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H Wajcman

Publications and source records attributed to H Wajcman.

At least 91 records · Page 5Linked to original sources
PubMed1993

Hb Val de Marne [alpha 133(H16)Ser-->Arg]: a new hemoglobin variant with moderate increase in oxygen affinity.

Hb Val de Marne [alpha 133(H16)Ser-->Arg] was found in a French family during a neonatal hemoglobinopathy screening program. The abnormal hemoglobin was found, within a few months interval, in two newborn children who were first cousins. In the children as well as in the parents carrying this hemoglobin variant, the red cell parameters were normal. Hb Val de Marne, isolated by isoelectrofocusing, displayed a slightly higher auto-oxidation rate than Hb A. Its oxygen affinity was increased 1.7-fold in comparison to that of Hb A. The heterotropic effects were normal.

Adult↗
PubMed1993

Structural characterization of abnormal hemoglobins from dried blood specimens in a neonatal screening program.

Blood specimens dried on filter paper are now widely used for neonatal screening of hemoglobinopathies. These samples are perfectly suited for electrophoresis studies and HPLC analysis. They may also be used for DNA analysis. The structural characterization of a hemoglobin variant is also possible using protein chemistry methods. After elution of the hemoglobin from the paper, the different components are fractionated by a microscale preparative isoelectric focusing. The structural modification of the abnormal hemoglobin is then determined through a series of techniques including chain separation, aminoethylation, trypsin digestion, analysis of the peptides and determination of their aminoacid composition. The efficiency of this strategy is demonstrated by the study of an alpha-chain variant (Hb Hasharon) and three beta-chain variants (Hb S, Hb D Punjab, Hb E). Unambiguous identification of the structural abnormality was obtained with samples stored for up to 18 months and with abnormal fractions amounting to only approximately 10% of the total lysate.

Chromatography, High Pressure Liquid↗
PubMed1992

Hemoglobin Rouen (alpha-140 (HC2) Tyr-->His): alteration of the alpha-chain C-terminal region and moderate increase in oxygen affinity.

Hb Rouen (alpha 140(HC2) Tyr-->His) is a moderately high oxygen-affinity variant that was found in coincidence with polycythemia vera in a French patient. This hemoglobin provides an example of an alteration of the C-terminus of the alpha-chain, a region involved in the mechanisms of allosteric regulation. The increase in oxygen-affinity and decrease in cooperativity of this variant is much smaller than that resulting from the same substitution in the beta-chain. This model provides additional evidence for the inequivalence between the alpha- and beta-subunits.

Aged↗
PubMed1992

Structure of the EF corner favors deamidation of asparaginyl residues in hemoglobin: the example of Hb La Roche-sur-Yon [beta 81 (EF5) Leu----His].

Some abnormal hemoglobins constitute models which allow one to evaluate the structural requirements for post-translational modifications in proteins, such as deamidation. Hb La Roche-sur-Yon [beta 81 (EF5) Leu----His] is an unstable hemoglobin variant displaying a moderately increased oxygen affinity. About half of the abnormal hemoglobin, in addition to the substitution at position beta 81, carries a deamidation of the neighboring asparagine residue, beta 80 (EF4). The histidine at position beta 81 cannot fit into the small hydrophobic pocket which normally accomodates the leucine residue. This structural change opens the heme pocket and modifies the general conformation of the EF segment, thus explaining the increase in oxygen affinity and the achievement of a three-dimensional structure favoring asparagine deamidation. Histidine beta 81 could also act as a catalyst in the deamidation reaction. Deamidation has already been reported for two other variants of the EF corner, Hb Providence [beta 82 (EF6) Lys----Asn] and Hb J Singapore [alpha 79 (EF8) Ala----Gly]. In all these cases it seems that a histidine may catalyze the deamidation of the asparagine residues and that disturbing the folding of the EF corner will provide an extra flexibility favoring the reaction.

Adolescent↗
PubMed1992

Two new human hemoglobin variants caused by unusual mutational events: Hb Zaïre contains a five residue repetition within the alpha-chain and Hb Duino has two residues substituted in the beta-chain.

With rare exceptions, the more than 600 human hemoglobin variants described are caused by a single point mutation. Other abnormal features, such as unequal crossing-over, frameshift mutagenesis or double mutations in the same polypeptide chain, have seldom been encountered. We report two new variants caused by such rare mutational events. Hb Zaïre [alpha 116(GH4)-His-Leu-Pro-Ala-Glu-117 (GH5)] is the second example in which a short amino acid sequence is inserted within the alpha-chain. This abnormal hemoglobin results from a tandem repetition of 5 amino-acid residues, from sequence 112 through 116, at the end of the GH corner. Hb Duino is an unstable hemoglobin. It presents within the same beta-chain, the association of two rare point mutations; these substitutions are those found in Hb Newcastle [beta 92(F8)His----Pro] and in Hb Camperdown [beta 104(G6)Arg----Ser]. Family studies demonstrated that the Hb Newcastle abnormality was a de novo mutation of a gene already carrying the Hb Camperdown substitution.

Adult↗
PubMed1992

Molecular adaptation of hemoglobin function in mammals.

Vertebrate hemoglobins are tetramers made of two pairs of alpha and beta subunits each containing a hydrophobic pocket where a heme molecule binds tightly and allows for the reversible binding of oxygen. Both tertiary and quaternary structures are ideally suited for the loading and unloading of oxygen necessary for the metabolic requirements of the organisms. Starting from a single ancestor hemoglobin subunit, evolutionary processes have led to heterologous tetramers exhibiting a moderate oxygen affinity due to heme-heme interaction (allosteric mechanism) which may be further modulated through electrostatic interactions with chloride and/or organophosphate anions present in the red cells. These effectors, which bind preferentially to the deoxy-Hb tetramers at a distance from the heme groups, play a major role in the adaptation of the respiratory properties of hemoglobin to either allometric-dependent oxygen needs or to various hypoxic environments such as altitude, burrowing, or foetal life. In most cases the existence or the strength of the effector-Hb complexes, hence the changes in the allosteric equilibrium, may be ascribed to one or a few mutations of residues at the effector binding sites. Typical examples of these mechanisms are described.

Adaptation, Physiological↗
PubMed1992

HB Al-Ain Abu Dhabi [alpha 18(A16)Gly----Asp]: a new hemoglobin variant discovered in an Emiratee family.

During a routine program of hemoglobin screening performed in the United Arab Emirates, we observed an electrophoretically fast-moving variant in a 9-month-old girl and in several members of her family. The structural determination, performed by reversed phase high performance liquid chromatography and amino acid sequencing, revealed a new variant that we named Hb Al-Ain Abu Dhabi [alpha 18(A16) Gly----Asp]. Its functional properties were normal.

Amino Acid Sequence↗
PubMed1991

Human erythrocyte band 3 polymorphism (band 3 Memphis): characterization of the structural modification (Lys 56----Glu) by protein chemistry methods.

Band 3 variants occur rather frequently in different populations. Based on sodium dodecyl sulfate (SDS)-polyacrylamide electrophoretic properties, a widespread polymorphism (band 3 Memphis) has been previously described. It corresponds to a protein that has been hypothesized to be elongated in its N-terminal cytoplasmic domain. Band 3 from a heterozygote subject for this polymorphism and that displays a normal reactivity towards stilbene disulfonates has been isolated and its primary structure determined by protein chemistry. Reverse-phase high performance liquid chromatography tryptic peptide mapping showed, as the only difference with controls, that the enzymatic cleavage between the two N-terminal peptides did not occur, yielding a 69 residue-long fragment. Further cleavages of this peptide (cyanogen bromide, V8 protease), amino acid composition, and sequence analyses demonstrated that the lysine at position 56 was replaced by a glutamic acid. Thus, surprisingly, a single amino acid change is responsible for the large difference in the electrophoretic behavior. This result suggests that single amino acid substitutions may similarly be involved in the structural modification of several other protein variants, described as elongated or shortened based only on SDS-polyacrylamide electrophoresis studies. When deletions/insertions were confirmed by sequence analysis, their extent was often different from that expected from electrophoresis.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1991

Three regions of erythrocyte band 3 protein are phosphorylated on tyrosines: characterization of the phosphorylation sites by solid phase sequencing combined with capillary electrophoresis.

The major part of band 3 phosphorylation was recently shown to concern the first tryptic peptide of the protein (Yannoukakos et al. (1991) Biochim. Biophys. Acta 1061, 253-266). Tyrosine 8 is the prevalent site of phosphorylation, but other phosphorylated regions were found which could not be analyzed with certainty. Direct characterization of the phosphorylated residues in all these phosphorylated fragments was made possible due to recent advances in protein chemistry techniques, such as solid phase sequence analysis and capillary electrophoresis. The present report establishes that band 3 phosphorylation occurs predominantly on tyrosines: besides tyrosine 8 already known in the N-terminal region, two other tyrosines are demonstrated to be targets for the tyrosine kinase, tyrosine 359 and tyrosine 904. These residues lie in regions of band 3 exposed to the cytoplasm, the junction of the cytoplasmic and the membrane-spanning domains, and the C-terminal end of the protein which is also cytosolic, respectively.

Amino Acid Sequence↗
PubMed1991

Phosphorylation sites in human erythrocyte band 3 protein.

The human red cell anion-exchanger, band 3 protein, is one of the main phosphorylated proteins of the erythrocyte membrane. Previous studies from this laboratory have shown that ATP-depletion of the red blood cell decreased the anion-exchange rate, suggesting that band 3 protein phosphorylation could be involved in the regulation of anion transport function (Bursaux et al. (1984) Biochim. Biophys. Acta 777, 253-260). Phosphorylation occurs mainly on the cytoplasmic domain of the protein and the major site of phosphorylation was assigned to tyrosine-8 (Dekowski et al. (1983) J. Biol. Chem. 258, 2750-2753). This site being very far from the integral, anion-exchanger domain, the aim of the present study was to determine whether phosphorylation sites exist in the integral domain. The phosphorylation reaction was carried out on isolated membranes in the presence of [gamma-32P]ATP and phosphorylated band 3 protein was then isolated. Both the cytoplasmic and the membrane spanning domains were purified. The predominant phosphorylation sites were found on the cytoplasmic domain. RP-HPLC analyses of the tryptic peptides of whole band 3 protein, and of the isolated cytoplasmic and membrane-spanning domains allowed for the precise localization of the phosphorylated residues. 80% of the label was found in the N-terminal tryptic peptide (T-1), (residues 1-56). In this region, all the residues susceptible to phosphorylation were labeled but in varying proportion. Under our conditions, the most active membrane kinase was a tyrosine kinase, activated preferentially by Mn2+ but also by Mg2+. Tyrosine-8 was the main phosphate acceptor residue (50-70%) of the protein, tyrosine-21 and tyrosine-46 residues were also phosphorylated but to a much lesser extent. The main targets of membrane casein kinase, preferentially activated by Mg2+, were serine-29, serine-50, and threonine(s)-39, -42, -44, -48, -49, -54 residue(s) located in the T-1 peptide. A tyrosine phosphatase activity was copurified with whole band 3 protein which dephosphorylates specifically P-Tyr-8, indicating a highly exchangeable phosphate. The membrane-spanning fragment was only faintly labeled.

Adenosine Triphosphate↗
PubMed1991

Hemoglobin Redondo [beta 92(F8) His----Asn]: an unstable hemoglobin variant associated with heme loss which occurs in two forms.

Hb Redondo [beta 92(F8) His----Asn] illustrates how post-translational structural modifications may modify the phenotypic expression of an unstable hemoglobin. This variant was found in a Portuguese patient suffering from a chronic hemolytic anemia. The electrophoretic pattern demonstrated that it occurred in two forms, both being semi-hemoglobins: the fastest one migrating like HbS and the other like HbA2; after a few days of storage at 4 degrees C the intensity of the slowest Hb fraction decreased while that of the other increased proportionally. In both cases, the RP-HPLC analysis of the tryptic digest of the aminoethylated beta chains demonstrated the presence of an abnormal beta T10 peptide carrying a His----Asx substitution. Microsequence study of these two peptides demonstrated that the slowest abnormal Hb fraction had a beta 92 His----Asn substitution and the fastest a His----Asp at the same site. All these results suggest that the beta 92 His----Asn variant loses readily its heme group and that a deamidation occurs rapidly in vitro, yielding a beta 92 Asp semi-hemoglobin. The oxygen affinity of the patient's red blood cells was increased, leading to a stimulation of erythropoiesis and to a macrocytic hemolytic disease.

Amino Acid Sequence↗
PubMed1991

Cleavage by protease from Staphylococcus aureus V8: an improvement in the sequence analysis of human hemoglobin variants.

Protease from Staphylococcus aureus V8 cleaves either at glutamic residues or at both aspartic and glutamic residues, depending on the experimental conditions. In structural analyses of human hemoglobin variants, the specificity of this enzyme is of considerable interest to localize substitutions occurring in medium or large size peptides as it cleaves in smaller fragments which may be unambiguously characterized. It may also recognize the replacement of an acidic residue by the corresponding amide, or vice versa, avoiding protein sequence analysis. The various aspects of the use of protease V8 are illustrated by the study of four alpha chain hemoglobin variants concerning peptides alpha T-9 and alpha T-12b.

Amino Acid Sequence↗
PubMed1990

Hemoglobin Calais [beta 76 (E20) Ala----Pro]: a hemoglobin variant with decreased intrinsic oxygen affinity.

Hb Calais [beta 76 (E20) Ala----Pro] is a new human hemoglobin variant displaying a decreased oxygen affinity. The only electrophoretical difference with Hb A was a slightly more acidic isoelectric point. A 2-fold decrease in the oxygen affinity was found by equilibrium measurements performed in a suspension of intact red blood cells and in the lysate. It was confirmed by kinetic studies of the purified abnormal hemoglobin. The rate of methemoglobin formation at 37 degrees C of Hb Calais was also increased relative to Hb A. The mechanism by which the Pro for Ala substitution of an external residue in the beta-chains results in these profound functional abnormalities is unclear. Subtle changes at the heme pocket, at a distance from the mutation, may be a plausible explanation for the effects observed.

Adult↗
PubMed1990

A new hemoglobin variant found during investigations of diabetes mellitus: Hb Pavie [alpha 135 (H18) Val----Glu].

Hb Pavie [alpha 135 (H18) Val----Glu], found during HbA1c measurement in a patient of Italian origin investigated for diabetes mellitus, exemplifies how the presence of an abnormal hemoglobin interferes with the measurement of glycated hemoglobin. This variant hemoglobin migrates as Hb A1c on polyacrylamide gel isoelectric focusing (IEF) and therefore hindered the estimation of glycated hemoglobin by this method. By ion-exchange high-performance liquid chromatography (IE-HPLC) Hb Pavie was eluted as a shoulder of the major component and the corresponding glycated fraction together with Hb A1b. Hb Pavie was purified in order to determine how its functional properties may modify red cell survival. The only functional abnormality observed was a slight decrease of the oxygen affinity, and therefore the total amount of glycated hemoglobin was not expected to be decreased by a shortening of the red cell survival.

Amino Acid Sequence↗
PubMed1989

Hemoglobin Nouakchott [alpha 114(GH2)Pro----Leu]: a new hemoglobin variant displaying an unusual increase in hydrophobicity.

The most striking fact in Hb Nouakchott [alpha 114(GH2)Pro----Leu] is the highly increased hydrophobicity of the abnormal chain. In comparison to other variants carrying the same amino-acid substitution, but at another position, the involvement of the environmental domains in the expression of the hydrophobicity is shown. Even though the substitution concerned a proline residue, it was without consequences on the oxygen binding and the stability of the molecule.

Adult↗