Genetic Variants of Hemoglobin and Other Blood Proteins in the San Francisco Bay Area

Studies of blood genetics in “normal healthy” persons and patients of different racial groups in the San Francisco Bay Area were carried out from January 1965 to April 1968. They show that diseases due to genetic abnormalities of blood are fairly common in this region. Frequencies for abnormal hemoglobins and erythrocyte enzyme deficiencies and variants were recorded and it was noted that abnormalities in hemoglobin metabolism that may lead to mild or severe clinical and hematological symptoms proved rather common. Accompanying other disease conditions, they may cause difficulties in diagnosis. Several diseases due to or associated with different enzyme abnormalities were encountered.     

Separation of asymmetrical hybrid hemoglobins by anaerobic cation-exchange high-performance liquid chromatography

Asymmetrical hybrid hemoglobins formed from mixtures of two structurally different hemoglobins were found to be readily separated by cation-exchange high-performance liquid chromatography under anaerobic conditions. When oxyhemoglobins A and S were mixed and deoxygenated, the resulting HPLC chromatogram showed three peaks. The distribution of the three components follow the binomial expansion a² + 2 ab + b² = 1, where a and b are the initial fractions of parent hemoglobins. The middle peak was collected in a test tube saturated with CO gas and reanalyzed under the same experimental conditions. This middle component gave two peaks of equal areas with retention times identical to those of the CO-form of the parent hemoglobins without the appearance of the hybrid hemoglobin band. No intermediate peak was observed in solutions of mixtures of liganded hemoglobins under aerobic conditions. Hybrid hemoglobins AC and SC were also formed when oxyhemoglobins A and C, S and C were mixed, respectively. The separation and the identification of hemoglobins and hybrid hemoglobin employing cation-exchange HPLC can be achieved within 30 min by gradient elution. In addition, the ability to isolate hybrid hemoglobins may be a valuable tool for the study of physical and chemical properties of hybrid hemoglobins.     

Structural study of hemoglobin Hazebrouck, beta 38(C4)Thr----Pro. A new abnormal hemoglobin with instability and low oxygen affinity

A new beta-variant has been detected and structurally defined in a French male, with a life-long history of hemolytic anemia. This variant is moderately unstable and has a low oxygen affinity. The abnormal hemoglobin was not detected by standard electrophoretic procedures. It moved slightly slower than Hb A during isoelectric focusing (IEF). Two minor fractions were also seen; the first migrated just cathodal to Hb F, as did partially oxidized Hb A or hemichrome derivatives of some unstable hemoglobins; the second in the position of free alpha-chains. The abnormal beta-chain was readily separated from both beta A- and alpha A-chains by acid-urea-Triton globin chain electrophoresis. Structural study was conducted simultaneously by fingerprinting and high-performance liquid chromatography (HPLC) of tryptic peptides. A new mutation beta 38(C4)Thr----Pro was found, which was named Hb Hazebrouck.     

Separation of asymmetrical hybrid containing hemoglobin F by anaerobic anion-exchange high-performance liquid chromatography

Asymmetrical hybrid hemoglobins formed from mixtures of oxyhemoglobins S and F and A and F were separated by high-performance liquid chromatography on a 4.6 X 250 mm wide-pore polyethyleneimine-silica gel column under anaerobic conditions. The resulting HPLC chromatogram showed three peaks, with the middle peak representing the hybrid hemoglobin. The areas of these three peaks were quantified and the amount of hybrids formed was less than that predicted theoretically. We found that the deviation was due to the equilibrium constant of the FS hybrid hemoglobin differing from that of the parent hemoglobins. In this report, we introduce the anaerobic recycle ion-exchange HPLC method to determine the rate of dissociation of AS and FS hybrid hemoglobins at constant pH buffer conditions. The results obtained by this method demonstrate that FS hybrid hemoglobin is more unstable than AS hybrid hemoglobin. The free energy of association for asymmetrical hybrids containing hemoglobin F is approximately 0.6 Kcal/mol greater than that of the symmetrical parent hemoglobins.     

Practical applications of paper electrophoresis

Paper electrophoresis of proteins is a simple, economical method well adapted to routine laboratory use. It can give important diagnostic information concerning serum proteins, and is invaluable in the differential diagnosis of diseases in which there are abnormal hemoglobins.     

Hemoglobin components and plasma proteins in Pitymys duodecimcostatus

Six different hemoglobins have been demonstrated by polyacrylamide disc gel electrophoresis in a species of vole Pitymys duodecimcostatus. Plasma proteins of the Pitymys duodecimcostatus were analyzed by polyacrylamide disc gel electrophoresis and there was no significant difference with the electrophoretic patterns of the rat (Rattus norvegicus). The hematological values, hematocrit, hemoglobin concentration and erythrocyte number have been analyzed.     

Hemoglobin variations on a small bahamian island

Hemoglobin characteristics were investigated in 492 blood specimens collected in a small island community (1,450 inhabitants) in the Bahamas. Using two different methods, abnormal variants were detected in 20.3% of the sample, including genotypes AS, AC, AF(A/HPFH), SC, and SF. Biodemographic evidence suggests that the origin and distribution of the hemoglobinpathies in this population have been influenced by historical migrations and genetic drift. The opportunity for drift has been particularly heightened by the “founder effect,” the maintenance of small population size, a relatively endogamous mating structure, and restricted immigration. Health survey results reveal a wide discrepancy between the actual prevalence of abnormal hemoglobins and that indicated by informant reports.     

Hemoglobin Tilburg: alpha 2-beta 2 73 (E 17) Asp----Gly. A new hemoglobin with reduced oxygen affinity

A new hemoglobin variant has been found in a Dutch Caucasian girl and detected also in members of three generations of her family. This variant is characterized by the substitution of an aspartic acid at position 73 (E 17) of the beta-chain with a glycine residue. Hemoglobin Tilburg makes up to 42% of the total hemoglobin in the blood of the proposita, it is stable at the isopropanol test, and not associated with significant hematological abnormalities in heterozygous carriers. The oxygen dissociation curve of the purified variant, carried out at different pH values, shows a definite reduction of the affinity for oxygen and a normal alkaline Bohr effect. Three more hemoglobins with a single amino acid substitution at the same site have been previously described: Hb Korle-Bu (Asp----Asn), Hb Mobile (Asp----Val) and Hb Vancouver (Asp----Tyr). In all these proteins the affinity for oxygen is lowered to an extent which is variable and characteristic of each mutant. In this paper we discuss the possible mechanism responsible for the abnormal behaviour of hemoglobins substituted at beta 73.     

Biosynthesis of rainbow trout (Salmo gairdnerii) hemoglobins in vitro

Cell free systems were established to analyze the biosynthesis of trout hemoglobins I, II and III. HbI, II and III were synthesized in trout erythroid cell lysates as well as in a mRNA dependent rabbit reticulocyte lysate supplemented with trout erythroid cell polyA-RNA. The newly synthesized hemoglobins apparently contained the N alpha-acetyl modification at their alpha-chain amino terminals since they co-migrated with carrier trout hemoglobins that are known to contain the modification. This observation suggests that the acetylation is determined by the information encoded in the mRNA.     

Hemoglobin tilburg: α2-ß2 73 (E 17) Asp→Gly: A new hemoglobin with reduced oxygen affinity

A new hemoglobin variant has been found in a Dutch Caucasian girl and detected also in members of three generations of her family. This variant is characterized by the substitution of an aspartic acid at position 73 (E 17) of the ß-chain with a glycine residue. Hemoglobin Tilburg makes up to 42% of the total hemoglobin in the blood of the proposita, it is stable at the isopropanol test, and not associated with significant hematological abnormalities in heterozygous carriers. The oxygen dissociation curve of the purified variant, carried out at different pH values, shows a definite reduction of the affinity for oxygen and a normal alkaline Bohr effect. Three more hemoglobins with a single amino acid substitution at the same site have been previously described: Hb Korle-Bu (Asp→Asn), Hb Mobile (Asp→Val) and Hb Vancouver (Asp→Tyr). In all these proteins the affinity for oxygen is lowered to an extent which is variable and characteristic of each mutant. In this paper we discuss the possible mechanism responsible for the abnormal behaviour of hemoglobins substituted at ß 73.     

Reduction of methemoglobins M Hyde Park, M Saskatoon, and M Milwaukee by ferredoxin and ferredoxin-nicotinamide adenine dinucleotide phosphate reductase system

The reduction of hemoglobins (Hb) M such as Hb M Iwate, Hb M Boston, Hb M Hyde Park, Hb M Saskatoon, and Hb M Milwaukee by the ferredoxin and ferredoxin-NADP reductase system was studied systematically under anaerobic conditions. The enzyme system could not reduce the abnormal chains in methemoglobin M with an alpha chain anomaly but effectively converted the methemoglobin M with a beta chain anomaly to the fully reduced form. During the reduction of the methemoglobin M with a beta chain anomaly, the spectra showed a shift of the initial isosbestic points, indicating the possible formation of intermediate hemoglobins in the partially reduced state. On the reduction mode of the methemoglobin M, however, it was classified into three types. 1) Only normal chains were reduced (Hb M Iwate and Hb M Boston). 2) Sequential reduction from normal to abnormal chains occurred (Hb M Milwaukee and Hb M Hyde Park). 3) Normal chains were preferentially reduced, but the reduction of abnormal chains also started at the same rate when the reduction of normal ones had proceeded halfway (Hb M Saskatoon). These differences are discussed in relation to the redox potential of each abnormal chain in methemoglobin M.     

Two hemoglobin phenotypes in the American bison (Bison bison): A possible genetic explanation based on structural studies

A survey of blood specimens from 146 American bison, Bison bison, showed the presence of two hemoglobin phenotypes because the ratio of the two normally occurring hemoglobins differed. These two hemoglobins, which are readily identified by their electrophoretic properties and are referred to as Hb-fast and Hb-slow, have been found in all bison. Chromatographic analyses showed that in the majority of animals the ratio between the relative amounts of Hb-fast and Hb-slow was about 60:40, but in three animals from South Dakota this ratio was about 80:20. Structural studies were made on the chains of Hb-fast and Hb-slow from one animal with the 60:40 ratio and on those from a second animal with the 80:20 ratio. Four types of chains could be demonstrated which differ from each other by at least one to three amino acid residues. It is suggested that these four -chain types are the products of two nonallelic Hb structural genes and their alleles.This research was supported by United States Public Health Service Research Grant HL-05168.     

Identification of inherited protein variants in individual erythrocytes

Inherited electrophoretic variants of hemoglobin, carbonic anhydrase, and glucose-6-phosphate dehydrogenase in individual erythrocytes were separated by electrophoresis in ultrathin agar gels. By staining the electropherograms with specific fluorescein-conjugated antibodies against hemoglobins, relative proportions of two hemoglobins within individual erythrocytes can be estimated. The findings suggested that the intracellular proportions of HbA and HbS in heterozygotes are heterogeneous within a given population of cells. By this method cells containing hemoglobin F (F cells) as well as a minor variant of hemoglobin F were identified. This tool potentially offers an approach to monitoring distribution of inherited variants in individual erythrocytes for a large number of proteins.This work was supported by a grant from the John A. Hartford Foundation and from the National Institutes of Health, Grant No. HL 15160.     

Genetic hemoglobin abnormalities in 2363 Cuban newborns

Summary A total of 2363 Cuban newborns were screened for genetic hemoglobin abnormalities; 2187 (92.56%) had a normal electrophoretic pattern. Of the 176 samples with abnormal electrophoretic patterns, 102 (4.32%) had hemoglobins A, F plus Bart's; 54 (2.29%) had hemoglobins A, F and S; 3 (0.13%) had hemoglobins A, F, S plus Bart's; 14(0.59%) had hemoglobins A, F and C; 1 (0.04%) had hemoglobins A, F, C and Bart's. The frequency of Hb Bart's was 4.46% in AA phenotype, 5.25% in AS, and 6.67% in AC. Two newborns were found to have rare variants. A close correlation was found between the observed and expected phenotypes, which indicates the accuracy of the diagnostic methods used. The results of all hemoglobin abnormalities were entered on the infants' hospital records. In addition, these families received genetic counseling.     

Single amino acid substitution in important hemoglobinopathies does not disturb molecular function and biological process

Hemoglobin is an important protein found in the red cells of many animals. In humans, the hemoglobin is mainly distributed in the red blood cell. Single amino acid substitution is the main pathogenesis of most hemoglobin disorders. Here, the author used a new gene ontology technology to predict the molecular function and biological process of four important hemoglobin disorders with single substitution. The four studied important abnormal hemoglobins (Hb) with single substitution included Hb S, Hb E, Hb C, and Hb J-Baltimore. Using the GoFigure server, the molecular function and biological process in normal and abnormal hemoglobins was predicted. Compared with normal hemoglobin, all studied abnormal hemoglobins had the same function and biological process. This indicated that the overall function of oxygen transportation is not disturbed in the studied hemoglobin disorders. Clinical findings of oxygen depletion in abnormal hemoglobin should therefore be due to the other processes rather than genomics, proteomics, and expression levels.     

The primary structure of the hemoglobins of the adult jaguar (Panthera onco, Carnivora)

The primary structure of the hemoglobins from Jaguar (Panthera onco) are presented. Electrophoretic separations without and with a dissociating agent revealed the presence of two hemoglobin components, alpha 2 beta I2 and alpha 2 beta II2. The separation of the hemoglobin components was achieved by ion-exchange chromatography. The globin chains were separated by ion-exchange chromatography and also by reversed phase HPLC. The amino-acid sequences of the native chains and peptides were determined by liquid-phase and gas-phase sequencing. N-Acetylserine was detected by FAB-mass spectroscopy as N-terminal group of the beta I chain. The sequences are compared with that of human hemoglobin (Hb A).     

Functional and structural analyses on abnormal hemoglobins with impaired oxygen binding properties—To elucidate the allosteric mechanism of hemoglobin

The altered oxygen binding curves for various abnormal hemoglobins were analyzed according to a two-state allosteric model. Of three allosteric parameters computed for abnormal hemoglobins, K _R was nearly constant, but K _T and L varied with the correlation of log c=?0.4 log L, where c is K _R/K _T. This correlation indicates that the abnormal allosteric oxygen binding of hemoglobin is due to altered molecular properties of the deoxy-T state but not that of the deoxy-R state. To clarify the molecular basis of this idea, resonance Raman spectra in the low-frequency region of abnormal hemoglobins were measured under different solvent conditions. Varied frequencies of iron-histidine stretching Raman lines was found to correlate with varied oxygen affinities (K _T) of deoxy-T states. The strength of the iron-histidine bond of deoxy-T states was changed, depending upon the magnitude of the strain imposed on hemes by globin, and this bond presumably comprises an important part of the regulation mechanisms for hemoglobin oxygen binding and structure changes.     

Appearance of an arachidonic acid 15-lipoxygenase pathway upon differentiation of the human promyelocytic cell-line HL-60

The metabolism of arachidonic acid and 15-HPETE was studied in a human promyelocytic cell line (HL-60). Upon exposure to DMSO, HL-60 cells undergo differentiation and acquire a 15-lipoxygenase activity while undifferentiated cells challenged with either arachidonic acid or 15-HPETE did not enzymatically transform these precursors. Products of the arachidonic acid 15-lipoxygenase pathway were identified by HPLC. UV-absorption and gas chromatography-mass spectrometry. Results indicate that upon differentiation HL-60 cells express a 15-lipoxygenase activity as well as the ability to transform 15-HPETE to 8,15-DHETEs and 14,15-DHETE. Moreover, these findings suggest that products of the 15-lipoxygenase cascade may be generated by a single cell system.     

Hematological data and hemoglobin components in bats (Vespertilionidae)

1. Statistically significant variations were observed in the RBC counts and Hb concentration in pregnant females of Pipistrellus pipistrellus. 2. Basic hematological values in 59 animals of five species of insectivorous bats were estimated. 3. Electrophoretic separation of the hemoglobins of Plecotus austriacus, Myotis nattereri and Myotis myotis showed two components, whereas in Miniopterus schreibersi and Pipistrellus pipistrellus appeared three and four components, respectively.