Search

The binding and specific elution of Hb peptides from Hp was studied. Our results were confirmed by the study of inhibition of binding of alpha chains to Hp. In conclusion, a model of contact areas of Hp-Hb complex is proposed.

Hemoglobin La Desirade is a new unstable hemoglobin variant arising from the substitution of beta 129 (H7) Ala for Val. Hb La Desirade exhibits a low oxygen affinity and normal heme-heme interaction. The variant was found in two unrelated black families in association with Hb S, Hb C or beta o thalassemia.

Hb Knossos alpha 2 beta 2 27 Ala----Ser was first described in a Greek family as a silent beta(+) thalassemia variant. Reexamination of 5,000 isoelectric focusing patterns of patients with microcytosis allowed the presumptive identification of two additional propositi. The first originated in the French West Indies (Martinique) and the second in Algeria. A branch of the family of the second propositus was also investigated. Identification of Hb Knossos was made easily in the first family since one member was a double heterozygote for Hb S and Hb Knossos. In the second family HPLC elution of the peptide fragments obtained by tryptic digestion of the aminoethylated beta chain allowed the isolation and characterization of an abnormal beta T3 peak with expected beta 27 Ala----Ser substitution. The Hb Knossos heterozygote from Martinique, besides an elevated alpha/beta globin chain ratio, had an elevated Hb A2 concentration in contrast to the Greek and Algerian families in which it was normal. This difference in phenotypes may be explained by the occurrence in the Mediterranean cases of a delta gene abnormality, presumably delta(0) thalassemia, in position cis to the abnormal beta-globin gene.

A new electrophoretically silent hemoglobin variant is described that produces the classical phenotype of beta thalassemic intermedia in association with beta thalassemia trait. This variant has the expression of a silent beta thalassemia trait. The abnormal hemoglobin was detected by acid-urea-Triton-acrylamide electrophoresis and further demonstrated by isoelectric focusing. The amount of the variant in carrier is approximately 30% of the total hemoglobin. No instability was found. Absence of hemoglobin A in the propositus blood facilitated structural studies. Peptides maps were normal but analysis of individual peptide spots showed an Ala leads to Ser substitution in the beta T3. This variant has been previously called Hb Knossos (beta 27 (B9) Ala leads to Ser).

Suspensions of red cells containing Hb Marseille-Long Island showed decreased oxygen affinity and low interaction with 2,3-diphosphoglycerate. Oxygen equilibrium studies of the purified component confirmed these abnormalities. Oxidation rate measurements of carbonmonoxy-Hb Marseille and carbonmonoxy-Hb A by ferricyanide showed an increased rate for the former, suggesting an increased dissociation constant for carbon monoxide. Nuclear Magnetic Resonance spectra in the high field region revealed small changes in the proximal region of the heme pocket. These results indicated that the mutation causes a perturbation at a distance from the mutation site.

Hemoglobin A1c concentrations were measured in 107 out patients with non insulin treated diabetes. A good correlation was observed between Hb A1c and random post-prandial plasma glucose levels. Mean Hb A1c concentrations were slightly higher in these diabetics than in controls but markedly lower than values observed in insulin treated patients. Sixty three per cent of the patients had Hb a1c levels below three standard deviations above our normal mean value. Random post-prandial plasma insulin levels were correlated to the degree of overweight but not other parameters. The best results were observed in patients treated by diet alone (31 % of the whole population) and in those on diet and sulfonylureas (30 %) who were also the less older and the less overweight subjects.

In order to determine whether any heterogeneity exists in the human alpha-globin chain, i.e. whether the products of the duplicated genes are identical, we have determined the total sequence of 14 alpha-globin chains: seven of these were abnormal, while six were normal chains from the same individuals, with one additional sample which consisted of the alpha chains from a normal control. In the individuals heterozygous for an alpha-chain abnormality, the product of a single alpha-gene could be isolated from that of the three others using the differing physicochemical properties of the mutant haemoglobins. In the special situation of a double heterozygosity for an alpha-chain abnormality, the products of the two mutated genes were separated from each other and from the mixture of the products of the two normal genes. They were then investigated independently, this approach increased the precision of our work. During the course of the present investigation, sequence determinations were mainly performed on large fragments of the chains, which were purified exclusively by gel chromatography. In this way mixtures of products of several genes could be studied, thereby overcoming the risk of losing peptides differing slightly in sequence. Such loss may often occur when using ion-exchange procedures to purify small peptides. Our results show the absence of any heterogeneity at the level of the gene products of the duplicated alpha loci. Thus the human alpha-globin chain has to be considered as homogeneous.

A hemoglobin of high electrophoretic mobility was detected in a French male suffering from an acute leukemia; this hemoglobin was also present in his family. The variant was unstable and possessed an abnormal beta chain, in which a glycyl residue in position 64 (E8) was replaced by an aspartyl residue. This variant constitutes a new case of Hb J Calabria. Since the substituted E8 residue is in close spatial contact with that at B6, it was of interest to compare the properties of Hb J Calabria with those of other hemoglobins bearing substitutions at the same site.

Hb J-Cordoba [alpha 2A beta 2(95)(FG2)Lys----Met], is one of the few hemoglobin variants discovered in Argentina. The structure and functional abnormalities are described. Hb J-Cordoba exhibits a slightly increased oxygen affinity, low cooperativity, and normal interaction with heterotropic cofactors.

A 26-year-old French woman born in Saverne (France) was found to have Heinz body hemolytic anemia. Isoelectrofocusing showed the presence of an abnormal band amounting to 35% of the total hemoglobin concentration, suggesting a beta variant. Structural analysis of the abnormal beta chain showed an elongated C-terminal segment. Histidine 143 is replaced by a proline and the C-terminal sequence is identical to the corresponding segment of Hb Cranston. This new variant, named Hb Saverne, has beta chains composed of 156 amino acid residues. Studies of its functional properties showed that Hb Saverne is an unstable, high affinity variant with low cooperativity.

Hb Fontainebleau [alpha 21(B2)Ala----Pro] was found in a family of Italian origin. This new variant has electrophoretic properties identical to those of Hb A with the exception of isoelectrofocusing in which it migrates like Hb A1c. The introduction of a prolyl residue at the beginning of the B helix in the alpha chain does not lead to a change in the stability or oxygen binding properties of the hemoglobin molecule.

A new abnormal hemoglobin was found in a diabetic Maltese woman by citrate agar electrophoresis. This variant was undetectable by isoelectric focusing. No hematological abnormalities were observed. The structural analysis included isolation of the abnormal beta chain, high pressure liquid chromatography of the corresponding tryptic peptides and then microsequencing of the abnormal T1. These procedures revealed a double abnormality: the presence of a methionyl residue extending the NH2 terminus and a histidine to proline substitution in position NA2.

A new case of Hb Providence was discovered in a French caucasian family presenting a mild polycythemia. Structural and functional studies of the two abnormal fractions (Hb Providence Asn and Asp) have been performed. These confirm the abnormal characteristics of Hb Providence described previously. Red cells containing Hb Providence were fractionated with a Percoll Albumin density gradient. The respective amounts of the two components were determined in the youngest and oldest cells. We observed a slight increase of Hb Providence Asp from 34 to 37% during the life span of the erythrocytes which confirms that the deamidation of Hb Providence Asn to Hb Providence Asp is a fast process, already present and close to its maximum, in the reticulocytes. Both abnormal components are stable in the presence of isopropanol.

Hemoglobin Kenitra is a new variant of the beta chain alpha 2 beta 2 69 (E13) Gly----Arg which does not produce any clinical symptoms. It is a slow-moving hemoglobin with a distinctive pattern of electrophoretic mobilities. The stability test was negative. Oxygen affinity studies were not performed. It was found in association with alpha-thalassemia and microcytosis, but paradoxically a high expression of the variant (55%) was observed.

A third case of Hb J Iran is reported. The propositus is of Russian-Armenian origin and was investigated for hematuria. The electrophoretic behavior and the characterization of primary structure are described. Hb J Iran is stable and has normal functional properties. High resolution Nuclear Magnetic Resonance spectra suggest the presence of structural perturbations in the heme pocket of the variant. Solubility studies of Hb S/Hb J Iran mixture indicated that His beta 77 belongs to a contact region of deoxy Hb S polymers.

A new method for the purification of human erythrocyte phosphoglycerate-kinase involving affinity chromatography on dye-ligand media (Red A), in the presence of 3-phosphoglycerate and ATP, is described. The method is rapid and technically simple. The purity of the enzyme was verified by electrophoresis in polyacrylamide gel in the presence of sodium dodecylsulphate, by amino acid analysis and by immunoprecipitation in Ouchterlony plates. Peptide mapping of tryptic digests of the purified enzyme was performed and the immunoneutralization of the enzyme activity evaluated with rabbit antibodies.

A double mutant hemoglobin possessing both the Hb S (beta 6 Glu leads to Val) and the Hb Stanleyville II (alpha 78 Asn leads to Lys) mutations has been purified from the blood of a donor heterozygous for both of the mutations. The purification required two chromatography steps, with the second permitting resolution of the double mutant from Hb A2 remaining after the first step. Measurement of the competence for fiber formation by the double mutant hemoglobin was carried out by the centrifugation of gels to obtain Csat. The double mutant was found to have a greatly elevated Csat, 26.4 gm/dl, compared to 15.2 gm/dl for the Hb S control. The concentration of the pellet of the centrifuged gel for the double mutant was in the range 48-50 gm/dl, suggesting that no major rearrangement in the structure of the fibers had been induced by the Stanleyville II mutation. Electron microscopic observations on the fibers of the double mutant confirmed that a normal appearance was maintained.

A second case of Hb Henri Mondor is reported. The subject, homozygous for Hb Henri Mondor, is of Algerian origin. The electrophoretical behavior and structural characterization are given and discussed. Hb Henri Mondor, which is characterized by the replacement of the lysine residue in position beta 26, as is the case for Hb E, has normal functional properties and is normally expressed.

The amino acid sequence of a circulating thymic factor (FTS, facteur thymique sérique) isolated from pig serum has been established as less than Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH. This sequence was obtained by Edman analysis both on the intact peptide and after tryptic digestion. The COOH-terminal residue was identified by carboxypeptidase A digestion. A synthetic peptide was prepared which showed the same biological activities as native pig circulating thymic factor, thus confirming the proposed primary structure.

Variants of p 30 (iso-p 30s), the 30,000 dalton major polypeptide of murine C-type retraviruses, have been characterized in all virus strains by isoelectric focusing. Several of these iso-p 30s have been found to coexist in a given virus strain. In the present study, two iso-p 30s, characteristic of the Friend-Rauscher subgroup, separated by preparative isoelectric focusing of p 30 in thin layers of polydextran gel, were subjected to tryptic peptide mapping and aminoacid analysis. The two iso-p 30s, with isoelectric points (pI) of 6.5 and 7.1, were found to possess a partially different primary structure, with about 50% homology. The results are discussed.

A new abnormal hemoglobin, Hb Villejuif [beta 123(H1) Thr----Ile] has been discovered during the exploration of a polycythemia in a 87-year-old patient of French origin. The isoelectric focusing of the lysate revealed the presence of a variant hemoglobin with an isoelectric point very close to that of HbA. The oxygen binding properties of the patient's red blood cells being normal, it was clear that the polycythemia was not a consequence of the presence of this hemoglobin. In fact, the red blood cell morphology and the involvement of the other blood cell lines, demonstrating excessive hemopoiesis, led to the diagnosis of polycythemia vera.

Bisphosphoglycerate mutase (EC 5.4.2.4.) is a trifunctional enzyme which displays synthase, mutase, and phosphatase activities. The purification, characterization, and structural study of an abnormal form of the enzyme, isolated from a patient which we reported earlier (Rosa, R., Prehu, M. O., Beuzard, Y., and Rosa, J. (1978) J. Clin. Invest. 62, 907-915), is described. The abnormal enzyme, present at 50% of the level of the normal enzyme as estimated by immunological methods, showed elevated electrophoretic mobility and hybridized with erythrocyte phosphoglycerate mutase (EC 5.4.2.1.) in the same manner as the normal control. The mutant enzyme was unstable at 55 degrees C and could be protected against thermal instability by 0.5 mM glycerate 2,3-bisphoshate but not by either glycerate 3-phosphate or glycolate 2-phosphate. Two of the three functions of the mutant enzyme were distinct from those of the normal protein. The specific activity of the synthase was 0.57% of normal and that of the mutase 4.1%. By contrast, the specific phosphatase activity was not affected by the mutation. However, the phosphatase activity of the mutated protein was markedly less stimulated by glycolate-2-phosphate than that of the control. High performance liquid chromatography analysis of tryptic peptides derived from the mutant enzyme showed an abnormal profile with the absence of two peaks normally containing the T12 and T13 peptides and without the appearance of a supplementary peak. Amino acid sequence and mass spectrometric analysis demonstrated the substitution of Arg----Cys residue in position 89 producing an uncleaved T12-T13 present in the same peak as the T6. Considered together, our data suggest that Arg-89 is located at or near the active site of bisphosphoglycerate mutase and that this residue is probably involved in the binding of monophosphoglycerates.

High-voltage paper electrophoresis and analytical and preparative amino acid ion-exchange chromatography were used to investigate the products resulting from strong acid treatment and strong acid treatment and subsequent alkalinization of alpha-amino-delta-hydroxyvaleric acid. After strong acid treatment, alpha-amino-delta-hydroxyvaleric acid and proline, as well as several unidentified components, presumably lactones, were recovered. No definitive identification of the putative unstable lactones was accomplished either by physical-chemical means or by ion-exchange chromatography or high-voltage paper electrophoresis. Nevertheless, after acid treatment and alkalinization, only alpha-amino-delta-hydroxyvaleric acid and proline were recovered, in approximately equal amounts. The formation of such large amounts of proline from the acid treatment and alkalinization of alpha-amino-delta-hydroxyvaleric acid is therefore an important consideration in quantitating the number of gamma-glutamyl phosphate residues present in various proteins.

We have found a sickling variant, Hb S Antilles, alpha 2 beta 2(6 Glu----Val, 23 Val----Ile), that has the same electrophoretic mobility as Hb S but a distinct isoelectric focus and produces sickling in the carriers of the Hb A/S Antilles trait. The carriers' erythrocytes tend to sickle at O2 partial pressures similar to those that induce sickling in Hb S/C disease. Pure deoxy-Hb S Antilles is 50% as soluble as deoxy-Hb S (saturating concentration = 11 g X dl-1 compared to 18.4 for Hb S). Dilute solutions of pure Hb S Antilles have a lower oxygen affinity than those of Hb A or Hb S (partial pressure for 50% binding is 9 mm Hg compared to 5.5 mm Hg for Hb A or S at pH 7.00). A/S Antilles erythrocytes have a much lower oxygen affinity than A/S cells; this is further decreased in dense cells fractionated on a Percoll density gradient. Their oxygen equilibrium curves had anomalous shapes like those of S/S cells. Fiber formation in the erythrocytes of Hb S Antilles carriers is clearly due to its low solubility and oxygen affinity, showing that heterozygosity for this hemoglobin presents another sickle cell syndrome and suggesting that Hb S heterozygotes who exhibit symptoms of sickle cell disease should be carefully screened for double mutations.

The kinetics of the change from the carboxy to the deoxy conformation of the mutated hemoglobins mentioned in the title and of normal human adult hemoglobin were determined from measurements of light absorption changes occurring up to 50 microseconds after nanosecond-laser photodissociation of the corresponding CO complexes. The spectral evolution of the mutated hemoglobins was found to be similar in its main features to that of normal hemoglobin. The kinetics could be decomposed into two phases with rates 1.1-1.8 x 10(6) s-1 and 0.17-0.34 x 10(6) s-1 (except Hb St. Mandé which displayed only the faster phase). Study of the mutated subunits of HbJ Mexico (alpha subunit) and Hb Hôtel Dieu (beta subunit) showed that they convert exponentially to the stable deoxy state after photodeligation at the same rates as the corresponding subunits of normal Hb: 1.1 x 10(6) s-1 (alpha) and 0.3 x 10(6) s-1 (beta). The results indicate that there is no direct correlation between the kinetics of spectral relaxation in the time range studied and the oxygenation properties for these hemoglobins. However, there is some indication that the kinetics are dependent upon the region of mutation.

Biorex chromatograhpy of haemoglobin has been compared to the standard chromatographic separation of radioactive globin chains in 60 fetal blood samples obtained for the antenatal diagnosis of haemoglobinopathies. Biorex chromatography of haemoglobin permitted two measurements, the optical density at 418 nm and the radioactivity incorporated into fetal and adult haemoglobin. The two measurements were highly correlated (r2=0.96) and enabled a distinction between homozygous from heterozygous states of the diseases to be made, particularly in beta thalassaemia. A single column was used for 50 analyses. This fast and very sensitive method is proposed for the antenatal diagnosis of haemoglobinopathies using fetal blood.

Oxygen equilibrium studies of purified Hb Camperdown [beta 104(G6) Arg----Ser] have revealed an increased oxygen affinity at acid pH, while it is decreased for pH values above 7.4. This accounts for an almost 40% reduction in the alkaline Bohr effect. The effects of chloride and organophosphate effectors on the oxygen affinity of Hb Camperdown are inhibited by 40-50%. In chloride-free Hepes buffer, Hb Camperdown exhibits a lower oxygen affinity than normal Hb A. The present results confirm the important role of the positively charged residues lining the beta 1 beta 2 interface in regulating the functional properties of hemoglobin.

A new minor Hb fraction initially designated Hbx, has been found in the hemolysate of an erythremic patient that we have previously described with a complete erythrocyte bisphosphoglycerate mutase (EC 5.4.2.4) deficiency. Hbx (3.5% of the total) was detected by isoelectric focusing and exhibited electrophoretic and chromatographic properties similar to those of several variants of the Hb central cavity. By density fractionation of red cells, it was demonstrated that Hbx was an aging hemoglobin as in the case of glycated Hb A1c. Functional studies revealed a low oxygen affinity and almost complete inhibition of the allosteric effect of the organic phosphate effectors. Structural studies demonstrated an absence of tryptic cleavage between the peptides beta T9 and beta T10 suggesting the presence of an adduct on Lys beta 82 or on a neighboring residue. Fast atom bombardment mass spectrometry and a specific enzymatic assay with glyoxylate reductase demonstrated that the beta 82 adduct was a glycerate moiety. It was concluded that Hbx was a glycerylated Hb, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine, to our knowledge the first example of glycerylated protein. The mechanism of formation of glyceryl Hb, which was found in the four studied subjects with a bisphosphoglyceromutase deficiency, remains to be determined.

A remarkably severe spontaneous amyloidosis involving multiple organs has characterized the inbred PS mouse strain since the 25th generation. The amyloid substance was extracted with H2O and purified by successive gel filtrations on Sephadex G100, Sephadex G10 and Biogel P4. It was submitted to biochemical, immunochemical and histochemical analysis in order to determine its origin. Potassium permanganate resistance of the affinity for Congo Red dye, aminoacid composition, cross-reactivity with anti-mouse light chains antisera suggested an amyloidogenic process comparable to that described for the AL substance in man. However, even if abnormal lymphoid infiltrates were present in several organs, the presence of M component could not be demonstrated in serum or urine of these mice. This indicated either a limited tumoral mass or a tumor poorly secreting a precursor that would be strongly amyloidogenic. Alternatively, the existence of a so far unidentified precursor could not be excluded.

The human erythrocyte 2,3-bisphosphoglycerate mutase (BPGM) is a multifunctional enzyme which controls the metabolism of 2,3-diphosphoglycerate, the main allosteric effector of haemoglobin. Several cDNA banks were constructed from reticulocyte mRNA, either by conventional cloning methods in pBR322 and screening with specific mixed oligonucleotide probes, or in the expression vector lambda gt 11. The largest cDNA isolated contained 1673 bases [plus the poly(A) tail], which is slightly smaller than the size of the intact mRNA as estimated by Northern blot analysis (approximately 1800 bases). This cDNA encodes for a protein of 258 residues; the protein yielded 34 tryptic peptides which were subsequently isolated by h.p.l.c. Our nucleotide sequence data were entirely confirmed by the amino acid composition of these tryptic peptides and reveal several major differences from the published sequence; the revised amino acid sequence of human BPGM is presented. These findings represent the first step in the study of the expression and regulation of this enzyme as a specific marker of the erythroid cell line.

Hb Luxembourg [alpha 24(B5)Tyr----His] was found in association with mild hemolytic anemia and increased indirect bilirubinemia in a family originating from the Netherlands. The slight instability of this variant may be the consequence of an indirect effect of the substitution on the alpha 1 beta 1 contact since position alpha 24 (B5) is internal and in contact with several residues involved in this interface.

The present report describes structural and functional studies of a new hemoglobin variant, Hb J Pontoise. This abnormal hemoglobin was discovered in a 36 year old Spanish man. The substitution of alpha63 (E12) Ala replaced by Asp is situated at an external site and appears to have no influence on the function of the hemoglobin. Nevertheless, it presents some instability in vitro, as demonstrated by the isopropanol test. This instability could partially explain the low percentage of the abnormal hemoglobin in the hemolysate (12%), since its rate of synthesis was normal.

We report the first case of Hb Boumerdes, an alpha chain variant alpha 2(37) (C2) Pro----Arg beta 2, in an Algerian family. The propositus was also homozygous for the sickle cell gene. The abnormal hybrid Hb alpha 2Boum. beta 2S had an electrophoretic mobility on cellulose acetate pH 8.7 electrophoresis between those of Hb S and Hb A2. Its expression was about 16%. The alpha 2Boum. beta 2A fraction has a mobility between those of Hb F and Hb S. The effects of this mutation on Hb oxygen affinity and deoxy Hb S polymer formation were not studied. The propositus' sickle cell phenotype was benign.

The characterization of two abnormal human haemoglobins by fast atom bombardment (FAB) mapping is presented. The first variant, called 'R', exhibits a tryptic FAB map identical to that of normal haemoglobin. However, using Staphylococcus protease V8, a peptide containing the carboxyl end of the beta-chain exhibits a mass shift down to 300 mass units. This clearly indicates the deletion of the two last amino acids of the beta-chain. The second variant, called 'Grenoble', is due to two different modifications of the beta-chain. The location of the Pro----Ser exchange on peptide T5 is achieved by the collisionally activated dissociation mass analyzed ion kinetic energy spectra of the corresponding [MH]+ ion. The m/z value of that peptide indicated a supplementary acid----amide modification, which was located by amino acid sequencing using chemical methods. This work concludes with the necessity of using complementary methods for achieving rapid determinations of abnormal proteins with minute amounts.