CONFERENCE ON HEMOGLOBIN 2-3 MAY 1957 (1958) / Chapter Skim
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From page 144... ...
Other investigators will discuss the application of zone electrophoresis elsewhere in these Proceedings, and I shall confine my remarks to practical applications of the moving boundary method in the study of human hemoglobin. In order to facilitate further discussion and clarify the notation on the figures which accompany this presentation, I shall review briefly the nomenclature of the human hemoglobins as agreed upon by interested investigators.4 5 Normal adult hemoglobin is the form found in most humans and is called hemoglobin A
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From page 145... ...
It is therefore conceivable that two forms of human hemoglobin have the same amino acid composition and yet have different net charges at the same pH. It is also possible for two hemoglobins to differ in their content of charged
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From page 146... ...
R.: Blood 10: 389, 1955. Hemoglobins A and S were resolved in phosphate buffers of ionic strength 0.1, and their charge difference was estimated to be two to four units from titration and electrophoretic data.t These hemoglobins are also well resolved in cacodylate buffer of pH 6.5 and ionic strength 0.1.
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From page 147... ...
4. Comparison of moving boundary patterns of carbonmonoxyhemoglobin in cacodylate buffer of pH 6.5, ionic strength 0.1, with the results of paper electrophoresis in barbital buffer of pH 9.2, ionic strength 0.01.
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From page 148... ...
Comparison of the components of carbonmonoxyhemoglobin in three types of sickle cell disease. To the left, analyses in cacodylate buffer of pH 6.5, ionic strength 0.1.
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From page 149... ...
and C are synthesized under the control of allelic genes at the locus designated Hb.'3 24 The electrophoretic patterns of carbonmonoxyhemoglobin in cacodylate buffer of pH 6.5, ionic strength 0.1 corresponding to the six possible combinations of the three genes are shown. The X component is probably the same as hemoglobin A.,.5' 6 The presence of fetal in homozygosity for the sickle cell gene is shown.
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From page 150... ...
Since the mother is heterozygous for the sickle cell gene and the father does not have this gene, the patient cannot be homozygous in the sickle cell gene However, the pattern closely resembles that seen in sickle cell anemia. The relative increase in hemoglobin S can be ascribed to the thalassemia gene, which was inherited from the father and which inhibits the synthesis of hemoglobin A
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From page 151... ...
In order to study the effect of this reaction on electrophoretic homogeneity, partially oxidized mixtures of carbonmonoxyhemc~globin were used since the components of such mixtures, carbonmonoxyhemoglobin and ferrihemoglobin, are stable compounds. Analyses of partially oxidized preparations of carbonmonoxyhemoglobin in phosphate buffer of pH 6.85 and ionic strength 0.01 revealed a mixture of components, including some with mobilities between those of carbonmonoxyLemoglobin and ferrihemoglobin ~ fig.
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From page 152... ...
The results summarized above indicate the reliability and relative simplicity of the electrophoretic method for the detection of small differences in the net charge of hemoglobin molecules. The fact that all of the known abnormal forms of human hemoglobin are electrophoretically abnormal is partially a consequence of the almost exclusive reliance of investigators on eliectrophoresis for the initial detection of abnormal forms.
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From page 153... ...
I Their demonstration in sickle cell anemia and other hematolo'~ric disorders by means of alkali denaturation.
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From page 154... ...
J.: The amino acid compositions of insulins isolated from beef, pork, and sheep glands, l.
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From page 155... ...
Unless the nitric oxide hemoglobin differs significantly in net charge from hemoglobin, I do not think we can do it by electrophoresis.
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From page 156... ...
It would be of interest to compare this material with the heterogeneous fraction of human hemoglobin, described by Dr. Kunkel, which also appears to increase with the age of the preparation.
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From page 157... ...
Homozygous C c. Sickle cell anemia d.
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From page 158... ...
Two specimens from individuals with sickle cell trait are also included. All of these showed the As component at approximately equal concentration.
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From page 159... ...
However, considerable 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 methemoglobin or cyanmethemoglobin was used.
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From page 160... ...
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
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From page 161... ...
It also resembles one of the chromatographic subfractions obtained 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.
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From page 162... ...
12. Shavit, N., and Breuer, M.: Electrophoretic heterogeneity of normal adult human hemoglobin at low ionic strengths and higher temperatures, Biochim.
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From page 163... ...
1) , paper electrophoresis 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
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From page 164... ...
Kunkel whether his results were obtained with recrystallized material or with the material as it came from the red cells.
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From page 165... ...
Fib D has the same mobility as sickle-cell hemoglobin.
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From page 166... ...
. were found, as the fetal pigment moves faster than the normal adult hemoglobin in chromatography and Hb H still much faster than Hb F
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From page 167... ...
too o 080 a' Us 060 A a: a: o 0.40 020 ,~ _ 1 0 20 30 co 50 TUBE NUMBER 60 70 FIG. I.- Chromatogram of hemolysates of normal adult red cells.
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From page 168... ...
This actually demonstrates two things: first, the column produced no multiple components, further confirming our data that a single protein gives but a single peak in this method, and second, that the genetic apparatus which gives rise to the multiple components in normal adult hemoglobin is completely altered in the homozygous sickle cell individual. After a number of abnormal hemoglobins had been investigated, a composite chromatogram such as is shown on figure j could be drawn.
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From page 169... ...
Dr. Itano, in his earlier talk, described the separation of hemoglobin molecules in which the iron atoms have been oxidized.4 If the major component of normal adult hemoglobin is oxidized into any of these types between methemoglobin and CO-hemoglobin, their separation can be achieved.
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From page 170... ...
The molecule with all the iron atoms in the ferrous form is least positively charged and is elated snore readily, while the methemoglobin carries the highest positive charge and is the last form to be eluted. Thus, the forms of hemoglobin may lead to erroneous results in the quantitative analysis of mixtures of Hb A and abnormal hemoglobins unless due care is exerted.
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From page 171... ...
DISCUSSION 171 monoxy- and ferclbemo~lobln by movlDg boundary electropbores~, J
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From page 172... ...
AMOZ I CHERNOFF All current techniques for the determination of fetal hemoglobin based upon the resistance of the embryonic pigment to denaturation by highly alkaline solutions stem from the observations made almost one hundred years ago by von Korber,~ who demonstrated that hemoglobin solutions prepared from cord blood erythrocytes were resistant to the destructive effects of NaOH whereas hemoglobin preparations obtained from normal adults were rapidly destroyed under the same experimental conditions.
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From page 173... ...
Baar and co-workers have shown that adult hemoglobin solutions prepared wild saponin often demonstrate two or more components on denaturation, whereas those hemolyzed with water behave ire a manner suggestive of a monomolecular reaction.3 These observations may explain the reports of a number of investigators that two or more hemoglobin components are present in normal adult hemoglobin solutions when studied by the denaturation technique's. Following what appears to tee ' complete denaturation of normal adult oxyhemoglobin solutions, a residual spectrophotometric reading of up to 1.8 per cent of the original hemoglobin concentration is noted.~ Although the material is benzidine positive, the evidence is inconclusive as to whether the compour~d causing this residual reading represents fetal hemoglobin, is a particularly al'~ali-resistant non-fetal hemoglobin fraction, is a denatured hemochromogen which is not precipitated by saturated (NH4~2SO4 or is, indeed, a heme pigment at all.
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From page 174... ...
In pathologic states, however, there is some disagreement as to the identity of the alkali-resistant compound and fetal hemoglobin. Thus, Larsen and co-workers believe tl-~at the alkali-resistant material in untreated pernicious anemia is probably related to a stromal factor found in the macrocytes of this disease.'' The alkali-resistant pigment in sickle cell anemia is claimed by van der Schaaf and Huisman to be different from fetal hemoglobin on the basis of careful amino-acid analyses of the prote~n when separated by ion exchange chromatography.13 On the other hand, there is considerable evidence that the alkali-resistant fraction in sickle cell anemia, as well as in all other hereditary hemoglobin diseases, is identical with the fetal compound.
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From page 175... ...
I Their demonstration in sickle cell anemia and other hematologic disorders by means of alkali denaturation, Blood 6: 413~28, 1951.
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From page 176... ...
ABNORMAL HEMOGLOBINS DISCUSSION Mr. Jbuer R
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From page 178... ...
Note the presence of small amounts of fetal hemoglobin in the mother, Gus, and the father, as well as a hemoglobin-like component in the hemoglobin A position in the two children with sickle cell thalassemia disease. Figure 5 illustrates a typical paper electrophoretic pattern at pH 8.6 of two typical AS individuals and two individuals whose hemolysates had previously been shown to contain hemoglobins A and D, by solubility tests, lack of sickling, and free electrc~phoresis method.
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From page 179... ...
Most investigators now agree, however, that human hemoglobin, though weakly antigenic, can induce the formation of specific antibodies in a number of animal species. The immunologic specificity of normal adult and fetal hemoglobin precipitins was first described in 1940 by Darrow and associates)
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From page 180... ...
180 PART III. ABNORMAL HEMOGLOBINS similarities between these two proteins.
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From page 181... ...
( Hbg in the illustration is an alternate usage of the more familiar Hb for Hemoglobin.) weakly with many normal adult hemoglobin specimens.
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From page 182... ...
H.: Differences in antigenic specificity of human normal adult, fetal, and sickle cell anemia hemoglobins, Blood 8: 422033, 1953.
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From page 183... ...
1. Above Salting-out curves for carbonmonoxy hemoglobins of normal adults and newborn children.
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From page 184... ...
3. Salting-out curves and corresponding derivative curves for carbonmonoxyhemoglobin of normal adult and of subjects with hemoglobin D trait or sickle cell trait Chromoprotein concentration— about 1.5 per cent.
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From page 185... ...
~ 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 carbonmonoxyhemoglobin of newborn child and subjects with Cooley's anemia or hemoglobin C disease.
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From page 186... ...
6. Crystal solubility curves and corresponding derivative curves for normal adult carbonmonoxyhemoglobin No.
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From page 187... ...
i 1~ ' : tC4If,, · ~ \: _ Ida | I,,, 90 its FIG. 7.- Salting-out curves and corresponding derivative curves for carbonmonoxyhemoglobin of subj ects with thalassemia minor, hemoglobin C trait and hemoglobin C-thalassemia association.
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From page 188... ...
8. Salting-out curves and corresponding derivative curves for carbonmonoxyhemoglobin of patients suffering from lymphoid leukemia, myeloid leukemia and acute leukosis.
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From page 189... ...
so 80 85 90 C SS FIG. 10.—Salting-out curves arid corresponding derivative curves for newborn carbonmonoxyhemoglobin No.
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From page 190... ...
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.
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From page 191... ...
3—MM; veronal buffer of pH 8.8 and ionic strength 0.02S or phosphate 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
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From page 192... ...
No. 465: artificial mixture of protein X and normal adult carbonmonoxyhemoglobin in phosphate buffer of pH 8.2 and ionic strength 0.03.
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From page 193... ...
15. Salting-out curves and corresponding derivative curves for newborn and normal adult carbonmonoxyhemoglobins and for the alkali-resistant fraction isolated from the latter.
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From page 194... ...
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 pigments.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 components named 3 and 4, as shown in the top row of figure 17.
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From page 195... ...
. 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.
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From page 196... ...
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.
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From page 197... ...
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.
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From page 198... ...
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.
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From page 199... ...
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.
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From page 200... ...
In view of the electrophoresis diagrams of 1 per cent normal adult hemoglobin in very low ionic strength cacodylate buffer at pH 6.5, it is very difficult 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.
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From page 201... ...
Then, after the protein has been isolated, purified and submitted to fractionation, the isotopic composition i.e., the specific activity (S.A.) of each fraction is measured.
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From page 202... ...
It has been generally considered that our usual chemical fractionation methods should not separate isotopes. However, Piez and Eagles in i955 observed that incorporatior~ of C i-; ir~to amino acids slowed their movement on chromatographic columns with possible resultant errors.
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From page 203... ...
paper chromatography, which gave us, using a pH 4.3 solution as mobile phase, elongated spots of hemoglobin displaying a narrowing from which we cut and eluted the two parts of the spot ;~ and 4.) alumina chromatography, which enabled us to demonstrate the adsorption of about 25 per cent of rabbit hemoglobin which was easily eluted by phosphate solutions.
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From page 204... ...
Adult rabbit hemoglobins labelled in vitro with radioactive iron and subsequently fractionated by alkali denaturation or by alumina adsorptions showed ratios of specific activities which are the reverse of those of fractions obtained from hemoglobin similarly labelled za vivo. Thus the in vitro kinetics of iron incorporation is the reverse of that found in viva.
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From page 205... ...
Without effectors + bone marrow + liver extract + vitamin Bee folinic acid 22 7 4 s 3 TABLE INS 0.71 + 0.31 1.33 +0.35 1.20 + 0.16 1.19 ~ 0.11 1.34 ~ 0.02 INCORPORATION OF RADIOACTIVE GLYCINE ALTO RABBIT HEMOGLOBIN to in. by :~< A_ Ratio I / II Globin Hemin Powder Glycine .
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From page 206... ...
the two curves crossed and finally became parallel. Thereafter the ratio of specific activities remained constant.-7 (Fig.
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From page 207... ...
The synthesis of foetal hemoglobin is less active than that of adult hemoglobin, but this cannot explain the greater effectiveness of cord blood than adult blood in the incorporation of radioactive iron.3i 2. Adult hemoglobin.
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From page 208... ...
No evidence on the occurrence of foetal hemoglobin in adult rabbit blood is available. Less than ~ per cent of foetal hemoglobin may occur in normal adult human hemoglobin.33 On Amberlite XE-64 chromatography of adult human hemoglobin labelled in vivo with Few, less than 1 per cent emerged as a small preliminary fraction, which may represent the foetal hemoglobin.
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From page 209... ...
However, a distinction is to be made between red blood cells with a brief or aberrant span of life and those with a normal life span. The finding of a temporary decrease in the radioactive iron content of rabbit hemoglobin II leads to a major conclusion—the possible existence of erythrocytes levity a very short life span.
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From page 210... ...
H.: Incorporation in q~itro of labeled amino acids into proteins of rabbit reticulocytes, J
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From page 211... ...
C., and Schapira, G.: Recherches sur la biochimie de l'hemoglobine a ['aide de fer radioactif. NT—Biosynthese des hemoglobines z~z ~vitro Bull.
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From page 212... ...
In figure 1 are depicted our most recent data. The values for the normal adults are the results of single analyses, while those for the thalassemic parents are the means of paired analyses.
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From page 213... ...
2. Starch electrophoretic pattern of the hemoglobins in the "Lepore abnormality" and in sickle cell trait.
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From page 214... ...
Except for this, the findir~gs were identical with those of previously published reports; namely, a mild hemolytic anemia, anisocytosis and poikilocytosis of the red cells, formation of inclusions in the erythrocytes after vital staining with cresyl blue, and splenomegaly. The abnormal hemoglobin moved faster than Al at pH 8.6, had an isoelectric point below pH 6.5, and was denatured by freezing.
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From page 215... ...
Kunl~el, H G., and Wallenius, G.: New hemoglobin in normal adult blood, Science 122: 288, 1955.
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From page 216... ...
1~. This component was markedly diminished in cord blood.
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From page 217... ...
The second peak is in the region of the principal component of normal adult hemoglobin. The last peak is in the area of the final peak of normal adult hemoglobirl, but differs in being significarltly larger than in the normal.
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From page 219... ...
The percentages may be off by virtue of the partial oxidation of Hb A, let us say one iron atom per molecule of normal A, which might give us a small increase. That is, if the major component A is partially oxidized, it would move in the same region as the third component.
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From page 220... ...
has an amino acid composition markedly different from that of the adult hemoglobins. It has also become clear that normal adult hemoglobin and the various abnormal varieties are very similar in composition, although Huisman has claimed that hemoglobin C contains slightly more lysine than do the others.
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From page 221... ...
AMINO ACID COMPOSITION STEIN 221 ._ .g ~ ' '' 'C " "" ' 'a Cat..,,, -..0 ~3 ~C:':,,'' C''~.
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From page 222... ...
Apparently, this sample of electrophoretically purified hemoglobin A is devoid of isoleucine, because the constancy of the base line, which is characteristic of the recorder procedure, coupled with the log scale of the plot, renders the method very sensitive to small quantities of amino acids. In fact, an amount of isoleucine equivalent to one residue per molecule of hemoglobin, less than 0.2 per cent, would appear as a peak about 173 of the size of the methionine peak.
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From page 223... ...
H., and Moore, S.: Observations on the amino acid composition of human hemoglobins, Biochim. Biophys.
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From page 224... ...
tend to be slightly lower than, but are in general quite similar to, other values to be found in the literature. It should be emphasized, however, that the single analyses performed on each 22- and 70hour hydrolysate are insufficient to provide definitive information relative to the amino acid composition of hemoglobin A
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From page 225... ...
'e He · me of c-)
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From page 226... ...
S., Schroeder, W A., and Pauling, I.: N-terminal amino acid residues of normal adult human hemoglobin: A quantitative study of certain aspects of Sanger's dinitrophenyl (DNP)
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From page 227... ...
Electrophoretically, there should be no difference between both of these hypothetical hemoglobin S molecules but, as will be clear below, both represent different chemical structures. Near the physiological pH probably all of the charges on hemoglobins come from their constituent amino acids.
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From page 228... ...
Rb-NHm = Rb-N pH 9.5 = pH 11.5 R., is lysine or arginine ;Rb-NH~ = Rb-N pH 6.0 = pH 8.0 Rb is histidine Ra—COO— = Ra—COOH pH 5.0 = pH 3.0 Ra is asp.artic or glutamic acid FIG. 3.—The approximate ranges of pH in which various charged groups of amino acids ionize.
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From page 229... ...
.~ ,^ FIG. 5.- Paper electrophoresis experi- :3: : ment in a phosphate buffer of pH 11.7.
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From page 230... ...
8~. These results indicate that the differences in net charge between the three hemoglobins are due to differences in their content of free carboxyl groups because the differences in charge are abolished at a pH at which only the carboxyl groups of the proteins' amino acid residues are uncharged.
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From page 231... ...
The observed differences in net charge between these hemoglobins do not depend on differences in small-ion binding since complete removal of smallions by ion-exchange resins results in different isotonic points for the three proteins.5 Differences in tyrosine or sulfhydryl content should not contribute to differences in charge except at pH values of about 10 and higher.4 The phosphorus content of hemoglobins A and S is not large enough, according to Havinga, to account for the difference in charge.6 Pauling and his co-workers calculated, on the basis of the difference in isoelectric points between hemoglobins A and S that hemoglobin S possessed two to four more net positive charges per molecule than hemoglobin A.i On the basis of that calculation, and the results reported above, it appears that hemoglobin S possesses about two to four less free carboxyl groups, and hemoglobin C possesses perhaps five or six less free carboxyl groups than normal adult human hemoglobin.7 Several possible structures could account for these differences as shown below.
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From page 232... ...
6. Havinga, E.: Comparison of the phosphorus content, optical rotation, separation of hemes and globin, and terminal amino-acid residues of normal adult human hemoglobin and sickle cell anemia hemoglobin, Proc.
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From page 233... ...
Accordingly, trypsin was allowed to digest samples of heat-denatured haemog;lobin A and S since it splits specifically those bonds in the polypeptide chains which are formed by the carboxyl groups of the amino acids lysine and arginine.
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From page 234... ...
It must therefore have a different structure and will represent the portion of the polypeptide chains where the chemical difference between the two proteins lies. The structures of these two peptides, the Hb A and Hb S no.
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From page 235... ...
Since there are two identical half molecules, this change occurs twice in the whole haemoglobin molecule and the fact that haemoglobin S has about 2 carboxyl groups fewer is now explained. The two haemoglobins differ very little; only one out of nearly 300 amino acids in the half molecule changes.
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From page 236... ...
Neel has shown that a single mutation of a haemoglobin gene produces the abnormal "sickle cell" gene.7 It appears now from the results briefly presented here that what is presumably an alteration of a portion of the gene results in an alteration of a portion of the polypeptide chain of the corresponding protein,- in this case haemoglobin. In the "sickle cell" mutation the change in the protein is very small indeed, indicating that this mutation is extremely localized in the gene.
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From page 237... ...
C.: Sickle cell anemia, a molecular disease, Science 110: 543, 1949.
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From page 238... ...
Neel, J V.: Inheritance of sickle cell anemia, Science 110: 64, 1949.
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From page 239... ...
Ingram studied, you will notice that he indicates that bovine has four sulfhydryl groups by silver titration, but he finds that two mercuries will block all four. Similarly, he finds eight in human hemoglobin by silver titration which can be blocked by six mercuries.
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From page 240... ...
TABLE I -SH GROUPS AND STABILITY OF HEMCGLOBIN Tris buffer pH 7.4 To decrease time in ~ 576 Tris buffer pH 7.4 Urea 4 M To decrease time in ~ 576 Human Hb control 20 furs. 2 20 furs.
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From page 241... ...
There are groups that bind both mercury and silver even though there is no -SH group under some conditions. It was found that there are specific binding sites for silver and specific binding sites for some mercurials.
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From page 242... ...
In the light of his experiments it would appear that there are no more than six -SH groups in normal human hemoglobin. My earlier findings that eight silver atoms and six mercury atoms are bound by the denatured protein seem to need a new explanation.
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From page 243... ...
Dr. Ingram's work would thus impose quite definite limits on the heterogeneity-of the hemoglobin he employed, and would indicate that the source of the gross heterogeneity found by other procedures must reside in structural differences which are not reflected in alterations in the sequences of the amino acid residues.
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From page 244... ...
1.) In the case of the amperometric silver titration, the specificity is further enhanced by the following factors: Owing to the high sensitivity of the rotating platinum electrode, the absolute concentration of protein, and therefore of silver, is extremely low (about 2 x 10-;'M>.
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From page 245... ...
that a deoxygenated sickle cell hemolysate has a negative temperature coefficient of "elation, i. e., a deoxygenated , ~ sickle cell hemoglobin solution of sufficient concentration gels at 38° C.,~ but the gel liquifes upon being cooled to 0° C
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From page 246... ...
I will summarize the data obtained by argentometric-amperometric titratior~ methods, as well as the mercurimetric-amperometric titration technique on Hb A and Hb So ~ and Hb C and Hb F.4 ~ It was found that the maximal number of titratable -SH groups is the same for Hb A, lIb S and Hb C; however, the mercurimetric-amperometric titration data suggest that their spatial arrangement appears to be different. The fetal hemoglobin molecule has fewer titratable -SH groups than Hb A and the mercapto group spatial arrangement is also different from that of the normal adult hemoglobin molecule.
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From page 247... ...
It can be seen that two -S-Hg-S- linkages are possible in this molecule. Dialyzed sickle cell hemoglobin binds about five mercury atoms per mole
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From page 248... ...
Pauling's steric hindrance theory of heme-heme interaction in hemoglobin provides an obvious explanation of the action of oxygen in preventing the sickling of sickle cell anemia erythrocytes as well as the gelling of the sickle cell hemolysates.8 He has visualized the sickling process as one in which complementary sites on adj acent hemoglobin molecules combine. It was suggested that oxyhemoglobin and carbonmonoxyhemoglobin do not aggregate because of steric hindrance of the attached oxygen or carbon monoxide molecules.
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From page 249... ...
Studies of Perutz~ and Perutz, Liquori' and Eirichi° have shown that the molecules of horse and human hemoglobin possess dyed axis of symmetry. Furthermore, -ray diffraction measurements of horse hemoglobin with four equivalents of silver showed only one slightly elongated peak in the electron density map for each pair of silver atoms, indicating directly that two -SH groups must be close together.
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From page 250... ...
C.: Amino acid composition of hemoglobins of normal negroes and sickle cell anemics, J
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From page 251... ...
In order to prove the first hypothesis some investigations were carried out by reducing this disulfide bridge with thioglycolic acid according to the method of Lindley.S With this technique it is possible, as shown for instance for insulin, to split a protein, which is built up by polypeptide chains linked by one or more -S-S- bridges, into the separate chains. The reduced protein was therefore studied after coupling of the free -SH groups with iodoacetamide both by paper electrophoresis and by moving boundary electrophoresis.
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