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STUI)IES ON THE HETEROGENEITY Of ADULT AND FETAL HEMOGLOBINS BY SALTING-OUT, ALKALI DENATURATION AND MOVING BOUNDARY ELECTROPHORESIS DIFFERENTIATION OF HEMOGLOBIN FROM NEWBORN CHILD AND FROM PATIENTS WITH COOL1EY'S ANEMIA YVES DERRI1:N During the past ten years we have studied, in collaboration with Roche and other co-workers, the heterogeneity of hemoglobins, using chiefly salting-out, alkali denaturation and electrophoresis techniques. With the salting-out method applied under carefully controlled conditions, three fractions Oft, f2, f3) have been distinguished in Hb ~ and at least two (~ and as groups, the latter in most cases splitting further into a'2 and a',2) ire Hb A, as shown in figure 1.0 3 4 The upper curves express the solubility 1Eo it_ CDH: /1~1~_ 05 ~ \ 2 _ ; \~ ~ - ! \,'., ~ ~OA=6% ~ I O Trove - I- i~ ~o ~_, 80 Is c 90 20E ~A' _] 1SO E 10 05 C - 80 HE Boo 1sO 100 50 - ma COW Novena By ~ ! ~ , I _ __\ ~ _, _ ~ ~ ~ Nf' ~ \7~ ~~ '.- ,?0~.. B2% ~^ Groupe, A= 69 % ~ I ~ ~~' , 1 ! ' ' : ~ ! ! ! 1 85 SO c 1 ~ ! , FIG. 1. Above Salting-out curves for carbonmonoxy hemo- globins of normal adults and new- born children. Chromoprotein con- centration = about 0.3 per cent; pH6.7; temperature - 24 C.; R.D.~. = alkali-resistant fraction; C - salt concentration, expressed in per cent by volume of tile stock salt solution (equimolar mixture of 3.5 M mono- and dipotassium phos- phate); E -~ optical density of fil- trates at ~ 500 my. Below: Derivative curves. /\E decrease of optical density for each increment ( /\C) of rne salt concentration C. /! C 1. Ordi- r~ate AE multiplied by 103. E as a function of salt concentration C; the lower curves are derivatives of the corresponding upper curves.' ~ The changing of fetal into adult: fractions has been followed during the embryonic life and after birth, as partly shown in figure 2. Data of this kind have been obtained for man and for cattle.` s Some abnormal human pigments have been studied by the same method and our chief results are summarized in figures 3 and 4. Hemoglobins S or D, identified by electrophoresis and by Itano's test,9 are easily detected by salting- i8:

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184 PART III. ABNORMAL HEMOGLOBINS 20 10 o AS x2/3 10 5 GS _ ~ 80 p1 90 c Ma 90 c I AS Adu/f e ! 1 \ t2 \/j | at fit O ~\V~Ob ~ it. IS 80 90 C 80 90 C 15 10 ( TV _ ._ _ _~ 5 E ~5 JEg 150 1 FIG. 2. Derivative curves for carbonmonoxyhemoglobin of a normal adult, a new- born child and 3 6-, 74-, 80- and 90-day-old children. Conditions of salting-out as in figure 1. _~03 Adulte Normal , ~ Hemoglobinose I) DA (~6O9J,' [% ~ R DA Labor ) ~ 6 % (D D'~D_54 % . \ 1 1 O ~ Aim__ Aim"._ 50 B5 90 C So es So C TOO- - :! _ , , ,. ~ , A' h\ SCT \ S RDArdhO,J 2% ~ ~ S'- S.,.. 5' ~ ~ ..\ ~ 65 C 90 dS 90 C - - 80 AS YU 1 1 FIG. 3. Salting-out curves and corresponding derivative curves for carbonmonoxy- hemoglobin of normal adult and of subjects with hemoglobin D trait or sickle cell trait Chromoprotein concentration about 1.5 per cent. Eoptical density of fil- trates at ~ = 550 my. Other details as in figure 1.

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HETEROGENEITY OF HEMOGLOBINS A AND FDERRIEN lgS out experiments. Both are salted out in an identical way, at a slightly lower salt concentration than the fraction a~, and yield two components each (S' and S. D' and D), the second-named (S and D) occurring in much larger proportion.~ ii Hemoglobin F in the newborn child' the fetal-like pigment of patients suffering from Cooley's anemia, and hemoglobin C have practically the same solubility but only hemoglobin C is alkali-labile.)'' i3 The indi- viduality of hemoglobins A, F. S. D and C can be defined by salting-out as well as by electrophoresis to j Ha l to I j E ~~: Solved -AYe E, ~ '4ne~m/e E ~ = ffemog/ob/nose: V9 ~ - N~ ! ~ :, 1- , ~ _:, . . . _ I\ I I N~ 1 ~ t ~ ~ ~ ' ~ t ~ ~ I \ ................. - N , I ~ ~ _ ~ _ At, (5 _ ~ ~~-~ 05 _~,;_ . ~ \~2 1 ~ ~l \~ 1 ~~ . ~ __ [__ ~ ~ I ~ AD A 4% ~C(ft~ ~~. - Grou,oe C. 66? i ~ . 05 _ . I , /'D A /6 % i - G'oa,oef; I'd i 0 . I . ~ I a:, :' , 1 . ! . - ~ 0 65 90 C 9 of; 0 65 0 C 9 i ~ 0 86 90 C ~ i AL ~ it it, Af ~ ~ f, BE ~(,~,1~ I /50 - 1~24.~2 /50 - ~ I50 I ~ fOl 56 700 ~~ 50t - 11 ~ oft ~ 90 c 9 j OS :~: - ~ ~ 9 - C - 1 OBO 55 SO C - s FIG. 4.- Salting-out curves and corresponding derivative curves for carbonmonoxy- hemoglobin of newborn child and subjects with Cooley's anemia or hemoglobin C disease. R.D.A. all~ali-resistant fraction of oxyhemoglobin. Salting-out conditions as in figure 3. On the other hand, the presence of fractions of different solubilities in hemoglobins A and :F has presented a quite new and still unsolved problem. The objection has been made that such discontinuities in salting-out curves probably indicate changes in the nature of the solid phase, resulting either from the precipitation of different crystalline forms of hemoglobin or from ~ change in the type of aggregation of the same hemoglobin due to interaction with small molecules or ions in the solvent. Our results do not support such interpretations. The arguments in favor of true heterogeneity of hemoglobins A and :F will be considered under the fol- lowing five areas of discussion. 1. The discontinuities in salting-out curves define an identical number of fractions whatever the nature of the neutral salt (ammonium sulphate or potassium phosphates), or the value of pH (between 6.5 and 8.5), or the

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186 PART III. ABNORMAL HEMOGLOBINS ~ r 05 ~: 93 (J: _ - l~t ~ - ~N ~ _ . ~ .\ ~1 ~'.E ''~ ~ \,~91 90 1 ', , , 1: . ~ ' . ~' _ ' ~ PO 30 40 ~ ~ 60 ( . 01 _ 109S 3 '5- , /~- : ~,~ . , t,O- . \ n ~ . ~ :~ ~ \; Fo ' ~o 80 50 40 50 C 60 1.0 . /093 ,_. - . ' ~N -94S ~ -: ~ --03 \ ~s ~2  ~\ ._ . 1 , 1 , ~ 1_ S 20 logi 40 50 C 60 ~'\\ ~ ~S 2 ,3 /~ PS 20 '~.S ~: - PO 30 40 50 C 60 tO ~5 ~IG. 5. Crystal solubility curves for horse carbonmonoxyhemoglobin (no. 1 ) and for separated fractions (nos. 2, 3, 4). S and log S are both functions of C, salt con- centration. C = per cent saturation in ammonium sulphate; S solubility expressed in optical density of filtrates; pH 6.4; temperature 24 C. ro 0,5 o at 200 15O nn 5D I,0 f 0,' ~ ~o COHb Ad hum f Fractmn P76 \ I NV-53 ~ ~ de COH~ N V-53 _ ~ Solub//~te de cr~st.~u~ _ \~~ So/ub//ite ae cr~(~ux ~ a =; Jo/o \ a _ 65 0/o =:ai__ I 05 \ _ \ ' \t _ \\ ~ ~2 ~a ~ ~ _ < ~~, ,[! , , ~) o 75 80 C 85 75 80 C 85 |~ ~ _. _ 2aoE ~d___ ~ ~ _ ;~]1: (~\ 75 80 C 85 75 80 C B5 a, 66 F`IG. 6. Crystal solubility curves and corresponding derivative curves for normal adult carbonmonoxyhemo- globin No. V-53 and for fraction P 76 separated from it. pH 6.7; temp- erature 24 C. Other details as in figure 1.

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HETEROGENEITY OF HEMOGLOBINS A AND ~ DERRIEN 187 nature of the solid phase, whether amorphous (salting-out curves) or crys- talline (solubility curves or crystal suspensions). Neither the combination with oxygen or carbon monoxide nor the partial or total oxidation into ferribemo- globir~ changes the heterogeneity of a preparation.~5 On the basis of these experimental data, it is deemed unlikely that the discontinuities in salting-out curves represent transition points between the regions of stability of different solid phases of the same hemoglobin. 2. It is always possible to isolate, in a more or less pure state, some of the fractions individualized in the salting-out curves of hemoglobins. As is shown in figure 5, the two major components of horse hemoglobin have been isolated.] The overlapping of fractions al and as of human adult hemoglobin made their separation more difficult. However, figure 6 shows that the fraction al may be partly purified by a single fractionation procedure. On the other hand, it was shown by ultracentrifugal studies that, in a 1 M solution of the primary and secondary phosphate mixture of pH 6.7 used for salting-out experiments, normal adult hemoglobin is homogeneous and totally dissociated into half molecules.* All of these data contradict any interpretation that fractions a, or ~2 were formed by reversible molecular dissociation or association or by salt binding. The fact that one of the fractions keeps the same salting-out fea- ture after being isolated as was seen in the initial mixture leads to the same 1,0 100 50 j Dhd/dsse~m/e ~0 demo9/o61i70se C to /iemo>7/ob/nose C- :05 ~05 'me Na. \\ i, ! P- -ma- SKI I . ~ ~\C(f,] PAD `4. 36;/ ~ Grau,ae C.PQ~ :2l_c,f~ con BO B5 90 C 0~ ! I ! , ~~ 5 08 C ~; go: C 9 5 _ ~ l !\a2 1\ . ~,.~,,,,~C Grau,ae C. PQ~ :2~_C.fs . ' . ! ! ! ~ :1~r l ADA, 6` If, Gro~,oc C. Big ~ j . ~ ! . ^. A A A , 150 ~ ~ 150 ~ a ~ LIC to o . i 1~ ' : tC4If,, ~ \: _ Ida | I,,, 90 its FIG. 7.- Salting-out curves and corresponding derivative curves for carbonmonoxy- hemoglobin of subj ects with thalassemia minor, hemoglobin C trait and hemoglobin C-thalassemia association. RD.A. ~ alkali-resistant fraction of oxyhemoglobin. Salt- ing-out conditions as in figure 3. Tonnelat, l., and Derrien, Y.: Unpublished studies.

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188 PART III. ABNORMAL HEMOGLOBINS 1, o 1 o,s 0 HE 150 inn .sn 0~ leucem. lymphoide ~ card) ~ .__ , Pa ~ I \ >a , ~ 2, ~[ so 85 C 90 1,0 l eucem. myeloide ~ _ ~ ~ /~iJJ ~ _ _ \ ~ ~1 _ \2 _ I \; O ! ~ ~ ~ ~ ~ 1 ~ ~ >_ 7 , 80 a, 65 ~ 9 AL 150 06 5L 80 85 vi 90 1 0 0,5 0 ,, I,, ,, I ,,: o 80 8s C TV at I ! 50 00 50 l Jocose Pique I {B/S ~ I to, _W \ I ' 1 ~ '~-- 1 1 W~ FIG. 8. Salting-out curves and corresponding derivative curves for carbonmonoxy- hemoglobin of patients suffering from lymphoid leukemia, myeloid leukemia and acute leukosis. Salting-out conditions as in figure 1. conclusion. Thus it has been established that normal human adult hemoglobin contains two components: a: and a2 (or a group). The absence or the very low proportion of al during the first two months after birth (fig. 2), in thal- assemia minor, in hemoglobin C trait (fig. 7), and in some cases of acute leukosis or myeloid leukemia (fig. 8) support the concept of the individuality of fractions al and a2. 3. Research on the blood pigment of the newborn child extends to hemo- globin F the evidence of heterogeneity shown by hemoglobin :'i. The per- centage of alkali-resistant pigment and the percentages of fractions f, identified bar salting-out, have been determined and compared in a large series of carbon- monoxyhemoglobin samples prepared from umbilical cord blood collected at 90 MA To ~0 70L ~0 fool 70t 60 \ o/ o . . \ / To Hip / ~ /o a\ / \ To o o o \ ~ Of 50~ J J AS OND J FMA~J JASONDJ F~A~J JASOND . . . ~ t953 t954 /95 5 FIG. 9. Comparative yield in alkali-resistant fraction (R.D.A. COO) and in fractions of the f group (f To ) of carbonmonoxyhemoglob- in from newborn children at dif- ferent times of year.

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HETEROGENEITY OF HEMOGLOBINS ~ AND FDERRIEN 189 various seasons of the year. As figure 9 shows, 80 + 5 per cent of the pigment is alkali-resistant in every season. On the other hard, the level of the fractions I in the salting-out curves reaches a minimal value of 55 per cent in Tanuary- February, and a maximum, nearly identical with the level of the alkali-resist- ant fraction, in summer. Such observations suggest that the alkali-resistant hemoglobin corresponds essentially to fractions f during the summer arid partly to fractions of the as group during the winter. This hypothesis is con- frmed by comparative studies of the total carbonmonoxyhemoglobin of 14 such I,0 ~ 04 ~ . . . ~32 ~ ~ COHbN-N `` ~Q.~.A. ~ - .~,1 ,12-S5 f ~ Cat, isolee : ~ I \ ! ~ \2 ~ ~ \jf; os I Sir, 0,5 _ ~ ~ - ~ tip - ~ ~ - | ~ _ ~ ~ off? _~.~.A. B4~o ~r i i ~ roupef 56% 1 AN? Groupe f 70% I $: I: ~ F ~ ~ I! ~ o o 80 85 90 C 95 80 Us 90 c 95 Af I ~ fief ~ f I 150 ~ A, I 150 t ~ ~ at ~ 11 t fl III . I , J0O _ IN ~ tocE - ~f ~~ 50 / ~ \~3- 50 _ - - ?1 ~ ~ O 4_?_l 1 ~ ~ ~ 11111 O 80 35 SO C 95 80 85 FIG. 11. Salting-out curves and ,, . . corresponding c er1vat1ve curves for newborn carbonmonoxyhemo- globin No. 455 (child born in June) and for its isolated alkali- resistant fraction (R.D.A.). Salt- ing;-out conditions as in figure 3. so 80 85 90 C SS FIG. 10.Salting-out curves arid corresponding derivative curves for newborn carbonmonoxyhemo- globin No. 255 (child born in January) and for its isolated al- kali-resistant fraction (R.D.A.). Salting-out conditions as in figure 3. 1,0 j 1,G ~ ~~ COH.b SN H ~ ~ Proof. R D.A. ~~-~ ~ ~ ~ 1~ ~ NO i, \t,I 0~5 . ~ ~ At as ~ \l tiroupef: 73% | `\~5 Groupe ~ 93% ~ \\f3 9S oh do R 4 - ~ , _ \ to I 1 1 ~ 1, 1 ~ 1 1,,,~1 o , 11 1 ~ l l l l 80 85 90 ~ 95 SO . 85 90 C 95 .~^ ~ ~ I Her ton v So 85 90 C 95

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190 PART III. ABNORMAL HEMOGLOBINS samples of blood from newborns and of their alkali-resistant fraction isolated by our technique. As shown in figures 10 and 1l, the alkali-resistant pig- ment can yield up to 30 per cent of fractions as in January and may be prac- tically free of fractions other than f in [une. The reliability of the salting-out curves method can be seen from the quantitative data assembled in table I: the ratio of the fractions f to the total alkali-resistant fraction of the pigment is nearly the same whether the fractions f are determined by salting-out of the total pigment or of the isolated alkali- TABLE I Carbonmonoxyhemoglobin of Newborn Children | Isola ted R.D.A^. Fraction Components ~0 No. Components % a', + at | f Group |~.D.A. Fract. f Group % oFf R D A | f GrouP ~ a ~ ~ a 13 18 21 24 27 29 31 1-55 2-55 4-55 8-55 1-56 1-5/ 4-57 47 31 28 17 21 32 27 42 41 24 35 44 43 26 53 69 72 82 73 64 73 56 56 73 63 55 57 74 77 83 80 86 83 75 77 80 84 77 78 79 80 86 69 83 90 95 88 85 95 70 67 95 ~1 70 71 86 70 85 94 93 90 87 93 72 70 93 70 73 87 26 11 6 5 10 13 7 28 30 6 10 28 27 13 resistant fraction. Such data give experimental evidence of the significance of the proportions of components as estimated by the salting-out method and show that the fractions a., of newborn children contains in some cases two kinds of pigments one of the adult type and the other of the fetal type- as determined by their resistance to alkali denaturation. The individuality of the alkali-resistant pigment of the as group can also be tested by experiments whose results are reported in figure 12. A partial fractionation of the isolated alkali-resistant hemoglobin can be achieved by salting-out and leads to an enrichment either in fractions as or in fractions f. Therefore, both types of pigments have to be considered as different. Furthermore, preliminary attempts to separate the components of the f group allowed us to partly purify some of these, especially fir. 4. The alkali-resistant fraction of normal human adults, isolated by a suitable technique,iS is spectrophotometrically identical to the whole pigment

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HETEROGENEITY OF HEMOGLOBINS A AND ~DERRIEN 191 't t o,; u . . . 80 85 90 C 95 ~ __,'2 ; f88 = )_ ~ ~ X,- _._ V _ 1 i O 1( 1111 1'11 85 90 C -- ~ PS9-92 : I I , . . ~ I \~? , 85 So C 95 FIG. 12.Salting-out curves for the alkali-resistant fraction (~.D.A.) iso- lated from newborn carbonmonoxy- hemoglobin No. 2 5 5 and for its fractionation products F 88, P 88 and P 89-92. Salting-out conditions as in figure 3. and contains about 3 per cent isoleucine.iS This fraction is a mixture of colorless protein, called X, and an alkali-resistant hemoglobin, as shown by its content of iron and nitrogen and by paper electrophoresis followed by brom- phenol stainingi9 (Sg. 13~. In veror~al buffer the protein ~ is slightly slower than hemoglobin C while the mobility of the alkali-resistant pigment is similar to the mobility of hemoglobin A. Beginning with the eleventh month after birth, the spot of protein X is visible in the whole hemoglobin of all subjects FIG. 13. Paper electrophoresis, using apparatus of the Grassman and Hannig type, W-hatmann No. 3MM; veronal buffer of pH 8.8 and ionic strength 0.02S or phos- phate buff en of pH 6.5 and ionic strength 0.02; bromphenol blue staining. I ~ normal adult carbonmonoxyhemoglobin; IIalkali-resistant f raction isolated from I. III - alkali-resistant fraction isolated from the oxy-derivative (O2TIb) of the same pigment; IV - protein X isolated from III by elusion of its spot.

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192 PART III. ABNORMAL HEMOGLOBINS ifs either normal or pathological condition, including that of Cooley's anemia. Like the slow hemoglobin A2 discovered by Kunkel and Wallenius,20 protein X is not detected in the hemoglobin of newborn children. The hemoglobin of normal adults contains about 1.5 per cent of protein X, which passes almost totally into the alkali-resistant fraction. This fraction contains about 50 per cent of alkali-resistant pigment when isolated from oxyhemoglobin and about 75 per cent when isolated from carbonmonoxyhemoglobin. Such data are in accord with earlier observations showing that, for a given hemoglobin prepara- tion, the carbonmonoxy-derivative contains a greater proportion of alkali-re- sistant hemoglobin than the oxy-derivative.2i 2~ O3 ~4 By submitting the isolated alkali-resistant fraction to oxidation, and again to fractionation by alkali denaturation, both types of components are separated as follows: alkali resistance of the hemoglobin is lost by oxidation to ferri- hemoglobin~4 and this is removed as alkaline ferrihemochromogen precipitate; protein X remains in solution. As shown in figure 14, the isolated protein X is nearly homogeneous as shown by electrophoresis in the Tiselius-Svensson apparatus. In phosphate buffer of pH 6.5 and ionic strength 0.2, its mobility is 1.7, a value very close to that of -globulin of serum. In cacodylate buffer of pH 6.5 and ionic strength 0.018, protein X shows the same mobility as the component 1 observed in the hemolysates of all red cells (see table II). In ~ x7 PROTf INS X A OUt TE (466, 1 (136) Pompon I phosphor/qua ~ I r/2= 0,2 . dSC./+I ~SC./-J 360 ma 234 ran PROTEINS X +A D UL OF (4 6 5) tampon AX A phosphoriq,~e I x FJ \< r/2= 0,0 3 ~ As c. /- _ ~ 7300mn canyon c~codyl/q(,e r/2 = Otto Id PR O TEI NE X +ADVL TE (464) ~sc.tJ ~ 27s mn ~ am Con c~codyllque [~/2= 0,018 FrG. 14.Moving boundary electrophoresis, using a Tiselius-Svensson apparatus. No. 466: isolated protein X in phosphate buffer of pH 7.8 and ionic strength 0.2. No. 465: artificial mixture of protein X and normal adult carbonmonoxyhemoglobin in phosphate buffer of pH 8.2 and ionic strength 0.03. No. 138: normal adult carbonmon- oxyhemoglobin in cacodylate buffer of phi 6.5 and ionic strength 0.018. No. 464: arti- ficial mixture of protein X and normal adult carbonmonoxyhemoglobin. Electrophoretic conditions as in experiment No. 138.

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HETEROGENEITY OF HEMOGLOBINS A AND FDERRIEN 193 TABLE II Components Nature 2 2' 3 ) s' ) s' Apparent mobility U' .10 ( Mean value ) Protein "X" Accompanying protein ( ?) Hb R.D.A. (Thalassemia) Hb A and Hb R.D.A. (Thalassemia ) Hb A and Hb R.D.A. (Hb F) Hb R.D.A. (Hb F`) - 1.7 +4.4 t5.0 +5.1 +5.2 alkaline as well as in acid buffer, this protein moves more slowly than hemo- globir1 A either in moving boundary or paper electrophoresis (fig. 13~. The isoleucine content of protein X is of the order of 4 per cent. The isoleucine content of the isolated pigment is very close to one per cent instead of the zero to 0.3 per cent in hemoglobin A and the 1.5 to 1.9 per cent in hemoglobin F'.~5 ~6~7 Therefore, the alkali-resistant fraction of adult hemoglobin must be different from hemoglobin ~ at least for a major por- tion. Such alkal:-resistant fractions contain components of the adult type fat, a'~. and ads)partly changed into products of lower solubility Gil, be, 63~- C,5 ~'2 a~ . it, 5.p V,J ~ _ ~1 E C0f/6 Ad 3M E ~ ~r~chona/c~/mores/st~ ~ 1~ EN ~ C0~6 N-~. | o Dam , <,2 p~ A_3~N t. Grou,oe by= 9%~6 ~ ..... ..... ...... f O ~ ~ ~ . ~ ~ O ~ ! ! ~ ~ ~ ~ ~ ! ' . ~ ! ! ! ~ O ~ ! ! ' ! ! ! ' ' ! ~ 60 65 C 90 75 BE B5 C 90 80 85 90 C 95 150 d~ . = /501 1 ~ 1 i HE 1\ 5.p as !_ /00 06 7 ! ' ~ ' 'C ' ~ ' 50 ~ ~ _/so/Be Be ccJ,7t At. ~ ! _ ~51 ~ \a 2 - ~ ~ ) Grope fig 8 % - ~ ( f I ) | ~ Grouse, 73 % ! 1 h' ~ ~ - C'Y To 1 ~~9OC9s 65 C 90 80 B5 90 C 95 FIG. 15. Salting-out curves and corresponding derivative curves for newborn and normal adult carbonmonoxyhemoglobins and for the alkali-resistant fraction isolated from the latter. SaIting-out conditions as in figure 3. ~ This change in solubility is due to the great dilution of the soluble pigment ob- tained by the denaturation method.

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194 PART III. ABNORMAL HEMOGLOBINS o,s o 50 HE 50 ~r~c~ior'~/ca/mores~s~ E Croci. ~/ca//r~ores/s~ E is Iso/ee dame/~;e. _ i~o/ee dome/~ge ~ ~ ;, `85 |(C9/lb AdS1{ ~ ff:O//b Adders In, ~ ~ w-/1 BY. 05 -~A ~ W-NtO% Q5 I dam, _ 42' | \tai' | ~ Na' 1 - ,,,,,: 1 t --\ ~ - ~ - Gyp 'Poe i, 30 % ~ , Groove fop 3& ,~: , Grove fop 60 O O ! ~ I ~ 1 1 . ~ ~ 1 1 1 ! 1 BY 85 90 C B5 90 C 80 85 90 C 95 tOO ~ s30(~,~' 'l lid 150 AL 100 So 1 1 .~ 1,, /5o HE ~50 50 `~a1 ~r~ct/o/7 d/cd/iito~es/s '' _~_ Jso/ee dume/d~7~re. I' (6086 Ad809( ,l ~ WN~x . drama)_ ~:~ Aid Otter: ~ ~ ~ ~59 4~50i ~~ O BE 90 C 65 90 C 85 90 C 95 J FIG. 16. Salting-out curves and corresponding derivative curves of alkali-re- ~istant fractions isolated from mixtures of various proportions of normal adult and newborn carbonmonoxyhemoglobins Salting-out conditions as in figure 3. and very little, if any, fetal hemoglobin (fig. 15), as checked by experi- ments which show that, after addition of only 5 per cent of newborns' hemo- globin to the whole pigment of an adult, the proportion of components f reaches 30 per cent of the alkali-resistant fraction isolated from the hemoglobin mixture (fig. 16~. This invalidates the initial assumption of Roche, Derrien and Roques3 7 that 5 to 10 per cent of most soluble fractions of the total normal adult hemoglobin corresponds to fetal hemoglobin. Almost the whole of these fractions are alkali-labile and the alkali-resistant hemoglobin of normal adults are mainly salted-out as components of the a type. Studies of the alkali-resistant hemoglobin of subjects suffering from sickle-cell anemia lead to a similar conclusion.~3 2s 5. Electrophoresis of hemoglobins in the Tiselius-Svensson apparatus, in cacodylate buffer of pH 6.5 and very low ionic strength (~2 0.018), provides a new set of arguments supporting the heterogeneity of these pig- ments.29' 30 In a concentration of 1.0 per cent and with a potential gradient of 7 voltsicm., normal adult hemoglobin gradually separates into two major com- ponents named 3 and 4, as shown in the top row of figure 17. Peak 3 is fol- lowed by very small peaks 1 and 2 of slower components, of which the slower has been identified as protein X (fig. 14~. Peak 4 is heterogeneous. As electro- phoresis proceeds, peak 4 separates into 4' and 4, and a small peak named 5, slightly more rapid, appears., T Berry and Chanutin3t have recently confirmed the resolution of two components (A and B) in human adult hemoglobin. According to these authors, a third faster boundary (I), presumably identical to our component 5, represents the concentration gradient of the cacodylate ions.

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HETEROGENEITY OF HEMOGLOBINS A AND FDERRIEN 195 ADVLTE (1387 N-N (444) 3 ~ 3, 3 150mn 2 1 ~ ~ . 234 mn 211 _ _ -~L 24 0 me ~5 ~ 25mn 31 ~5 A.COOlEX il 5 3 (423) 2 Jo 2 me' 1017mn 111 arc I-) 2 41- l330 mn 3 ; 55 a sc.~-' FIG. 17. Moving boundary electrophoresis of human carbonmonoxyhemoglobin No. 138, from normal adult; No. 444, from newborn child; No. 423, from patient with Cooley's anemia. Patterns recorded at different times in the L. K. B. Tiselius-Svensson apparatus. Standardized experimental conditions: concentration of hemoglobin solu- tions ~ 1 per cent; cacodylate buffer of pH 6.5 and ionic strength 0.018; potential gradient = 7 ~roltlcm.; temperature - 2 C. Diagrams obtained with hemoglobin of newborn children (fig. 17, middle row N-N) show only a small amount of component 3 and always record im- portant quantities of a component which often separates into peaks 4 and 5 during long-run experiments. Hemoglobin of subjects suffering from Cooley's anemia has an electrophoretic behavior very different from that of the new- born child, as is shown in the bottom row of figure 17. Component 3 pre- dominates, whereas component 4 is usually absent. Peak 5 splits into 5 and 5' and ~ new component 2' appears. Thus this pathological hemoglobin can Basil be differentiated from both hemoglobin :F and hemoglobin A in dilute cacody- late buffer. Peak 2' had previously been found only in hemoglobin preparations from tl~alassemia blood (thalassemia major or minor). Components 3, 4 and 5 show apparently identical mobilities in the hemoglobin of the adult, the rlew- born child and patients with Cooley's anemia (table Il). Additional evidence of this identity is furnished by electrophoretic analysis of artificial mixtures of normal adult hemoglobin with the hemoglobin of newborn children or of patients suffering from Cooley's anemia (fig. 18~. The components of each hemoglobin are superimposed according to their respective mobilities, without appearance of a new peak and without disappearance of any peaks present in

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196 PART III. ABNORMAL HEMOGLOBINS ~ s 1 N-N (444) 133 Omn 3 ADUL HE I ' A.COOLEX (44n (4 5 1) ADUL TE + N-N (455) 1310rnn 3 ADULTE 5 +A.COOLEY 4+s 3 1~0 An s asc.(_)_ FIG. 18.Moving boundary electrophoresis of human carbonmonoxyhemoglobin No. 444, from newborn child; No. 451, from normal adult; No. 447, from patient with Cooley's anemia; No. 455, a mixture of carbonmonoxyhemoglobins Nos. 444 and 451; No. 449, a mixture of carbonmonoxyhemoglobins Nos. 451 and 447. Standardized experimental conditions as in figure 17. the initial hemoglobin preparations. All these preparations have been sub- mitted to moving boundary electrophoresis in the form of 100 per cent car- bonmonoxyhemoglobin. They remain in this form during the electrophoresis run, as checked spectrophotometrically at the end of the experiment. They were homogeneous in the ultracentrifuge in the same buffer used for the electrophoresis. The observations in the foregoing five sections support the hypothesis of a true heterogeneity of hemoglobins. Furthermore, they present some evidence that hemoglobins of the fetal type in newborn children and in patients with Cooley's anemia are not identical, even though these cannot be differentiated by electrophoresis either in cacodylate buffer of pH 6.5 and ionic strength 0.1 or in phosphate buffer of pH 8.2 and ionic strength 0.03, or by salting-out curves or by chemical methods. Whatever the nature of the difference for which immunological evidence has just been reported* comparative electro- phoretic studies of newborn and Cooley's-anemia hemoglobins of similar yield of an alkali-resistant fraction lead to the conclusion that resistance to denatura- tion by bases can be common to different hemoglobins. On the other hand, the same electrophoretic component can be common to various hemoglobins (table II). For example, the peak 3 includes an alkali- ~ Diacono, fI.: Compt. rend. Soc. de Biol. (in press).

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HETEROGENEITY OF HEMOGLOBINS A AND ~ DERRIEN 197 resistant hemoglobin in the blood of Cooley's anemia patients and art alkali- iabile hemoglobin in normal adult blood. According to these data it appears that, as sensitive as is the technique of electrophoresis in dilute buffer, it does not allow a precise definition of the exact number of components of any nor- mal or pathological mixture in red cells. As seen in sections 3 and 4 above, solubility and alkali denaturation experiments lead to a similar conclusion about the sensitivity of the salting-out method by which resolution of alkali- labile and alkali-resistant fraction of the a type are not obtained. However, it is of interest to point out that the degree of heterogeneity shown in normal adult or newborn hemoglobin is of the same order of magnitude whether de- termined by salting-out or by electrophoresis. For example, the resolution of two main components bat and a2 or 3 and 4) in adult hemoglobin is obtained by both methods. Summary. 1. Herx~oglobin A contains at least two components and hemo- globin ~ includes fractions of the a., and f solubility types, the f type in much larger proportion. 2. The alkali-resistant hemoglobins of normal adults and of subjects with sickle cell anemia are of the a solubility type, at least for a considerable por- tion. Their isoleucine content is significantly lower than that of hemoglobin F. 3. Hemoglobins of newborn children and of patients with Cooley's anemia which yield similar amounts of alkali-resistant fraction are definitely different as shown by electrophoretic behavior in cacodylate buffer of very low ionic strength. REFEREN CES 1. Roche, J., Derrien, Y., Reynaud, J., Laurent, G., and Roques, M.: Sur l'heterogene- ite des hemoglobines. I. Technique d'etablissement des courbes de solubilite et premiers essais de fractionnement, Bull. Soc. Chim. biol. 36: 51, 1954. 2. Derrien, Y., and }loche, J.: Etude comparee des hemoglobines du nouveau-ne, de l'enfant et de l'homme adulte par la methode des courbes de relargage (salt- ing-out), First International Congress of Biochemistry, Cambridge 19-25 Au- gust 1949, Abstracts of Communications, p. 368. 3. Roche, J., Derrien, Y., and Roques, M.: Sur l'heterou eneite des hemoglobines . . . . ~ , ~ humaines chez l'adulte et le foetus, Compt. rend. Soc. biol. 146: 689, 1952. 4. Roche, J., and Derrien, Y.: Les hemoglobines humaines et les modifications phy- siologiques et pathologiques de leurs caracteres, Le Sang 21: 97, 1953. 5. Derrien, Y.: Individualisation, characterization and fractionation of the serum proteins by salting-out, Svensk. Kem. Tidsl~r. 59: 139, 1947. 6. Derrien, Y., Laurent, G., and Reynaud, J.: Individualisation et caracterisation des constituents proteiques du serum par la methode des courbes de relargage, J. de chem. phvs. 48: 651, 1951. 7. Roche, J., Derrien, Y., and Roques, M.: Sur le remplacement des hemoglobines de type adulte par celles de type foetal au cours du developpement embryonnaire et apres la naissance, chez l'homme et chez le boenf, Bull. Soc. chim. biol. 35: 933, 1953. 8. Roche, J., Derrien, Y., and Roques' M.: Sur les hemoglobines du boeuf et cur

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198 PART III. ABNORMAL HEMOGLOBINS leurs transformations au cours du developpement foetal et apres la naissance, Compt. rend. Soc. biol. 146: 694, 1952. 9. Itano, H. A.: Solubilities of naturally occurring mixtures of human hemoglobin, Arch. Biochem. 47: 148, 1953. 10. Roche, J., Derrien, Y., Gallais, P., and Roques, M.: Sur les hemoglobines des sang a drepanocytes (hematies en faucille ou falciformes), Compt. rend. Soc. biol. 146: 889, 1952. 11. Derrien, Y., Cabannes, R., Laurent, G., and Roche, J.: Sur l'individualisation de l'hemoglobine D, Compt. rend. Soc. biol. 149: 1350, 1955. 12. Derrien, Y., Laurent, G., and Roche, T.: Sur l'indi~ridualisation de l'hemoglobine C chez des porteurs homozygotes et heterozygotes, Compt. rend. Soc. biol. 149: 641, 1955. G., and Borgomano, M.: Identification des hemoglobines et natholo~ioues Dar leurs courbes de relar~a~e et leur 13. Derrien, Y., Laurent, humaines normales <~ alcalino-resistance, XVme Congres des Pediatres de Langue Francaise, Mar- seille 23-25 May 1955, Communications p. 69. 14. Itano, lI. A.: The hemoglobines, Ann. Rev. Biochem. 25: 331, 1956. 15. Laurent, G., Bouscayrol, S., Dunan, J., and Borgomano, M.: Influence de la methemoglobinisation sur les courbes de relargage des hemoglobines humaines de type adulte et de type foetal, Compt. rend. Soc. biol. 150: 738 (no. 4), 1956. 16. Derrien, Y., Laurent, G., and Bouscayrol, S.: Sur une variation saisonniere de la teneur en fractions f (relargage) de l'hemoglobine de nouveau-ne, Compt. rend. Soc. biol. 150: 397 (no. 2), 1956. 17. Derrien, Y., Laurent, G., and Borgomano, M.: Isolement par denaturation frac- tionnee et etudes des courbes de relargage de la fraction alcalinoresistante des carboxyhemoglobines de nouveau-nes, Compt. rend. Soc. biol. 149: 137, 1955. 18. Derrien, Y., Laurent, G., and Roques, M.: Recherches sur la fraction alcalino- resistante de l'hemoglobine de l'homme adulte normal, Arch. sci. biol. 39: 650, 1955. 19. Derrien, Y., Laurent, G., and Borgomano, M.: Sur une proteine accompagnant l'hemoglobine de l'homme adulte et sa concentration dans la fraction alcal- inoresistante isolee de cette derriere, Compt. rend. Acad. Sci. 242: 1538, 1956. 20. Kunkel, H. G., and Wallenius, G.: New hemoglobin in normal adult blood, Sci- ence 122: 288, 1955. 21. lIelpern, M., and Strassman, G.: DifFerentiation of fetal and adult human hemo- globin, Arch. Path. 35: 776, 1943. 22. Betke, K., Richarz, H., Schubothe, H., and Vivell, O.: Beobachtungen zu Krank- heitsbild, Pathogenese und Aetiologie der akuten erworbenen hamolitischen Anamie (Lederer-Anamie), Klin. ~7ochnschr. 31: 373, 1953. 23. Singer, K., and Fischer, B.: Studies on abnormal hemoglobins. 7. The composition of the non-S fraction in sickle-cell anemia bloods. A comparative quantitative study by the methods of electrophoresis and alkali denaturation, [. Lab. Clin. Med. 42: 193, 1953. 24. Derrien, Y., Laurent, G., and Roche, J.: Sur la resistance a la denaturation alcaline des hemoglobines et de leurs derives, Compt. rend. Soc. biol. 147: 1934, 1953. 25. van der Schaaf, P. C., and Huisman, T. H. J.: The amino-acid composition of human adult and foetal carbonmonoxyhemoglobin estimated by ion exchange chromatography, Biochim. et biophys. acta, 17: 81, 1955. 26. Rossi-Fanelli, A., Cavallini, D., De Marco, C., and Trasatti, F.: Emogrlobina fetale. I. Analisi quantitative degli amino-acid) della emoglobina umana fetale