persons were nearly the same, indicaing that Hb A and Hb S are glycosylated in vivo to about the same extent. In vitro studies confirm these findings. It was also confirmed by in vitro studies that the glycosylation of hemoglobin involves both specific and nonspecific binding of carbohydrate to α and β chains.

Heterozygosity and Homozygosity for the High Oxygen Affinity Hemoglobin Tarrant or α126 (H9) ASP→ASN in two Mexican Families

Two Mexican families from the State of Jalisco have been studied in which 11 members were carriers of Hb Tarrant. Ten subjects were Hb Tarrant heterozygotes producing about 25% of the abnormal hemoglobin. One 9-year-old boy was homozygous for Hb Tarrant. About 50% of his hemoglobin was of the variant type. The heterozygotes had mild erythrocytosis which was considerably more severe in the homozygote. The average P50 value for blood of the heterozygote was 15.1 mm Hg (controls: 22.5 mm Hg) while this value was decreased to 9 mm Hg in the homozygote. The clinical condition of the homozygote is compatible with a mild chronic tissue hypoxia.

High-performance liquid chromatographic separation and quantitation of glycosylated hemoglobin A2 as an alternate index of glycemic control

By a combination of DEAE-cellulose chromatography and cation-exchange high-performance liquid chromatography glycosylated components of hemoglobin (Hb) A2 were separated and quantitated from persons with diabetes and some common hemoglobinopathies. Hb A2Ic values correlated well with total glycosylated Hb levels assayed by affinity chromatography, and Hb AIc, Hb SIc and Hb CIc levels, determined by high-performance liquid chromatography. The results indicate that Hb A2Ic may serve as an alternate index of glycemic control.

Affinity Chromatographic Quantitation of Glycosylated Hemoglobin in Newborn Infants

Levels of glycosylated hemoglobin (glyco Hb) were determined in 45 newborn infants by affinity chromatography on phenylboronate-agarose gel and found to correlate well with the results obtained by two chemical methods. However the percentages of Hb FIa+b and Hb FIc determined by Bio-Rex 70 chromatography did not correlate with the glyco Hb values. The glyco Hb levels in the newborns were significantly lower than in the adults. Rechromatography of the glyco and nonglyco Hb fractions on Bio-Rex 70 columns indicated that Hb FO-like glyco Hb (presumably formed by glycosylation of the alpha chain amino terminus and epsilon-amino groups of lysyl amino acids) constituted about 60% of the glyco Hb fraction, whereas both Hb FIc and Hb FIa+b components contained only small portions of the glyco Hb.

Synthesis of Hemoglobin chains in Adult and Newborn Goats: Possible Influence of the βc Synthesis on the Production of α CHAINS

The rates of in vitro synthesis of hemoglobin a and non-a chains were determined in adult goats during blood loss anemia and in newborn goats during postnatal development. the reticulocytes were incubated in a medium supporting protein synthesis and containing [14C] leucine. the hemoglobin chains were separated by CM-cellulose chromatography. the adult animals responded to the phlebotomy with a production of nearly 100% βc chain about 10 days after the start of the experiment. Severe anemia and the production of βC chain was accompanied by a significant increase in the α/non-α-synthesis ratio. the y chain production in the newborns declined rapidly after birth and was completely replaced by that of the βc chain in 7-10 days. No significant difference in the relative synthesis of the three types of α chain was observed during this period. Goats with either a homozygosity for the Iα or Iα-chains synthesize the Iα (or Iβ) and IIα chains at a ratio of 3:1. When a heterozygosity for IαB chains is present the I...

Modification of sickle hemoglobin by acetaldehyde and its effect on oxygenation, gelation and sickling

Erythrocytes from sickle cell anemia patients and chromatographically purified Hb S0 were incubated with 0.25 to 120 mM acetaldehyde for 15 min to 6 h at different temperatures and pH. Several hemoglobin adducts stable to dialysis were separated by Biorex 70 chromatography and the proportions of the adducts formed were dependent on the period of incubation, acetaldehyde concentration, pH and temperature. Acetaldehyde treatment showed an increase in solubility, minimum gelling concentration and oxygen affinity and a decrease in sickling which showed a dependence on Hb S modification. With 0.25 to 1 mM acetaldehyde, significant inhibition of sickling was observed without any effect on the physical characteristics of the hemoglobin molecule. Acetaldehyde may act as a gelation inhibitor as well as a cell sickling inhibitor.

Oxygen equilibrium analyses of isolated hemoglobins A2, Lepore-Washington and P-Nilotic

Oxygen equilibrium studies have been carried out on hemoglobins A2 (alpha2delta2), Lepore-Washington (alpha2(deltabeta)2) and P-Nilotic (alpha2(beta2delta)2) using the beta chain containing hemoglobins A and S as controls. This investigation was initiated mainly because of controversial data that have been published on the oxygen affinity of hemoglobin (Hb) A2 and because samples containing the rare Hb P-Nilotic became available. Each hemoglobin was isolated in pure form by anion exchange chromatography; the samples used in the equilibrium analyses contained 100 mg Hb/dl with less than 5% ferrihemoglobin and no 2,3--diphosphoglycerate. Oxygen equilibrium analyses were made at 37 degrees C with the method of Benesch et al. (1965) Anal. Biochem. 11, 81--87; Anal. Biochem. 55, 245--248 (1973). A slight, but definite increase in oxygen affinity was observed for Hb A2 as well as for Hb P-Nilotic while the increase for the Hb Lepore-Washington was somewhat greater. The values for n, the Hill coefficient, and the Bohr effects were the same for all hemoglobin types. The differences in oxygen affinity of these hemoglobins apparently result from the differences in primary structure that are characteristic for those proteins.