Electrophoresis of hemoglobin

Fig ure 3. Starch gel electrophoresis of hemoglobins. Tris-EDTA-boric acid buffer, pH 9.0. O-Dianisidine stain. 

Figure 4. Cellulose acetate electrophoresis of hemoglobins. Variations in the quantities of Hb-Ag are hardly detectable (cornpare the samples 2 and 3 from top). Hb-N Baltimore is a p-chain variant in which lysyl residue in position 95 is replaced by a glutarnyl residue.

Figure 5. Citrate agar electrophoresis of hemoglobins. The plate is stained with O-dimethyb...

Fig. 13. Starch gel electrophoresis of hemoglobin of cord blood samples from newborns with various types of a-thalassemia. Tris-EDTA-boric acid buffer, pH 8.6. Stained with o-dianisidine. From Pootrakul et al. (P22) with permission of the authors and publisher.

Matioli, G., and Niewisch, H., Electrophoresis of hemoglobin in single erythrocytes. Science 150, 1824-1826 (1965). 

Jenkins, M., and Ratnaike, S. Capillary electrophoresis of hemoglobin. Clin. Chem. Lab Med., 41,... 

Detection of Met(Ferrl-)Hemoglobins (Hb-M) Detection of these variants can be made by starch gel electrophoresis of the ferrl-derlvatlves of hemoglobins In red cell hemolysate using a phosphate buffer, pH 7 0 (25) However, some methemoglobln variants can be separated from normal Hb-A at pH 9 0 (40) ... 

Structural Analyses of Hemoglobin Variants It has become Impossible to characterize nearly any abnormal hemoglobin by Its electrophoretic and/or chromatographic mobility only This Is most strikingly demonstrated by the fact that over fifty different variants behave similar to Hb-S In electrophoresis Characterization, therefore, often requires detailed structural analyses or the demonstration of a property unique to a specific variant Some of the techniques used In these studies will be... 

Experiment 62 Separation of Hemoglobin and Cytochrome C by Horizontal Agarose Gel Electrophoresis... 

Prepare 100 mL (500 mL if the gel is to be run under the buffer) of an electrophoresis buffer that is 20 mM tris-(hydroxymethyl)amino methane (TRIS or THAM), 6 mM sodium acetate, and 1 mM disodium EDTA. Adjust the pH of this solution to 7.9 using concentrated HC1. Also prepare small volumes of solutions of hemoglobin and cytochrome C in the buffer (the concentration is not important) and also a mixture solution of these two solutes. Add a quantity of sucrose to each. 

A. Photograph of a gel prior to electrophoresis. B. Diagram of hemoglobins A, S, and C after electrophoresis. 

Figure 8-14 SDS-polyacrylamide gel electrophoresis of human erythrocyte ghosts. (A) From untreated cells. (B) From cells digested externally with chymotrypsin. (C) Inside-out vesicles prepared from cells pretreated with chymotrypsin. (D) The same inside-out vesicles after further treatment with chymotrypsin. (E) Polypeptides released hy the chymotryptic treatment of the inside-out vesides. The peptides are numbered according to the system of Steck232 Hb, hemoglobin. From Luna et al233...

M20. Motulsky, A. G., Paul, M. H., and Durrum, E. L., Paper electrophoresis of abnormal hemoglobin and its clinical applications. A simple semi quantitative method for the study of the hereditary hemoglobinopathies. Blood 9, 897 (1954). 

OTFLEXES Figure 3. Haptoglobin separation schematic. A schematic illustrating typical results of a Hp determination run in 8 mm, 10% starch gel prepared with tris citric acid buffer at pH = 8.6. The tank buffer is boric acid, pH — 7.9. The electrophoresis is run at 100 V for 17 hr at 4°C. The Hp-Hb complexes are stained by virtue of the peroxidase reaction of hemoglobin which gives a o color reaction with benzidine. 

Figure 7.17 Gel electrophoresis of haptoglobins. Haptoglobin 1-1 moves as a single component, whereas the other two types show genetically derived polymorphisms. Hemoglobin-binding activity is identical in the three types.

Fig, 23. Quantitation of pepsin by paper electrophoresis and hemoglobin digestion in 165 lyophilized gastric juices of humans. From Class (C3). 

This rapid denaturation of hemoglobin affords this method for determining the presence of an unstable hemoglobin variant even when starch gel electrophoresis of hemolysate shows no evidence of an abnormality. The heat stability of the hemoglobin variant is compared with that of a normal control the two are incubated simultaneously in a phosphate buffer at 60°C (K9). 

The concentrations are 4 moles per mole of hemoglobin, which corresponds to 15 mg of PMB in 10 ml of 0.05 Af Tris-HCl buffer, pH 7.5 and 12 moles per mole of hemoglobin, which corresponds to about 45 mg per 10 ml of Tris buffer. These solutions are mixed with an equal volume of red cell hemolysate (6.5 g of Hb per 100 ml) the reaction is allowed to take place for at least 4 hours at room temperature. Identification of subunits can be made by starch gel electrophoresis at pH... 

Fig. 23. Starch gel electrophoresis at pH 9.0 and 4°C of hemolysates treated with PMB. Samples 1 and 8, untreated hemoglobin from subject with the unstable Hb-Savannah and her normal father, respectively samples 2 (propositus), 3 (normal mother), and 4 (normal father) were treated with 12 moles of PMB per mole of hemoglobin samples 5 (propositus), 6 (mother), and 7 (father) were treated with 4 moles of PMB per mole of hemoglobin. Gel is stained with Amino Black lOB. NHP denotes nonhemoglobin protein fraction, X the abnormal hemoglobin. From Huisman et al. (H55) with permission of the authors and publisher.

Application of FT-ICR-MS for on-hne capillary electrophoresis-MS (CE-MS), e.g., in the analysis of single cells [78]. A erythrocyte cell was lysed in a CE colunm. The a- and P-chaitrs of hemoglobin, ca. 450 amol present in the cell, were detected with isotopic resolution. 

Capillary isoelectric focusing coupled to mass spectrometry has gained popularity in recent years (by analogy to two-dimensional gel electrophoresis). Additional information obtained from mass spectrometry includes not only precise molecular-weight determination but also the possiblity for peptide sequencing. Analysis of hemoglobin variants, recombinant proteins, and monoclonal antibodies have been demonstrated [1,7,8]. 

Fig. 2 Separation of hemoglobins by CIEF in blood from a patient suffering from Hb S/p thalessemia. Capillary 27 cm (20 cm to detector) X 50 /xm i.d. DB-1 (J W Scientific) ampholytes 2% pH 6-8 10-3 (10 1) Pharmalytes (Pharmaceia Biotech) and 0.375% methylcellulose catholyte 20 mM sodium hydroxide anolyte 100 mM phosphoric acid in 0.375% methyl-cellulose focusing 5 min at -30 kV mobilization low pressure (0.5 psi) for 10 min at —30 kV detection UV, 415 nm. [Reprinted from Electrophoresis 78 1785 (1997), copyright (1997) Wiley-VCH.j...

Figure 20-6 The common phenotypes of haptoglobin, as shown by sieving gel electrophoresis of haptoglobin-hemoglobin complexes.

Cotton F, Lin C, Fontaine B, Guibu B, Jansens J, Ventongen F. Evaluation of a capillary electrophoresis method for routine determination of hemoglobins A2 and F. Clin Chem 1999 46 237-43. 

Gerritsma, Sinnige D, Drieze C, Sittrop B, Houtsma P, Hulshort-Jansen N, Huisman W. Quahtative and quantitative analysis of hemoglobin variants using capillary zone electrophoresis. Ann Clin Biochem 2000 37 380-9. 

Hempe JM, Graver RD. Separation of hemoglobin variants with similar change by capillary isoelectric focussing value of isoelectric point for identification of common and uncommon hemoglobin variants. Electrophoresis 2000 21 738-43. 

Sugano M, Hidaka H, Yamauchi K, Nakabayashi T, Higuchic Y, Fujita K, et al. Analysis of hemoglobin and globin chain variants by a commonly used capillary isoelectric focussing method. Electrophoresis 2000 21 3016-9. 

Two modifications of classical hemoglobin electrophoresis are also useful for studying these proteins isoelectric focusing and electrophoresis of individual globin chains, with or without the heme. These techniques can be used to identify some hemoglobins that are not separable by electrophoresis of the complete hemoglobin tetramers. 

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