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OCR for page 199
HETEROGENEITY OF HEMOGLOBINS A AND F DERRIEN 199
cristallizzata ed alcuni accorgimenti tecnici, Boll. Soc. it. Biol., sper. 31: 328
(fasc. 3-4), 1955.
27. Lissitzky, S., and Laurent, G.: Dosage de la leucine, de l'isoleucine et de la
phenylalanine des proteines par chromatographie sur papier, Bull. Soc. Chim.
biol. 37: 1177 (No. 11), 1955.
28. Roche, J. Derrien, Y., and Laurent, G.: Sur les fractions alcalinoresistantes des
hemoglobines dans les enemies drepanocytaires, Compt. rend. Soc. biol. 147:
957, 1953.
29. Derrien, Y., and Reynaud, J.: Sur l'heterogeneite electrophoretique de l'hemo-
globine humaine (sujets adultes normaux), Compt. rend. Soc. biol. 2~47: 660,
1953.
30. Derrien, Y., and Reynaud, J.: Etude comparee des hemoglobines humaines nor-
males et pathologiques par electrophorese en veine liquide en tampon cacodylique
dilue. Distinction des hemoglobines d'anemiques de Cooley et de nouveau-nest
XVme Congres des Pediatres de Langue F`ran~aise, Marseille, 23-25 Mai 1955,
Communications p. 178, and Compt. rend. Soc. biol. 149: 1595, 1955.
31. Berry, E. R., and Chanutin, A.: Electrophoretic studies of red cell extracts of
stored blood, J. Clin. Invest. 36: 225, 1957.
DISCUSSION
Dr. H. J. Itano: Some time ago I suggested the possibility that these dis-
continuities in the salting-out curve might arise from phase transitions.) ~ Dr.
Perutz obtained different crystalline forms of human hemoglobin at different
concentrations of phosphate.3 ~ think this possibility as well as heterogeneity
should be considered in the interpretation of salting-out curves. Unfortunately,
this possibility complicates the interpretation considerably. Perhaps both heter-
ogeneity and phase transitions are contributing to these curves.
The other point has to do with the electrophoresis. I showed that in two or
three hours we w-ere able to separate components which differ by one charge.
The diagrams of Dr. Derrien show separation after 20 hours. The amount
or separation shown must correspond to a minute fraction of a charge. On a
structural basis a fractional change in charge can result from a small change
in pK of one of the groups. However, experimentally one must consider the
fact that in these very prolor~ged runs convective disturbances may occur
either from diffusion of salt from the electrodes, from the delta boundaries
being pushed into the bottom section during reverse compensation, or from
electrolysis at one of the electrodes.
REF`EREN CES
1. Itano, H. A.: Solubilities of naturally occurring mixtures of human hemoglobin,
Arch. Biochem. Biophys. 47: 148, 1953. (See also No. 2j
2. Oc:ston. A. G.. and Tombs, M. P.: An ambi~uity in the variable-solvent solubility
~ , ,
.
test: tiomogene~ty of p,-lactoglobulin, Nature 178: 200, 1956.
3. Perutz, M. F~., Liquori, A. M., and Eirich, F.: X-ray and solubility studies of the
haemoglobin of sickle-cell anaemia patients, Nature 167: 929, 1951.
Dr. Derrien: The objectior~s formulated by Dr. Itano on the interpretation
OCR for page 200
200
PART III. ABNORMAL HEMOGLOBINS
of the salting-out curves and electrophoresis diagrams I presented should not
be accepted, in view of the following facts.
In view of the electrophoresis diagrams of 1 per cent normal adult hemo-
globin in very low ionic strength cacodylate buffer at pH 6.5, it is very dif-
ficult to deny the individuality of the two major constituents 3 and 4 of Hb
A. These begin separating within two hours and the process of their separation
as a function of time does not show any convection disturbances. In accordance
with Dr. Svensson, with whom I discussed this problem, the electrophoretic
individuality of these two constituents has to be considered as certain under
the experimental conditions. The difference in net charge seems to be only
Q.3, which would exclude the possibility of the faster moving component
(No. 4) being an intermediate form of oxidation.
Furthermore, our experimental results are not in favor of the hypothesis
that: discontinuities in salting-out curves represent changes in the solid phase
of the same hemoglobin. As I said earlier the solubility curves of crystals show
exactly the same discontinuities as the salting-out curves of solutions (precini-
tation of solid amorphous phases>. Therefore, I think that if it were a matter
of transition between different crystalline forms, it would be surprising that
we should get exactly the same pictures in both types of experiments.
Representative terms from entire chapter:
human hemoglobin
