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Hemoglobin A2 is a batch fractionated on a column containing diethylaminoethyl-cellulose (DE52, Whatman) with a discontinuous buffer system at pH 8.9 and 7.3. A short micro-column method is also presented, which allows separation in less than 30 min with no interference from hemoglobin S. Both methods clearly distinguish normal from thalassemia specimens, and are simpler and more rapid than previously published methods.
Hemoglobin A(2) (alpha(2)delta(2)), a minor (2-3%) component of circulating red blood cells, acts as an anti-sickling agent and its elevated concentration in beta-thalassemia is a useful clinical diagnostic. In beta-thalassemia major, where there is a failure of beta-chain production, HbA(2) acts as the predominant oxygen delivery mechanism. Hemoglobin E, is another common abnormal hemoglobin, caused by splice site mutation in exon 1 of beta globin gene, when combines with beta-thalassemia, causes severe microcytic anemia. The purification, crystallization, and preliminary structural studies of HbA(2) and HbE are reported here. HbA(2) and HbE are purified by cation exchange column chromatography in presence of KCN from the blood samples of individuals suffering from beta-thalassemia minor and E beta-thalassemia. X-ray diffraction data of HbA(2) and HbE were collected upto 2.1 and 1.73 A, respectively. HbA(2) crystallized in space group P2(1) with unit cell parameters a=54.33 A, b=83.73 A, c=62.87 A, and beta=99.80 degrees whereas HbE crystallized in space group P2(1)2(1)2(1) with unit cell parameters a=60.89 A, b=95.81 A, and c=99.08 A. Asymmetric unit in each case contains one Hb tetramer in R(2) state.
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A polyacrylamide gel electrophoresis technic for the determination of hemoglobin A2 is presented. Rapid separation is an advantage. The use of a diluted hemolysate as a 4 percent standard avoids overestimation of the A2 fraction by densitometry and also provides a reference for visual comparison. Normal range of A2 hemoglobin by this method is 1.12 to 4.13 per dkg. Coefficient of variation was 5.84 percent.
To understand determinants for hemoglobin (Hb) stability and Hb A2 inhibition of Hb S polymerization, three Valdelta6 Hb A2 variants (Hb A2 deltaE6V, Hb A2 deltaE6V,deltaQ87T, and Hb A2 deltaE6V, deltaA22E,deltaQ87T) were expressed in yeast, and stability to mechanical agitation and polymerization properties were assessed. Oxy forms of Hb A2 deltaE6V and Hb A2 deltaE6V,deltaQ87T were 2- and 1.6-fold, respectively, less stable than oxy-Hb S, while the stability of Hb A2 deltaE6V,deltaA22E,deltaQ87T was similar to that of Hb S, suggesting that Aladelta22 and Glndelta87 contribute to the surface hydrophobicity of Hb A2. Deoxy Hb A2 deltaE6V polymerized without a delay time, like deoxy Hb F gammaE6V, while deoxy Hb A2 deltaE6V,deltaQ87T and deoxy Hb A2 deltaE6V,deltaA22E,deltaQ87T polymerized after a delay time, like deoxy Hb S, suggesting that beta87 Thr is required for the formation of nuclei. Deoxy Hb F gammaE6V,gammaQ87T showed no delay time and required a 3.5-fold higher concentration than deoxy Hb S for polymerization, suggesting that Thr effects on Valdelta6 Hb A2 and Valgamma6 Hb F variants are different. Mixtures of deoxy Hb S/Hb A2 deltaE6V,deltaQ87T polymerized, like deoxy Hb S, while polymerization of Hb S/Hb A2 deltaE6V mixtures was inhibited, like Hb S/Hb F gammaE6V mixtures. These results suggest alpha2betaSdelta6 Val, 87 Thr hybrids and Hb A2 deltaE6V,deltaQ87T participate in Hb S nucleation, while only 50% of alpha2betaSdelta6 Val hybrids and none of the Hb A2 deltaE6V participate. These findings are in contrast to those of mixtures of Hb S with Hb F gammaE6V or Hb F gammaE6V,Q87T, which both inhibit Hb S polymerization. Our results also suggest participation in nucleation of some alpha2betaSdelta hybrids in A2S mixtures but not alpha2betaSgamma hybrids in FS mixtures.
The DIAMAT Analyser System is a fully automated high performance liquid chromatographic (HPLC) instrument originally designed for the quantification of glycated hemoglobin (HbA1c). Buffers were developed for the separation and quantification of hemoglobin A2 on the DIAMAT. Also studied were the retention patterns of various hemoglobin variants on the DIAMAT using the buffers developed for the hemoglobin A2 quantification.
Hemoglobin A2 (Hb A2) and hemoglobin F (Hb F) are important analytes in the diagnosis and follow up of Hb diseases. We evaluated a new capillary zone electrophoresis (CZE) kit for Hb A2 and Hb F measurements. The imprecision ranged from 3% to 6% for Hb A2 and Hb F at physiological and pathological concentrations. The method compared well with cation-exchange HPLC for Hb A2 and Hb F and with anion-exchange chromatography in microcolumns (MAEC), for Hb A2. Nevertheless, higher results were obtained [Hb A2 CZE (%) = 1.233 Hb A2 HPLC - 0.2; Hb A2 CZE (%) = 1.190 Hb A2 MAEC + 0.1; Hb FCZE (%) = 1.118 Hb FHPLC + 0.4], and new reference values had to be determined (Hb A2 2.7-3.8%; Hb F <1.2%). Quantification of Hb A2 was not influenced by Hb S. Measurement of Hb F was accurate and precise except at low concentrations in Hb AS patients. This new CZE kit is rapid, precise, and reliable, and seems appropriate for use in clinical laboratories.
High-resolution proton nuclear magnetic resonance spectroscopy at 300 and 600 MHz has been used to investigate the conformation of a minor hemoglobin component of human blood, hemoglobin A2 (alpha 2 delta 2), in solution. We have found that (i) the replacement of the beta chains by the delta chains in hemoglobin A2 conserves the alpha 1 delta 2 interface but slightly perturbs the alpha 1 delta 1 interface, and (ii) one surface histidine residue in the deoxy form and one in the carbonmonoxy form of hemoglobin A2 have local conformations and/or electrostatic environments which are different from the corresponding ones in human normal adult hemoglobin. By comparing the proton nuclear magnetic resonance titration of individual histidine residues in hemoglobin A2 and in human normal adult hemoglobin, we can conclude that in human normal adult hemoglobin, both beta 116 and beta 117 histidine residues are titratable in both the deoxy and the carbonmonoxy forms. Thus, these two histidine residues can contribute to the Bohr effect of human normal adult hemoglobin. The present nuclear magnetic resonance data on hemoglobin A2 and those previously obtained in our laboratory on sickle hemoglobin suggest that the antisickling property of hemoglobin A2 does not originate from an alteration of the intermolecular contact site at the beta 6 position, but involves additional amino-acid residues which are different in the beta and delta chains. We have found that the replacement of the beta 116 and beta 117 histidine residues in the delta chains does not play a significant role in the antisickling effect of hemoglobin A2 and, thus, these amino-acid residues do not participate in the intermolecular interactions responsible for the polymerization of sickle hemoglobin.
To critically assess the method of capillary electrophoresis, we examined 97 clinical samples submitted for hemoglobin electrophoresis by both conventional methods (electrophoresis on cellulose acetate and citrate agar for detection of abnormal variants, ion-exchange chromatography for quantification of HbA2, alkali denaturation for quantification of HbF, supravital stains for detection of HbH) and CE. CE was performed using a 72 cm (50 cm to detector) x 50 microns i.d. fused-silica capillary with detection of absorbance at 200 nm. Of the 97 samples examined, 34 contained a hemoglobin variant. Migration time, the difference of the variant peak to that of HbA, was used in an attempt to identify the hemoglobin variant by CE. We found the method to be suitable for the quantification of HbA2, useful as a screening technique for the presence of hemoglobin variants, but unsuitable for the quantification of HbF.
Partially purified hemoglobin A2 has been examined for the existence of glycosylated components by isoelectric focusing and by acid agar gel electrophoresis. Bands analogous to the glycohemoglobin derivatives of hemoglobin A, hemoglobin-A1.a.b.c, were readily detected. Evidence that these minor bands are in fact glycohemoglobins was obtained by showing that 14C-glucose bound to hemoglobin A2 moved with these minor bands. The amounts of glycohemoglobin derivatives of hemoglobin A2 were increased in the blood of diabetic patients.
While no significant physiologic function of hemoglobin A2 (Hb A2), the minor basic component of human hemoglobin, has been recognized, only its oxygen equilibria have been studied in detail. Since hemoglobin A2 and its oxidative denaturation product, hemichrome A2, bind to the red cell membrane, particularly to band 3, to a greater extent than do Hb A or hemichrome A, some of the properties of Hb A2 that might influence hemoglobin-membrane association were examined. Hemoglobin A2 exhibited slightly increased susceptibility to autoxidation to methemoglobin. No differences were noted between methemoglobins A and A2, including the rates of enzymatic reduction and stability of the heme-globin linkage. Oxyhemoglobin A2 had a slightly lower solubility in phosphate buffer than did hemoglobin A. While the hemichromes (prepared with phenylhydrazine) of hemoglobins A2 and A had the same optical spectra, the A2 hemichrome exhibited greater stability. It is suggested that the differences in products of oxidative denaturation may provide the basis for functional differences between hemoglobins A2 and A.
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Commercial microcolumns introduced in 1976 by Helena Laboratories ("Hb A2 Quik Column") and by Isolab, Inc. ("Quik-Sep") provide a rapid, simple, accurate method for quantitation of hemoglobin A2 (Hb A2). However, these kits cannot be used for the quantitation of Hb A2 in the presence of slow-moving variants such as Hb S. Recently, Isolab, Inc., produced a new kit ("Quik-Sep Improved Hb A2 Test") for quantitation of both Hb A2 and Hb S. We compared results obtained with the new Isolab kit to results obtained with the original Tris/HCl method for quantitation of Hb A2 and Hb S. Blood was drawn from persons with sickle cell trait (A/S), sickle cell anemia (S/S), sickle cell/beta+ thalassemia (S/beta+ thal) and sickle cell/beta 0 thalassemia (S/beta 0 thal) and percentages of Hb A2 and Hb S were determined by each method. We found no significant differences in Hb A2 percentages by the two methods, and the coefficients of variation were similar. Both methods showed only slight overlap of Hb A2 values from subjects with some form of beta thalassemia and those with A/S or S/S. However, the Tris/HCl method consistently gave values for Hb S that were higher and closer to those expected, suggesting that the Isolab kit does not accurately quantitate Hb S.
Erythrocyte hemoglobin A2 (Hb A2) was quantitated in 28 hyperthyroid patients prior to antithyroid therapy and at intervals during therapy. Fetal hemoglobin (Hb F) levels and red cell mean corpuscular volume (MCV) were also monitored. Before therapy, Hb A2 was significantly elevated (mean +/- SD, 3.3 +/- 0.5%: normals, 2.5 +/- 0.3%: p less than 0.001). Hb F levels exceeded 1.0% in 10 cases. A tendency to red cell microcytosis was also observed (mean MCV 81.1 fl). To-date 10 of these subjects have had adequate studies following appropriate and sustained responses to antithyroid therapy. All showed a fall in Hb A2, usually accompanied by a rise in red cell MCV. A hematologic profile for hyperthyroidism is proposed.
Hemoglobin A2 (HbA2) comprises about 2.2% of the total hemoglobin in the erythrocytes. The separation and quantitation of this minor hemoglobin by capillary electrophoresis (CE) using an arginine Tris buffer is described. Some of the variables affecting the accuracy and precision of HbA2 quantification are investigated. Furthermore, the quantification of this hemoglobin by CE is compared to that of a microcolumn chromatography method. The CE method is better suited than the microcolumn method for measuring HbA2 in the sickle cell trait.
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