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OCR for page 162
162
PART III. ABNORMAL HEMOGLOBINS
of Berry and Chanutin14 would appear to be similar although these workers
observed a disappearance of their second component in stored cells rather
than an increase as in the present study. Considerable further work is neces-
sary to resolve these results obtained in different laboratories with different
techniques.
Summary. Evidence has been presented for the presence of two minor
hemoglobin fractions in normal human blood. Tile one designated Fib As is
a clearly defined entity which is characteristically elevated in thalassemia.
The other represents a heterogeneous fast fraction which may be produced
from Hb A. Studies with Few indicate that it has biological significance.
REFERENCES
1. Kunkel, H. G., arid Wallenius, G.: New hemoglobin in normal adult blood.
Science, 122: 288, 1957.
2. Kunkel, H. G., Ceppellini, R., Muller-Eberhard, U., and Wolf, J.: Observations
on the minor basic hemoglobin component in the blood of normal individuals
and patients with thalassemia, J. Clin. Invest. (in press).
3. Schapira, G., Dreyfus, I.-C., and Kruh, J.: Heterogeneous metabolism of haemo-
globins, Ciba Foundation Symposium on Porphyrin Biosynthesis and Metab-
olism. Churchill Ltd., London, and Little Brown Boston 1955.
. . . . .
4. Stern, K. G., Reiner, M., and Silber, R. PI.: On the electrophoretic pattern of
red blood cell proteins, J. Biol. Chem. 161: 731, 1945.
5. Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C.: Sickle cell anemia, a
molecular disease, Science, 110: 543, 1949.
6. Singer, K., Chapman, A. Z.. Goldberg, S. R., and Rubenstein, H. M.: Studies on
abnormal hemoglobins. X. A new syndrome: hemoglobin C-thalassemia disease,
Blood, 9: 1032, 1954.
7. Morrison, M., and Cool;, J. L.: Chromatographic fractionation of normal adult
oxyhemoglobin, Science, 122: 920, 1955.
8. Gerald, P.: to be published.
9. Aksoy, M., Lehman, H., and Luan Eng, L. I.: The recognition of haemoglobins
A2 and E, Lancet, 272: 792, 1957.
10. Josephson, A. M., and Singer, E.: to be published.
11. Derrien, Y., and Reynaud, J., Sur l'heterogeneite electrophoretique de l'hemo-
globine humaine (so jets adultes normaux), Compt. rend. soc. biol. 147: 660,
1953.
12. Shavit, N., and Breuer, M.: Electrophoretic heterogeneity of normal adult human
hemoglobin at low ionic strengths and higher temperatures, Biochim. et
Biophys. Acta, 18: 241, 1955.
13. Hoch, H.: The steady state, a test for electrophoretic homogeneity, ]3iochem. I.
46: 199, 1950.
14. Berry, E. R., and Chanutin, A.: Electrophoretic studies on red cell extracts of
stored blood, J. Clin. Invest. ]6: 225, 1957.
DISCUSSION
Dr. Yves Derrien: In connection with Dr. Kunkel's very interesting talk,
I should like to comment briefly about researches in the same field, partly
OCR for page 163
DISCUSSION
163
published in 1955 and described in more detail in my paper later in this
Conference.
~.~ ~
::::
I.,< ..
:::~:~::~:::~:~:::~:~:~::::::::~:~:~:~:~:~:::
· ,. ,,, f .
..H,.~,,..h,.,.,. Act...
FIG. 1. I;lectrophoresis of normal adult hemoglobin (I) and "protein X" (II).
As can be seen in the upper part of the illustration (fig. 1), paper elec-
trophoresis of normal adult hemoglobin in veronal buffer of pH 8.8 and
ionic strength 0.025 always shows a small component in front of the major
spot of Hb A and another one slightly slower than Hb C. This slow minor
component, only visible after staining and corresponding to a colorless pro-
tein we called "protein X" in 1955, is not removed by a single crystallization
of the hemoglobin preparation nor by passing it through absorbents like celite.
It is resistant to denaturation by bases and passes into the all~ali-resistant
fraction of hemoglobin. Paper electrophoresis of this fraction (II) shows a
large spot of protein X, a small spot of a slightly faster nonchromogenic com-
ponent called X~ and the alkali-resistant hemoglobin. Protein X can be iso-
lated by elusion of its spot and contains from 15 to 20 per cent hemoglobin,
as determined by iron/nitrogen ratio and spectrophotometric analysis.
I should like to ask Dr. Kunkel if it is to be excluded that his As minor
component corresponds to hemoglobin A adsorbed on the so-called proteins X.
This hypothesis is suggested by the additional fact that, as is A2, proteins X
are always present in the hemoglobin of adults and of patients with Cooley's
anemia but are never detected or only in extremely small amounts in
newborn hemoglobin. For normal subjects proteins X appear from 11 to 12
months after birth.
Dr. Keel: I did not have time to mention the non-hemoglobin compo-
nents that orate observes following electrophoresis of red cell hemolysates.
There are at least three that are constantly present and can be observed as
clear bands or peaks. One of these which migrates slightly slower than Hb As
at pH 8.6 is present at a concentration of 1 to 3 per cent of the total protein.
This may well be Dr. Derrien's ~ protein. It has no relation to Hb A2 and
can be separated from it by electrophoresis. We know this protein rather well
because it has caused contamination or the Hb As when separation was not
completely optimal. There is no possibility that fIb As represents a complex
between Hb A and some other protein because it has the same sedimentation
OCR for page 164
164
PART III. ABNORMAL HEMOGLOBINS
coefficient as Hb A and also has a very similar heme protein and iron protein
ratio.
Dr. Jerome Vinograd: I Irish to ask Dr. Kunkel whether his results were
obtained with recrystallized material or with the material as it came from the
red cells.
Dr. Kunkel: We have had the opportunity of studying two crystalline
specimens and both of these showed the As component to be present at the
same 2.5 per cent concentration that was in the original hemoglobin solution.
However, most of our work was done with the whole hemoglobin solution.
Representative terms from entire chapter:
proteins x
