T. H. J. Huisman

Studies on the heterogeneity of hemoglobin: I. The heterogeneity of different human hemoglobin types in carboxymethylcellulose and in amberlite irc-50 chromatography: Qualitative aspects

Abstract Hemoglobin samples from normal adult individuals, from newborn babies and from patients suffering from some hemoglobinopathies were studied by different analytical procedures. A close relation between the heterogeneities found by starch block electrophoresis and by chromatography on CM-cellulose as well as on Amberlite IRC-50 was demonstrated. The hemoglobin of a normal adult individual was found to consist of six Hb-fractions named Hb-A1A, Hb-A1B, Hb-A1C Hb-A0 Hb-F and Hb-A2, while next to these fractions a slightly yellow fraction was found consisting of the methemoglobin reductase (about 10 %) and other unknown proteins. Some data on the Chromatographic behaviour of the recently discovered abnormal Hb-A2 fraction (named Hb-A2) are also presented. Both by CM-cellulose and by Amberlite IRC-50 chromatography, two fetal hemoglobin fractions were found in the hemoglobin of cord blood erythrocytes. The two Hb-F fractions were present in blood samples obtained from human embryos and premature infants as well as from babies some months after birth. In case of sickle-cell anemia four minor Hb fractions were present next to the main Hb-S component. These minor Hb-components are Hb-A0 and Hb-A1C (probably derived from transfused red cells of a normal blood donor), Hb-F0 and a Hb-S1 fraction with the same low solubility in concentrated salt solutions as the Hb-S0 component when both Hb fractions were converted to the reduced state. The Hb-E was found to have a similar heterogeneity as Hb-S. The previously described CM-cellulose and Amberlite IRC-50 Chromatographie techniques were modified, and a simple technique for the concentration of dilute hemoglobin solutions is described. The methods employed permit the isolation of many hemoglobin components on a preparative scale. Rechromatography, cross-experiments and investigations of the fractions by other techniques were readily carried out.

Hemoglobin C in newborn sheep and goats: a possible explanation for its function and biosynthesis.

Extract: Chromatographic analyses of the hemoglobins of newborn kids over a postnatal period of 120 to 140 days have shown that fetal hemoglobin is replaced by βC chain-containing hemoglobin types rather than by normal βA chain-containing adult hemoglobin components. These βC chain-containing hemoglobins are the same as those observed in adult goats subjected to severe blood-loss anemia. A similar, but quantitatively different, phenomenon was also observed in newborn lambs. The increase in βC chain production is preceded by an elevation of an erythropoietic stimulating factor (ESF) in blood plasma. When ESF extract of human urinary origin was injected into either young lambs or a young kid, an increased production of βC chain-containing hemoglobins was observed.Speculation: It is postulated that the production of βC chain-containing hemoglobin types in the newborn kid is a physiological adjustment that allows these fast growing animals to supply oxygen to the tissues in an easy fashion. The effects observed as a response to an increased ESF activity in serum are interpreted to be the result of two separate mechanisms that differ qualitatively and quantitatively. The low sensitivity of sheep to the stimulus of an elevated ESF activity is explained by a possible formation of an activator-inhibitor complex resulting in a decreased activation of the silent HbβC structural gene.

Postnatal Changes in the Chemical Heterogeneity of Human Fetal Hemoglobin

Extract: The fetal hemoglobin (Hb-F) of blood samples from 11 newborn babies (two normal infants, two sickle cell trait carriers, two Hb-C heterozygotes, two infants with Hb-SG disease, one infant with Hb-Richmond heterozygosity, one β-thalassemia heterozy-gote, and one infant with a heterozygosity for the hereditary persistence of fetal hemoglobin) and from 16 adults (eight normals, two Hb-S heterozygotes, one Hb-C heterozygote, and five SC patients) has been examined to determine the ratio of the two structurally different γ chains, namely the Gγ and Aγ chains. This ratio is about 2:3 in the Hb-F of the adults and, therefore, significantly different from the 3:1 ratio in the Hb-F of the newborn. This newborn ratio undergoes a considerable change between the 3rd and 4th months of life, at which time it approaches that of the Hb-F of adults.Speculation: The mechanism by which the gradual change from γ chain synthesis to β and δ chain synthesis is controlled remains unclear. However, the change in the ratio of production of structurally different γ chains as a function of postnatal age indicates a rather complex mechanism which probably involves an unequal repression of the γ chain structural genes. Any explanation of the mechanism must take into account the fact that the production of two genes, the Gγ and Aγ, is greatly decreased, whereas that of two other genes, the β and δ, is started. Perhaps a closely related or even identical mechanism controls not only the ratio of production of the Gγ and Aγ genes but also that of the β and δ genes.

Nature of fetal hemoglobin in the Greek type of hereditary persistence of fetal hemoglobin with and without concurrent beta-thalassemia.

The fetal hemoglobin in the affected members of three Greek families with the hereditary persistence of fetal hemoglobin has only gamma-chains of the type with alanine in position 136. Although certain Negro families had been considered to have only this type of gamma-chains in their fetal hemoglobin, further studies required that they be reclassified. Consequently, the Greek cases are the sole examples of this class among the heterozygotes for the hereditary persistence of fetal hemoglobin. In Greek double heterozygotes for beta-thalassemia and the hereditary persistence of fetal hemoglobin, fetal hemoglobin is increased above the level of hemoglobin F in simple heterozygotes and gamma-chains with glycine in position 136 become apparent. In these individuals, gamma-chains with alanine in position 136 apparently derive from the chromosome for the hereditary persistence of fetal hemoglobin and are present in the hemoglobin F with gamma-chains of both types from the chromosome for beta-thalassemia. When these data are correlated with earlier knowledge of the genetic state of the Greek individuals, modifications of our previous ideas about deletions as the genetic basis of the hereditary persistence of fetal hemoglobin must be considered.

The amino-acid composition of human adult and foetal carbonmonoxyhaemoglobin estimated by ion exchange chromatography☆

Abstract 1. 1. Haemoglobin from the blood of normal adult individuals and purified foetal haemoglobin have been analysed for seventeen amino-acids and ammonia using, with slight modifications, the gradient elution chromatographic method as recently developed by Stein and Moore . It was found that purified foetal carbomonoxyhaemoglobin contains more threonine, serine, methionine and iso-leucine and less proline, alanine, valine, tyrosine and lysine. The most striking differences between the two haemoglobins are the much higher content of serine and isoleucine and the lower amount of valine in the foetal haemoglobin. 2. 2. As the yields of valine and isoleucine were complete after a short time of hydrolysis and showed no increase with longer times as found for other proteins, it is unlikely that these two amino-acids are combined in peptide linkage in these two haemoglobins. 3. 3. Since for some amino-acids the number of residues could not be divided by four, it is unlikely that both adult and foetal globin consist of four identical polypeptide chains. Some possibilities concerning the structure of the protein molecule are discussed. 4. 4. It is pointed out that the differences in amino-acid composition of COHb-a and COHb-f will be great help for the detection of the presence of the foetal compound in the blood in different diseases.

A Homozygote for the HbGγ Type of Foetal Haemoglobin in India: A Study of Two Indian and Four Negro Families

Summary. The haemoglobin of a 9‐yr‐old boy from the area of Bombay has only haemoglobin F. The γ‐chains of this haemoglobin F are solely of Gγ type, and the child is considered to be a homozygote for hereditary persistence of foetal haemoglobin of the Gγ type. The same kind of haemoglobin F is also present in a second Indian family and in four American Negro families. One Negro family has an appreciably lower percentage of haemoglobin F in heterozygotes than the other five families. In some of the families certain individuals also have haemoglobin E or haemoglobin S in trans to the other condition and haemoglobin A is absent. Although it has been concluded that the hereditary persistence of foetal haemoglobin is present the condition is not expressed uniformly in all these families. Some of them have certain characteristics of F‐thalassaemia. It is possible that two conditions are, in reality, represented: perhaps some families have the hereditary persistence of foetal haemoglobin and others have F‐thalassaemia.

Investigations on two different hemoglobins of the sheep

Abstract In many Dutch sheep the incidence of two different hemoglobins is described. These proteins differ in many physical and chemical properties. Next to varieties in electrophoretic and chromatographic behavior (on Amberlite IRC-50) differences were found to exist in solubility, in resistance to alkali, and in amino acid composition. It was found that the “abnormal” hemoglobin II in the sheep contains higher amounts of glutamic acid, threonine, and serine, and lower amounts of aspartic acid, glycine, and alanine. The genetic basis of the occurrence of the two hemoglobins in this kind of animal seems to be similar to that in men. The differences in the amino acid compositions suggest that the genes responsible for the synthesis of the two proteins influence the formation of different polypeptide chains.

Further Studies of the Postnatal Change in Chemical Heterogeneity of Human Fetal Hemoglobin in Several Abnormal Conditions

Extract: The fetal hemoglobin of 10 infants has been examined serially from birth to several hundred days of age. In the normal child, the ratio of Gγ to Aγ chain alters from about 7:3 at birth to about 2:3 at 150–200 days. In children with abnormalities of hemoglobin synthesis, the ratio changes as expected from data from adults with the particular abnormality. Thus, little change in ratio was shown by two β-thalassemia homozygotes, whereas two β-thalassemia heterozygotes showed the same behavior as a normal child; the latter behavior is expected from one of the two classes of β-thalassemia heterozygotes. A heterozygote from the GγAγ class of hereditary persistence of fetal hemoglobin behaved much like a normal child, but two heterozygotes from the Gγ class showed the expected change from approximately 9:1 at birth to 10:0 postnatally. Two children with sickle cell anemia resembled a normal child in change of ratio whereas the third, not unexpectedly, did not show a change in ratio.Speculation: The mechanisms involved in the gradual change from γ chain synthesis to β and δ chain synthesis are complex and ill defined. The results of analyses of the ratio between the products of the Gγ and Aγ structural genes in infants with distinct genetic disorders at birth and during the postnatal period underscore the complexity of the changeover. However, differences in the postnatal change in the Gγ to Aγ ratio in infants with an apparent identical condition may help to further define these mechanisms, and to delineate the conditions involved.

Nature of Foetal Haemoglobin in F‐Thalassaemia

The nature of Hb‐F was studied in 32 heterozygotes for F‐thalassaemia, in four homozygotes, and in four persons who have F‐thalassaemia in combination with β‐thalassaemia or Hb‐S. Analysis of the cyanogen bromide fragment γCB‐3 indicated that, in all heterozygotes, both Gγ and Aγ chains were present in Hb‐F in an average ratio of about 2:3. In the homozygotes and the double heterozygotes, both Gγ and Aγ chains were observed in an approximate ratio somewhat higher than 1:1. This pattern of γ chain synthesis is nonspecific for F‐thalassaemia but similar to that observed in the traces of Hb‐F of normal adults. In conjunctionwith existing information from other genetic studies, it may be concluded that the mutation in F‐thalassaemia is associated with a complete deficiency of β and σ chains from cis position together with an increased synthesis of γ chains that is directed by both Aγ and Gγ loci.

The T gamma chain of human fetal hemoglobin at birth and in several abnormal hematologic conditions.

The T gamma chain of human fetal hemoglobin has a threonyl in stead of an isoleucyl residue in position 75. When the cord bloods from infants from varied ethnic backgrouds and geographic areas were tested for the presence of the T gamma chain, it was present in 28 or 98 samples. In some groups as many as 40% had the T gamma chain whereas none was detected in other. When the T gamma chain was present, its quantity was about 20% of the total gamma chains, but one case had 35%. Among beta-thalassemia homozygotes of the Mediterranean region, 70% and the T gamma chain in the amount of 20-50% of the total gamma chains, but seven Black beta-thalassemia homozygotes were negative for the T gamma chain. The fetal hemoglobin of 16 adult patients with sickle cell anemia had no T gamma chains, but 2 of 9 newborn children with sickle cell anemia had the T gamma chain. The frequency of the T gamma gene (16), the relationship of the T gamma gene to the G gamma and A gamma genes, and the significance of the T gamma gene are discussed.