Harvey A. Itano

The Human Hemoglobins: Their Properties and Genetic Control

Publisher Summary The variations of human hemoglobin provide a valuable tool for the investigation of protein homogeneity and heterogeneity, and of the genetic control of protein synthesis. A number of techniques for the detection of heterogeneity in hemoglobin have been evaluated, and some have been shown to provide ambiguous results. Their basic deficiency is either that they compare two observations taken under different conditions, or that they induce heterogeneity not present in the original sample. The heterogeneity of human hemoglobin results from the existence of adult hemoglobin, fetal hemoglobin, and a minor component, hemoglobin A 2 . The proportions in which two hemoglobins occur in an individual can be related to relative net rates of synthesis. The factors that govern the relative rates of synthesis of adult and fetal hemoglobin during transition from fetal to adult metabolism are not known. The term “thalassemia” is applied to conditions associated with marked inhibition of net rate of synthesis of adult hemoglobin without detectable alteration of physical properties. Total or partial inhibition may occur, and it is likely that mutations at more than one locus are grouped under the general category of thalassemia. The postulate of rate-controlling mutants at the Hb locus accounts for the existence of phenotypically normal red cells in individuals who carry a thalassemia allele.

Methanolysis of the pyrrolidone ring of amino-terminal pyroglutamic acid in model peptides

Abstract Treatment with HCl MeOH opened the pyrrolidone ring of the amino-terminal pyroglutamyl residue in several small peptides. Among the peptides tested, only Pyr-Gly and Pyr-Gly-Gly gave evidence of partial cleavage of peptide bonds. Several pyroglutamyl peptides, including new compounds Pyr-Lys, and Pyr-Asp-Ile-Gln-Leu, and some blocked derivatives of these peptides were synthesized. The chromatographic and staining properties of pyroglutamyl dipeptides and of the products of these peptides after HCl MeOH treatment were determined.

Genetic and biochemical studies of intermediate types of Cooley's anaemia.

IT is widely acknowledged that both parents of individuals having the most severe form of thalassacmia (Cooley’s or Mediterranean anaemia) carry the trait of the disease (Angeliiii, 1937; Caminopetros, 1938; Damcshek, 1943; Necl and Valentine, 1947; Smith, 1948; Chini and Valcri, 1949; Astaldi and Tolentino, 1952; Silvestroni and Bianco, 1952; March, Schlyeii and Schwartz, 1952; Gatto, 1953). The parents are asymptomatic, and on haematological cxamination their blood shows only slight deviations from normal. This state has been termed ‘thalassaeinia minima’ by Gatto (1953), ‘thalassaemia minor’ by Nee1 and Valentine (1947), and ‘microcytcmia’ by Silvestroni and Bianco (1952). On the other hand Smith (1948) has referred to this condition as the mildest form without anaemia. Between the clinical extremes of the severe anaemia and the trait, one finds individuals having the essential features of the disease but with clinical and haematological manifestations covering an ‘intermcdiate’ spectrum of severity (Dameshek, 1943; Smith, 1948; Chini and Valeri, 1949; Astaldi and Tolentino, 1952; Gatto, 1953). The term intermediate in this paper is employed primarily in a general clinical sense and is not intended to carry genetic connotations. Our concept of the clinical limits of ‘intermediate’ thalassaemia corresponds essentially to those dclineatcd by Astaldi and Tolentino (1952) for thalassaemia minor. Attciiding the Haematology Clinic of this Hospital are four families having members exhibiting ‘intermediate’ Cooley’s anaemia. The afflicted individuals vary in age from 3 to I 2 years. They are considered ‘intermediate’ primarily because they maintain haemoglobin coiiccntrations of 5.9 to 9.0 g. per IOO ml. without transfusion. Recently, additional knowledge pertaining to the nature of the ‘intermediate’ case has been acquired by electrophoretic, denaturation and solubility studies of the haemoglobins from the members of these families. In one of the families a unique feature has been found, and in other instances findings have differed from those in classical severe Cooley’s anaemia. This report is concerned with the above material.

Induction of haemolytic anaemia by substituted phenylhydrazines.

The induction of anaemia and reticulocytosis by arylhydrazines was influenced by substituents on the benzene ring of phenylhydrazine. Arylhydrazines with ortho substituents, which would hinder the binding by haemoglobin of a ligand derived from the arylhydrazine, resulted in the least anaemia and reticulocytosis. These results are consistent with a previous finding that hydrazinobenzoic acid, which did not produce a ferrihaemochrome from ferrihaemoglobin, did not induce anaemia. The parallelism between the formation of a ferrihaemoglobin compound and the induction of anaemia supports the hypothesis that destabilization of haemoglobin by the binding of a ligand derived from the aryl portion of an arylhydrazine is an essential step in arylhydrazine‐induced haemolytic anaemia.

2-Aminophenanthroimidazole, fluorescent product of the reaction of phenanthrenequinone with arginine

The fluorescent product of the phenanthrenequinone test for arginine and other monosubstituted guanidines was identified as a new compound, 2-amino-1H-phenanthro[9,10-d]imidazole. Identification was based on method of synthesis, elementary analysis, chemical properties, and mass spectrum. The mass spectrum of the fluorescent product is similar to that of 1H-phenanthro[9,10-d]imidazole in that both have significant peaks at me 163, 164, 190. Fragments at me 163, 164 are characteristic of phenanthro[9,10-d] heterocycles, and the me 190 fragment probably is analogous structurally to the me 90 fragment in the spectrum of benzimidazole. Differences in the two spectra can be accounted for by the presence of an amino group on the C2 atom of the fluorescent product. The acetyl derivative of this compound, N-1H-phenanthro[9,10-d]imidazol-2-ylacetamide, was also prepared.

Electrophoretic separation of intermediate compounds in two reactions of ferrihemoglobin.

Abstract The intermediate compound hypothesis for reactions of hemoglobin has been confirmed by electrophoretic separation of intermediate species. Partial oxidation of carbonmonoxyhemoglobin to ferrihemoglobin is accompanied by the formation of electrophoretic components which migrate more rapidly than carbonmonoxyhemoglobin and more slowly than ferrihemoglobin at acid pH. Comparative results by electrophoretic and spectrophotometric analyses indicate that these components are the intermediate species in the series of reactions: ( HbCO ) 4 →( HbCO ) 3 Hb +1 →( HbCO ) 2 Hb 2 +2 →( HbCO ) Hb 3 +3 → Hb 4 +4 Electrophoretic heterogeneity associated with partial saturation of ferrihemoglobin with cyanide ion apparently results from the reactions: Hb 4 +4 →( HbCN ) Hb 3 +3 →( HbCN ) 2 Hb 2 +2 →( HbCN ) 3 Hb +1 →( HbCN ) 1 . These separations were possible because of the charge differences among the species and the slowness with which they re-equilibrate.

Reactions of phenyldiazene and ring-substituted phenyldiazenes with ferrihemoglobin

Two simultaneous reactions take place between ferrihemoglobin and phenyldiazene in the absence of excess ferricyanide or of oxygen, namely the reduction of ferrihemoglobin to ferrohemoglobin and the binding of an exogenous ligand by ferrihemoglobin to form a compound with the optical spectrum of a ferrihemochrome. In the presence of excess ferricyanide, only the formation of a ferrihemochrome is observed. This compound differs from the ferrihemochrome induced by salicylate or by benzoate with respect to optical spectrum, concentration of inducer, and stability. One phenyl group is bound per heme, probably as phenyldiazene. Phenyl groups are also bound to globin, but phenyldiazene does not react anaerobically with thiols. Each ring-substituted isomer of methylphenyldiazene or bromophenyldiazene yields a different ferrihemochrome spectrum with ferrihemoglobin in the presence of ferricyanide. Only reduction of ferrihemoglobin occurs with 2- or 4-diazenylbenzoic acid in the absence of excess ferricyanide, but partial formation of ferrihemochrome occurs with 4-diazenylbenzoic acid in excess ferricyanide. The ability of an aryldiazene to bind quantitatively to ferrihemoglobin parallels the ability of the corresponding arylhydrazine to induce in vivo hemolysis.

Protein-protein interaction among hemoglobin subunits: A comparison of adult, fetal, and bovine carboxyhemoglobin

Abstract Alkali resistance, sedimentation velocity, and molecular weights of human adult, fetal and of bovine carboxyhemoglobin were compared. Sedimentation coefficients and molecular weights were determined at a number of pH values between pH 5.7 and 11.9. Bovine hemoglobin, the subunits of which do not recombine, was considerably more alkali resistant than fetal hemoglobin. Hemoglobin A was least resistant. It appears that there are no appreciable differences in the mode of dissociation of these three species of hemoglobin, except at pH 11.9. The differences seen at this pH presumably result from the alkali stability of the symmetric dimer subunit. Monomer formation is a necessary step for the occurrence of recombination after symmetric dissociation. From the close similarities in sedimentation and molecular weight data obtained with the three species of hemoglobin studied, it was concluded that appreciable amounts of monomer are not present in the equilibrium when dissociation is produced by an elevation of pH. The number of titratable groups essential for the integrity of the hemoglobin tetramer between pH 9 and 11.6 ( n ), together with dissociation constants for the three hemoglobins, were calculated. The average value for n obtained was 1.33. The biochemical and crystallographic data available indicate that either of the two tyrosine residues at position α42 or β (γ) 35 were the groups titrated.

Nonhydrolytic chemical conversion of octaethylverdochemocrome to octaethylbiliverdin

Abstract Octaethylverdohemochrome, a ferrous oxaporphyrin, was converted to octaethylbiliverdin with the incorporation of an oxygen atom from 18O2 into the latter; this result showed that hydrolysis is not essential for this conversion and that an oxygen-bridged compound cannot be excluded as a precursor of biliverdin.

Induction of Heinz Body Formation by Sodium Dithionite

Incubation of normal erythrocytes with sodium dithionite resulted in the formation of Heinz bodies, but incubation with sodium metabisulfite did not. Addition f superoxide dismutase to the incubation medium increased the formation of Heinz bodies by sodium dithionite. Addition of catalase to suspensions of erythrocytes in the presence and absence of superoxide dismutase inhibited the formation of Heinz bodies. These findings indicate that hydrogen peroxide, not superoxide, is the active oxidant in Heinz body formation.