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166 PART III. ABNORMAL HEMOGLOBINS d. The mobility of the rare Hb I is between those of Hb A and Hb F. e. It seems impossible to separate lIb ~ from the adult component. A comparison of the relative Nobilities of the different hemoglobins in paper electropl~oresis and in chromatography is given in figure 2. In general the same sequence exists. For the hemoglobins ~ and H important differences C ~ ' E o - ' , 5 ~ , D ~ ' F ~ _ ,. A q J l 1 H , O _ 1 ' I | ,_~ paper electrophoresis I I chromatography _ Flu. 2. Comparison of the relative mobilities of different types of human hemoglobin in paper electrophoresis (open blocks) and chromatography ( filled blocks ) . were found, as the fetal pigment moves faster than the normal adult hemo- globin in chromatography and Hb H still much faster than Hb F. With the other hemoglobin types, smaller differences are present for the hemoglobins E, I and I. Each method has some advantages. The identification of hemoglobins E and I, for instance, is much easier with paper electrophoretic techniques. The chromatographic estimation of Hb A, on the other hand, may be important; the characterization of this hemoglobin by paper electrophoresis is difficult. Hemoglobins H and I can also be distinguished by using the chromatographic method. The use of both methods at the same time is of importance for the definite identification of an abnormal hemoglobin type. REFERENCES 1. Huisman, T. H. J., and Prins, H. K.: J. Lab. Clin. NIed. 46: 255, 1955. 2. Prins, H. K., and Huisman, T. H. J.: Nature 177: 820, 1956. DISCUSSION Dr. Martin ILlorriso?z: I would like to outline briefly our procedure for the column chromatographic separation of human hemoglobins. The tech-

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DISCUSSION 167 nique eve employ is to adsorb the hemoglobin on a resin bed of XE 97 (IRC- 50 ~ which is in equilibrium with a phosphate-citrate buffer, pH 6.3, and which has a sodium ion concentration of 65 mEq. per liter. The hemoglobin solution itself has been previously dialyzed against the same buffer. The pro- tein is then eluted from a 1 ~ 50 cm. column by gradually increasing the sodium ion concentration. When the oxyhemoglobin obtained from a hemolysate of the red cells of a normal adult is investigated in this way, a chromatogram such as is shown in the figure 1 is obtained. Note that the hemoglobin is eluted in three fractions. too o 080 a' Us 060 A a: a: o 0.40 020 ,~ _ 1 0 20 30 co 50 TUBE NUMBER 60 70 FIG. I.- Chromatogram of hemo- lysates of normal adult red cells. The most rapidly eluted of these components represents approximately 10 per cent of the total hemoglobin contained in the l~emolysate. The second, or major, component represents about 86 per cent of the total hemoglobin, while the third and most slowly eluted component comprises 4 to 6 per cent of the hemoglobin and, as subsequent illustrations will show. moves chromato- graphically as Fib E. ~ . . . . More detailed studies of each fraction were undertaken. By employing dilute buffers, eve found that the first, or most rapidly eluted component, could be further separated into two colored components, the first of these being a protein fraction ravish methemoglobin reductase properties. When the major component, representing 86 per cent of the hemoglobin of the hemo- lysate of the normal adult, was purified by column chromatography or starch block electrophoresis, a single peak, shown in figure 2, was obtained. This demonstrates that tile fractions were neither column artifacts nor conversion products of the protein and further verifies that when a single hemoglobin is present, a single peak is obtained. Our efforts then naturally focused upon identification of the components which we found in the normal adult. Since there was substantial evidence i the literature that fetal hemoglobin occurs in the normal adult in small per- centages, she began with pooled cord hemoglobin, the chromatogram of which is shown in figure 3. The first component to come o* the column was re- sistant to alkaline denaturation. This, as well as spectral analysis, definitely

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168 60r ~50 E o ~ 4 0 z 30 c lo: 20 On q 10 PART III. ABNORMAL HEMOGLOBINS FIG. 2. Single peak obtained by chroma- tography of major fraction in figure 1. 1 ~ 1 !~1 20 30 40 50 60 70 TUBE NUMBER established it as fetal hemoglobin, which is in sharp contrast to the results obtained from the normal adult component which came off the column in the same region. The adult component is not resistant to alkaline denaturation and on electrophoresis at pH 8.6 moves more rapidly than Hb A, while fetal hemoglobin, which is resistant, moves behind fIb A on electrophoresis. 1 800 1600 ha. 10m to - Z ,200 - ~ 1000 be z 800 o A 600 o 0 4 00 200 1 C ) ! ! ~~ 1 10 20 30 40 50 60 TUBE NUMBER I POOLED CORD HEMOGLOBIN (5 ~ am pies) FIG. 3. Chromatogram of pooled cord blood hemo- globin. so so 6 o 501 40 SO 20 T :~> Z C3O o OCR for page 166
D1SCL; SSIO3N 1G9 ~ . A Zen i ! ~ ~ ~ FIG. 5.Composite chromato- 0 ~ :~ \, ~ ~ gram Of several types of hemo- 0 10 20 30 40 50 60 70 80 90 TUBE NUMBER tube 55, Hb S at tube 65 and Hb C at tube 7j. Of the hemoglobins investi- gated, Hb E moves chromatographically as the third component of normal adult. The movement of the hemoglobins in column chromatography is directly related to the charge carried by the protein.' This relationship is demonstrated as follows: Relative Mobilities of Human Hemoglobins on electrophoresis at pH 8.6 pH 6.5 On chromatography at pH 6.3 A ~ S E C C S E A F A E S C Below the iso-electric point, hemoglobin carries a positive charge. On elec- trophoresis, the compound with the lowest mobility has the smallest charge. This means it will be least strongly adsorbed on the column and will, there- fore, move off the column most readily. Conversely, the compound with the greatest positive charge will be most strongly adsorbed to the column and will be eluted last. It follows, then, that electrophoretic mobility and column chromatographic mobility are inversely related, and that this relationship is a direct consequence of the charge carried by the protein. Essentially, no anomaly has been found to this situation. (See, however, bibliographic ref- erence No. 3~. Dr. Itano, in his earlier talk, described the separation of hemoglobin mole- cules in which the iron atoms have been oxidized.4 If the major component of normal adult hemoglobin is oxidized into any of these types between methe- moglobin and CO-hemoglobin, their separation can be achieved. Boardman and Partridge,5 in their first study of the chromatography of hemoglobins by ion exchange resins shorted that they could separate bovine methemoglobin from bovine CO-hemoglobin. We have found that we car separate and spec-

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170 PART III. ABNOR'VIAL HEMOGLOBINS trophotometrically demonstrate the separation of the various intermediates achieved by the partial oxidation of the iron atoms of hemoglobin. Table I shows the type of separation achieved, which is very good and has the ad- vantage of making possible spectrophotometric inspection of each f faction. When this is done, the spectrophotometric analysis shows that the fractions TABLE I COLUMN- CHROMAT~GRAPHIC MOVEMENT OF FORMS OF HEMOGLOBIN A Compound CO-hemoglobin Intermediates Methemoglobin Ratio Fe+'/Fe~ 3 4/0 311 2/2 1/3 0/4 Peak Tube No. 28 54 65 75 90 correspond to hemoglobin ire which the iron atoms were progressively oxidized. As was the case with the abnormal hemoglobins, the forms of Hb A move off the column according to the charge on the molecule. The molecule with all the iron atoms in the ferrous form is least positively charged and is elated snore readily, while the methemoglobin carries the highest positive charge and is the last form to be eluted. Thus, the forms of hemoglobin may lead to erroneous results in the quantitative analysis of mixtures of Hb A and abnormal hemoglobins unless due care is exerted. The procedure was also used to investigate the possible dissociation of Ilb A into components. Employing exactly the same buffers and chromato- graphic system, with the exception that all solutions were made four molar with respect to urea, we investigated the homogeneity of Hb A. It would appear that if Hb A did dissociate under these conditions, we might be able to separate the components from one another. However, under the experi- mental conditions we employed, we could detect no dissociation. I might mention that our results with thalassemia are, qualitatively at least, comparable with the results Dr. Kunkel~ 7 has obtained. However, we find that in thalassemia major there is a higher concentration of our third component which appears to move like Hb E on our column. Atcknowledgment: Figures 1, 2, 4 and 5 appear in Federation Proceedings 16: 764, 1957, and are reproduced by permission of the publishers, the Fed- eration of American Societies for Experimental Biology. REFERENCES 1. Morrison, M. and Cook, J.: Chromatographic fractionation of normal adult oxy- hemoglobin, Science 122: 920, 1955. 2. Morrison, M. and Cook, J.: Column chromatography of human hemoglobins, Fed. Proc. Vol. 16 (September) 1957. 3. Huisman, T. H. I. and Prins, H. K.: Chromatographic estimation of four different human hemoglobins, J. Lab. and Clin. Med. 46: 255, 1955. 4. Itano, H. A. and Robinson, E.: Demonstration of intermediate forms of carbon-

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DISCUSSION 171 monoxy- and ferclbemo~lobln by movlDg boundary electropbores~, J. A. C. S. 76: 641i, 1966. 5. Boardman, N. K. and Fartdd~e, S. Hi.: Sep~radoD of neutral proteins OD 1OD excbaDge reslDs, Plocbem. J. SP: 643, 1933. Kunket H. O. and ~allenlus, G.: Bed bemo~loblD ID DOrma1 adult blood, Science 722: 2SS, 19i5. 7. Junket H. O.: D~trlbutloD and sl~nlEcance of the minor bemo~loblD components of DormaI bumaD blood, Fed. Proc. Vol. 16 (September) 1937.