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ZONE ELECTROPHORESIS AND THE MINOR HEMOGLOBIN COMPONENTS OF NORMAL HUMAN BLOOD HENRY G. KUNKEL Filter paper electrophoresis because of its simplicity and widespread avail- ability has been of great value in elucidating the various abnormal hemo- globins. For population screening a simple technique of this type was essen- tial. However, a number of limitations have gradually become apparent; adsorption in the path of migration, inequality of migration in different parts of the paper and poor adaptability to preparative isolation represent a few of these limitations. As an alternative procedure we have been interested in the use of other supporting media such as potato starch and polyvinyl chloride particles, pare ticularly for the isolation of various hemoglobin fractions. Both of these media, employed in the form of a thin slab, although possessing inherent limitations of their own, have been of some value for these purposes. The starch block technique has the disadvantage that extraneous materials fro ~ the starch itself frequently contaminate the purified hemoglobin fractions. The polyvinyl chloride supporting medium avoids this limitation. However the ease of handling the starch, the homogeneity of migration, the low elect troosmotic flow and the ready visibility of minor colored components against the white starch background have made this procedure more generally useful Fig. 1 illustrates a photograph taken with transmitted light on orthochro~ matic film of the red components of various normal and~abnormal hemo- globins separated on a thin starch block. The separation is in general very ~7 .~ ~.:-~ .. ~ , . FIG. 1.Comparison of different Hbs separated on the same starch slab. Line at left is site of application. Anode.-to the right. Barbital buffer pH 8.6 I/2 0.05. a. Normal Hbs b. Homozygous C c. Sickle cell anemia d. Umbilical cord e. Sickle cell trait

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158 PART III. ABNORMAL HEMOGLOBINS similar to that observed with filter paper. The use of broader starch slabs permitted the separation of as many as thirty specimens in a single experiment with resolution similar to that seen in fig. 1. For detecting the abnormal hemoglobins, the hemoglobin solutions were usually diluted with buffer to a protein concentration of approximately 3 per cent. Quantitative elusion of hemoglobin from starch segments could be carried out by displacement filtra- tion over ground glass filters, thus permitting direct determination of rela- tive concentration from hemoglobin color. Quantitation was most accurate when the CO or cyanmethemoglobin derivatives were employed. c c ........ _: _ _ r _ _4 ............... FIG. 2. Photograph of multiple normal Hb specimens separated on a broad starch slab. Two specimens from indi- viduals with sickle cell trait are included for comparison. The minor Hb A`, is visible in all specimens. . _ ~ _ ~ ~ ~ ~ ~ ~~ ~ ~~ ~ a ................ ' ~ I"'"''' '' .~ ~'~,~' ' I, ,~ ''' ''"''''"'. ........ ... . . ~ ~ ~ ~~ ~ ~ ~ ~ ~ ~ .~ ~ ~ . ~,~.~., ~.~ .~ ~ ~ ~.~ All ~ ................................................ ~ :~.~.~ ~~ it . ~ ~ ~T .. ~~ ~ ~ . .~. .. ~ ................................................. - ... , ~.~ ~ ~ ~ ~ ~ i ~ ~ ,~ ...... ~ i ~ .. ~ ~ ..... , ... . i I.~.~ ..... ............ .. . ~ T ~ ~ i. ~ ............ I .. ~~ i. ~ ~: ~ ~ ~ C ~ i ... ~~ .~ .... i .~.. .. ... ~ ~.~ ~ g. :::::::: i::: FIG. 3. Comparison of the mobilities of the isolated normal Hb components separated at equal concentrations. a. Slow An b. Main A c. Fast fraction The normal hemoglobin sample shown at the top of fig. 1 shows two dis- tinct components. A minor fraction with a mobility just slightly greater than Hb C was apparent with all specimens from normal individuals. This frac- tioni 2 (termed Any was best visualized when the hemoglobin solution was separated at a 10 per cent protein concentration. Fig. 2 shows multiple normal specimens on a broad starch block. Two specimens from individuals with sickle cell trait are also included. All of these showed the As component at approximately equal concentration. In addition to the A2 fraction, the main A fraction always showed faster

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ZONE ELECTROPHORESIS OF HEMOGLOBIN A KUNKEL 159 migrating material projecting in front of the round A spot;) this is visible in both figs. ~ and 2. Some of this material migrated markedly faster than Hb A. Isolation and re-running showed that it kept its original fast mobility. Fig. 3 shows the result of one such experiment where the isolated Hb A2, Hb A from the main A peak, and fast material were examined at equal concentrations on the same starch block. The A2 Hb kept its original slow mobility. The fIb A no longer showed projecting faster hemoglobin and moved as a sharp band. The fast fraction was broad and retained the original mobility. The same components were observed with the polyvinyl supporting medium. Ordinary filter paper strips did not show the A., clearly but thicker paper, which permitted more hemoglobin to be applied, brought out this component. Derivatives of oxyhemoglobin, carbonmonoxyhemoglobin, me/hemoglobin, cyanmethemcglobin and reduced hemoglobin all showed a similar relative distribution for the minor components despite differences in over-all mo- bility. Different anticoagulants and different procedures of faking the red blood cells gave similar results. Numerous observations with the isolated A component indicated that it did not give rise to the A, fraction. However, con- siderable evidence was obtained that the fast fraction or material like it could be produced from Hb A. This transformation occurred slowly with oxy- and carbonmonoxyhemoglobin but was particularly rapid when methe- moglobin or cyanmethemoglobin was used. Old hemoglobin samples also were found to contain considerably more of the fast fraction. Although the fast fraction could not be quantitated, it appeared to be present at a con- centration somewhere between 4 and 12 per cent in fresh hemoglobin speci- mens. The accumulated evidence strongly suggested that the A2 component was a well-defined entity but that the fast fraction might be derived from Hb A. Some degree of quantitation of the A2 component was possible. Replicate analysis of one normal hemoglobin sample (separated 16 times) showed a mean value of 2.51 per cent with a standard deviation of + 0.31. Examina- tion of the blood of 65 normal individuals showed that the As level was 2.54 per cent + 0.35. In a larger series of normal individuals studied sub- sequently, no A' level above 3.3 per cent was encountered. However, in pa- tients with thalassemia considerable elevation was frequently found. The highest level observed was 11 per cent in an adult with an intermediate type of thalassemia. Four individuals classified as intermediate thalassemia were studied and all showed unusually high levels. The over-all results in patients with thalassemia excluding the Cooley's type (34 cases) was 5.11 per cent + 1.36. The infants with Cooley's anemia frequently had relatively normal levels. Two patients with thalassemia minor also showed levels in the normal range. The significance of the elevation in thalassemia was not apparent. No elevated values were obtained in any other condition studied.2 One possible

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160 PART III. ABNORMAL HEMOGLOBINS explanation is that a compensatory increase of lIb A2 similar to that found for fetal hemoglobin occurred in the presence of defective Hb A synthesis. However, the frequency of levels just twice the normal in thalassemia minor remains unexplained. To further determine the significance of the A2 arid fast hemoglobin fractions, comparative specific activities were obtained after administration of F'e59. Observations by Schapira and associates3 had indicated that this method gave evidence for the heterogeneity of human hemoglobin. The possibility also arose that such experiments would provide evidence regarding the presence of the minor fractions in all red blood cells or just in selected cells at high concentration. Table I shows representative results obtained in two essentially normal individuals at different times after the intravenous in- jection of approximately 32 tic of Few (0.024 ma. Fe). In the experiments with patient ELF. the separated hemoglobin was divided into three fractions designated A2, A and fast. With the hemoglobin of patient Y.R. further subdivision was carried out. The fast material was divided into two parts, a very fast fraction, designated fasts and a relatively slower fast fraction designated fasts. Hb A was also divided into a fast and slow part. In each case, all of the separated hemoglobin fell into one of these fractions and the relative percentage of each in the different experiments is indicated in table I. The most striking finding was the low specific activity of the fast fraction compared to that of Hb A. This was evident in all experiments and can be seen best from the relative specific activity calculated by assigning the value 1.0 to either the whole of Hb A, or to its least contaminated slower portion. No essential differences were observed whether the radioactivity was meas- ured in terms of hemin or as the whole hemoglobin solution. The experi- ments with the second patient are the more informative because the fasts fraction was completely free of Hb A. The fasts fraction as well as the whole fast fraction in the experiments with the first patient did not represent pure samples but probably contained some Hb A. FIb As showed a specific activity similar to but always slightly below Hb A. However, it was somewhat di~- cult to make an exact comparison because of the range in specific activities of the A and the overlapping fast fractions. These results appeared to indicate that the fast hemoglobin had special significance despite the fact that it could be produced in vitro from Hb A. The exact reason for the low specific activity of this fraction compared to the bulk of the Hb A is not clear. One possible explanation is that the fast component was more concentrated in old red blood cells and was formed in vivo from Hb A under the stress of survival in the blood stream. References to fractions which may have corresponded to these minor hemo- globins have previously appeared in the literature. The As component prob- ably had been observed by earlier investigators employing the classical Tiselius procedure in occasional specimens of blood from normal persons,3 4

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ZONE ELECTROPHORESIS OF HEMOGLOBIN A KUNKEL 161 TABLE I COMPARISON BETWEEN THE SPECIFIC ACTIVITY OF VARIOUS IEMCGLOBIN FRACTIONS AT DIFFERENT TIMES AFTER ADMINISTRATION OF FE59 Days after Hb % of Spec. Act. Ratio of Subj. Feo9 Fract. Total cts/100 fly F`e Spec. Act. . fast 5.2 152. .584 4~/2 A 92.3 2g6. 1.0 A, 2 5 282. .95 f ast 4.8 208. .49 M.F. 11 A 93.1 425. 1.0 A2 2.1 390. .92 fast 4.2 198. .56 21 A 93.2 354. 1.0 A2 2.6 297. .84 fast' 3.1 145. .32 fasts 13.6 397. .87 7 Af . ; 1.4 420. .92 Ash. 30.4 455. 1.0 A`, 1.6 446. .9 . fasts 1.2 178. .43 fasts 3.7 232. .56 Y.R. 14 Af . 59.4 353. .85 Asl. 33.6 417. 1.0 AS 2.3 333. .80 fast' 2.2 127. .64 fast., 4.5 179. .89 37 Af . 51.1 220. 1.1 Ash. 40.8 200. 1.0 An 1.4 180. .9 _ sickle cell patients and thalassemia patients6 but had not been defined as a hemoglobin. It also resembles one of the chromatographic subfractions ob- tained by Morrison and Cook: from normal human blood. Recently additional information concerning Hb A2 in thalassemia has been obtained by Gerald,8 Aksoy and co-workers9 and Josephson and Singer.~ The fast fraction, since it is impossible to quantitate, is not easily related to other fractions described in the literature. It seems possible that the heterogeneity noted by Derrien and his associates, Shavit and Breuer~2 and Hochi3 may be caused by this fraction. Also the recently described component