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OCR for page 183
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;
pH—6.7; 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:
OCR for page 184
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. E—optical density of fil-
trates at ~ = 550 my. Other details as in figure 1.
OCR for page 185
HETEROGENEITY OF HEMOGLOBINS A AND F—DERRIEN 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
OCR for page 186
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 N°V-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.
OCR for page 187
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.
OCR for page 188
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.
OCR for page 189
HETEROGENEITY OF HEMOGLOBINS ~ AND F—DERRIEN 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 ,1°2-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. 4—55 (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. 2—55 (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
OCR for page 190
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
OCR for page 191
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. 3—MM; 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; II—alkali-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.
OCR for page 192
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.t—J ~
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.
OCR for page 193
HETEROGENEITY OF HEMOGLOBINS A AND F—DERRIEN 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.
OCR for page 194
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 Ad§S1{ ~ 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.
OCR for page 195
HETEROGENEITY OF HEMOGLOBINS A AND F—DERRIEN 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
OCR for page 196
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).
OCR for page 197
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
OCR for page 198
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
Representative terms from entire chapter:
protein x
