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H Wajcman

Publications and source records attributed to H Wajcman.

At least 55 records · Page 3Linked to original sources
PubMed1997

Generation of tryptic maps of alpha- and beta-globin chains by capillary electrophoresis in isoelectric buffers.

A novel method for generating peptide maps, following tryptic digests of proteins, is reported here: capillary zone electrophoresis in the presence of isoelectric buffers as the sole buffering species. A typical buffer composition comprises 50 mM aspartic acid (pH = pI = 2.77), 0.5% hydroxyethyl cellulose (added as a dynamic coating agent for preventing peptide adsorption to weakly ionized silanols), 5% trifluoroethanol and 1% zwitterionic detergent (CHAPS). With this buffer composition, a high-voltage gradient can be applied (typically 600 V/cm in 75 microns I.D. and 900 V/cm in 50 microns I.D. capillaries), thus drastically reducing the analysis times. The method is applied to the generation of peptide maps of alpha- and beta-globin chains from human adult hemoglobin. In the case of beta-peptides, at an operative pH of 2.77, which represents a cross-over point in the titration curve of peptides T2 and T9, the two analytes merge into a single peak. However it is shown that it is possible to change the pH of the zwitterionic buffer by adjusting its concentration in solution. In 30 mM Asp (pH 3.0) or 20 mM Asp (pH 3.1) resolution of these two peptides is fully restored. Isoelectric, amphoteric buffers thus seem to represent a novel, powerful buffer system able to offer high resolution and high selectivity.

Adult↗
PubMed1997

Combined mass spectrometric methods for the characterization of human hemoglobin variants localized within alpha T9 peptide: identification of Hb Villeurbanne alpha 89 (FG1) His-->Tyr.

Mutation-induced amino acid exchanges occurring on the large T9 peptide of the alpha-chain of human hemoglobin (residues 62-90) are difficult to identify. Despite their high m/z value (around m/z 3000), collision-induced dissociation spectra of liquid secondary ion mass spectrometrically generated protonated alpha T9 peptides were performed successfully. In parallel electrospray mass spectrometry (MS) was used both to measure the molecular mass of the intact proteins and to determine the number of protonatable sites in the alpha T9 peptides. Peptide ladder sequencing using carboxypeptidase digestions and analysis of the truncated peptides by matrix-assisted laser desorption ionization time-of-flight MS confirmed the interpretation. This set of methods allowed the characterization of three hemoglobin variants, with amino acid exchanges located in the alpha T9 part of the sequence. Two of them, Hb Aztec [alpha 76(EF5) Met-->Thr] and Hb M-Iwate [alpha 87(F8) His-->Tyr] were already known. The third [alpha 89(FG1) His-->Tyr] was novel and named Hb Villeurbanne.

Amino Acid Sequence↗
PubMed1997

Structural and functional studies of hemoglobin Moabit (alpha 86(F7) Leu-->Arg.

An abnormal hemoglobin fraction was detected on high performance liquid chromatography profile performed for the measurement of glycated hemoglobin in a 55-year-old caucasian patient. The structural and functional studies were performed by standard techniques. Separation of hemoglobins by alkaline electrophoresis and by IEF revealed a slightly more rapid fraction than does Hb S. By acid electrophoresis, no abnormal Hb fraction could be observed. Separation of globin chains by electrophoresis demonstrated an alpha-chain variant and by chromatography, a fraction which eluted between beta and gamma globin chains. Tryptic digests and amino acid analysis have demonstrated a previously described substitution of Leu-->Arg alpha 86(F7).

Blood Protein Electrophoresis↗
PubMed1997

Alteration of an intersubunit contact in hemoglobin variants: comparative study of modifications at position alpha 126 Asp (H9).

Four human hemoglobin variants have already been described at position alpha 126 (H9), which is normally occupied by an aspartate: Hb Montefiore (-->Tyr), Hb Tarrant (-->Asn), Hb Fukutomi (-->Val), Hb Sassari (-->His). An additional variant, Hb West One (alpha 126 (H9) Asp-->Gly) is herein described. Aspartate alpha 126 (H9) is involved in a set of hydrogen bonds and salt bridges located at the C-terminal portion of the alpha-chains and of the C-helix of the beta-chains, which are broken in the oxy conformer, providing one of the most important sources of the difference in free energy between the T- and R-state in hemoglobin. A comparative study of four of these alpha 126 Hb variants is presented. An identical degree of alteration of the oxygen binding properties (increased oxygen affinity and decreased cooperativity) was found in all cases, when measured under standard experimental conditions (pH 7.2, 0.1 M NaCl). In contrast, the effect of L345 (a derivative of bezafibrate, which is a specific alpha-chain binding effector) on oxygen binding to Hb differed from one variant to another. When a bulky Tyr or His residue occupied the alpha 126 (H9) position, little effect of L345 was observed. Conversely, when this position was occupied by a residue of smaller size (Gly or Asn), normal heterotropic effects were observed. Molecular graphic modelling indicates that two classes of three-dimensional structure modifications may occur.

Allosteric Site↗
PubMed1997

Cation-exchange HPLC evaluated for presumptive identification of hemoglobin variants.

A battery of relatively simple tests allows the presumptive identification of hemoglobin (Hb) variants, making unnecessary structural analysis by protein chemistry methods or DNA sequencing. The primary step in this strategy involves the use of a matrix of electrophoretic mobilities obtained under various experimental conditions. This leads to an unambiguous result in approximately 90% of the cases. Additional tests are required to characterize with more confidence the remaining 10%. We describe here the use of cation-exchange HPLC on the Bio-Rad Variant automated analyzer with the "beta Thalassemia Short" program. By comparing the elution time of 125 human Hb mutants, we found that some variants with almost identical pI values or produced by the same type of amino acid substitution displayed different elution times. We present several examples in which use of the HPLC profile helped establish the diagnosis.

Cations↗
PubMed1996

Hb Bruxelles, deletion of Phebeta42, shows a low oxygen affinity and low cooperativity of ligand binding.

Functional studies of partially purified hemoglobin (Hb) Bruxelles, Phebeta42 (CD1) --> 0 indicate a major shift in the allosteric equilibrium toward the deoxy (T state) conformation. While Hb A shows a roughly symmetrical oxygenation curve with maximum cooperativity near half-saturation, Hb Bruxelles shows mainly properties of the low affinity (T state) form. The oxygen equilibrium curves for purified (>80%) Hb Bruxelles show little cooperativity and a P50 (without 2,3-diphosphoglycerate) about twice that of Hb A. The low cooperativity for Hb Bruxelles is partially compensated by an increase in oxygen affinity of the deoxy conformation and a lower 2,3-diphosphoglycerate effect. The beta chains of normal Hb have consecutive phenylalanine residues at positions 41 and 42. DNA sequencing studies of Hb Bruxelles showed a deletion of the codon TTT, which corresponds to residue Phe42. The CO rebinding kinetics after flash photolysis show mainly the slow phase, characteristic of CO binding to the deoxy conformation. In phosphate buffer at pH 7, the slow phase dominates even at low photolysis levels, where the main reaction is ligand binding to the triply liganded form. This indicates a switchover point, from the deoxy to oxy conformation, occurring beyond three ligands for Hb Bruxelles. There are few natural mutants that show a change in the oxygen affinity and cooperativity as large as that observed for Hb Bruxelles.

Adult↗
PubMed1996

Hb Montefiore (126(H9)Asp-->Tyr). High oxygen affinity and loss of cooperativity secondary to C-terminal disruption.

Hb Montefiore was found, in the heterozygous state, in a Puerto Rican female who had a slightly elevated total Hb level. Structural analysis revealed that Asp-alpha126 was replaced by Tyr. Hb Montefiore migrates close to HbF (at pH 8.6) and accounts for 20.3% of the hemolysate. Oxygen binding of red blood cells revealed a 40% decrease in the P50 (pH 7.4) and a low n value of 1.6 (normal: 2.6). Depletion of red blood cell 2,3-DPG did not change the results. Stripped Hb Montefiore at pH 7.2 showed an 8-fold reduction in P50 (0.6 versus 4.6 mm Hg) and very low cooperativity (n = 1.2 versus 2.9 for the control). Heterotopic effectors, as 2,3-diphosphoglycerate and inositol hexaphosphate had a normal effect and in addition, they increased cooperativity. The chloride ion effect and the Bohr effect were moderately reduced. A bezafibrate derivative (L345), known to bind alpha126, increases the P50 of HbA by 9-fold, but only by 1. 5-fold that of Hb Montefiore. Combining these functional studies with intrinsic fluorescence and Resonance Raman spectroscopy, we interpret the very low n value and the high oxygen affinity for Hb Montefiore as a result of both a destabilized T state that switches to R upon ligand binding and a deoxy T state that binds ligands with higher affinity than that of deoxy HbA. Hb Montefiore still binds ligands cooperatively, but the difference in ligand binding properties of the two quaternary states has been drastically reduced.

Allosteric Regulation↗
PubMed1996

Perfusion chromatography on reversed-phase column allows fast analysis of human globin chains.

Human globin chain analysis provides important information on the genetics and molecular pathophysiology of hemoglobinopathies. We propose using perfusion chromatography on the reversed-phase stationary phase to perform these studies. The technique, herein described, involves a high-velocity flow of the mobile phase through a porous chromatographic stationary phase made of microspheres of poly(styrene-divinylbenzene) having throughpores of 6000-8000 A diameter with short diffusive pores of 500-1000 A diameter connected to them. The composition of fetal hemoglobin (Ggamma:Agamma ratio) is determined, using this method, as an order of magnitude faster than with conventional HPLC. Elution is performed by developing a linear gradient of acetonitrile at a flow rate of 3 ml/min (or more), easily obtained on any HPLC machine. Analyses may be done on samples containing as low as 3.0% Hb F. Results are similar to those obtained with the reference HPLC technique, which uses a C4 column. In addition, reversed-phase perfusion chromatography, using a shallow curvilinear gradient, may help in the characterization of Hb variants. This technique allowed us to discriminate several alpha and beta chain mutants from variants that have closely similar patterns of electrophoretic mobilities.

Chromatography↗
PubMed1996

Priapism following splenectomy in an unstable hemoglobin: hemoglobin Olmsted beta 141 (H19) Leu-->Arg.

We report a case of severe priapism occurring in a patient with an unstable hemoglobin, Hb Olmsted (beta 141 Leu-->Arg) This is a rare hemoglobin variant, which until now has been reported only once. The clinical course of the 12-year-old boy was characterized by severe hemolytic anemia leading to splenectomy and cholecystectomy at the of 3.5 years. The priapism occurred 8 years after splenectomy, during a hemolytic febrile episode and required aspiration of the corpora cavernosa. This report raises the question of the benefit of splenectomy in patients suffering from a chronic hemolytic anemia such as that due to an unstable hemoglobin. This treatment lowers the frequency and the severity of acute hemolytic attacks, but several cases of vascular complications have been reported after splenectomy.

Anemia, Hemolytic↗
PubMed1996

Hemoglobin Debrousse (beta 96[FG3]Leu-->Pro): a new unstable hemoglobin with twofold increased oxygen affinity.

Hemoglobin Debrousse (beta 96[FG3]Leu-->Pro) is a new unstable variant, with high oxygen affinity responsible, in the steady state, for an apparently well-compensated chronic hemolytic anemia. The functional properties of this variant are due to the replacement of a leucine residue which is involved in the hydrophobic environment of the proximal side of the heme. This electrophoretically neutral hemoglobin was found as a de novo case in a 6-year-old girl suffering from severe anemia with hemolysis and transient aplastic crisis, following infection by parvovirus B19.

Adult↗
PubMed1996

Abnormal hemoglobins with high oxygen affinity and erythrocytosis.

More than 200 hemoglobin variants with high oxygen affinity have been reported since 1966. In about one third of these, the increase in oxygen affinity is responsible for a compensatory erythrocytosis. The degree of erythrocytosis depends primarily upon the molecular defect of the hemoglobin molecule. Measurement of the oxygen binding properties of the blood (or of the stripped lysate) is a convenient test both for the diagnosis of these variants and for evaluation of their severity. In the severe cases the affinity of the abnormal hemoglobin is identical to that of isolated subunits and the heme heme interaction is abolished; erythrocythemia is usually observed. Conversely, when the increase in oxygen affinity is moderate no clinical abnormal features are noticed. A high oxygen affinity variant has to be suspected when the hemoglobin level is at the upper limits of the normal in females or individuals with a mild iron deficiency. Several high oxygen affinity hemoglobins are also unstable and a hemolytic process may mask the increase of the Hb level. These patients, despite Hb levels close to the normal, are anemic from a functional view point. Examples have been reported in the literature in which the high oxygen affinity hemoglobin was found in coincidence with another red cell abnormality: such a situation lead usually to a more severe syndrome than the simple heterozygous state.

Erythrocytes↗
PubMed1996

Hemolytic anemias due to hemoglobinopathies.

Hemoglobinopathies responsible for hemolytic anemias may be divided into two groups. The first one corresponds to thalassemias and the second to the presence of a structurally abnormal hemoglobin (Hb). In thalassemia, the primary biochemical abnormality is a quantitative defect in the biosynthesis of one type of Hb chain. This defect leads to an overall deficit of Hb accumulation in the erythrocyte (hypochromia) together with the presence of an excess of the normally synthesized chains. The unpaired subunits which are less soluble than HbA precipitate, bind to the membrane and ultimately lead to hemolysis. In the second group, the hemolytic anemia is a direct consequence of the physicochemical properties of the structurally abnormal Hb. This molecule may polymerize, precipitate or crystallize within the red blood cell (RBC) leading to membrane alterations and to the destruction of the cell. This chapter will emphasize several examples of structurally abnormal Hbs, such as sickle cell disease and congenital Heinz body hemolytic anemia (CHBHA).

Anemia, Hemolytic↗
PubMed1996

Hb Uxbridge [beta 20 (B2)Val-->Gly]: a new variant with mild increase in oxygen affinity found during a neonatal screening program.

Hb Uxbridge [beta 20(B2)Val-->Gly] was found in an English family during a neonatal hemoglobinopathy screening program. In both the child and the parent carrying this hemoglobin variant, the red cell parameters were normal. By isoelectrofocusing Hb Uxbridge appeared to have an isoelectric point slightly higher than Hb A but was silent on cellulose acetate and acid gel electrophoresis. Structural modifications affecting position beta 20(B2) have been demonstrated to be responsible for a high oxygen affinity and polycythemia in Hb Olympia (Val-->Met) and Hb Trölhattan (Val-->Glu). In the case of Hb Uxbridge, despite an alteration of the same site, the oxygen binding parameters of the patient's hemolysate showed only a mild (ca. 20%) increase in oxygen affinity.

Female↗
PubMed1996

Hb Aubenas [beta 62(B8)Glu-->Gly]: a new variant normally synthesized, affecting the same codon as in Hb E.

Hb Aubenas [beta 26(B8)Glu-->Gly] is a mildly unstable variant that was found in a French family without hematological or clinical features. The structural abnormality was determined by protein chemistry methods, including tandem mass spectrometry, and was confirmed by Apa I digestion of a polymerase chain reaction-amplified DNA fragment. Although the substitution involves the same residue as in Hb E, the new nucleotide sequence does not create an additional out-of-frame splice site. The mutated chain is therefore normally synthesized.

Adolescent↗
PubMed1995

Hemoglobin Roanne [alpha 94(G1) Asp-->Glu]: a variant of the alpha 1 beta 2 interface with an unexpected high oxygen affinity.

In hemoglobin (Hb) Roanne, the aspartate residue alpha 94(G1) is replaced by a glutamic acid. This residue plays a key role in the structural changes affecting the alpha 1 beta 2 contact area during the deoxy- to oxy-state transition in the hemoglobin molecule. Aspartate alpha 94(G1) is involved in several contacts both in the deoxy- and oxy-structures. The most important of those is a hydrogen bond with asparagine beta 102 (G4), stabilizing the oxygenated structure. Alteration of this contact usually leads to a decrease in oxygen affinity. Hb Roanne is the first example in which an increased oxygen affinity was found as a result of a structural modification at this position. Functional data suggested that the mechanisms responsible for this altered property are a destabilisation of the T-structure and a modification of the allosteric equilibrium.

Aged↗
PubMed1995

Germline mosaicism for an alanine to valine substitution at residue beta 140 in hemoglobin Puttelange, a new variant with high oxygen affinity.

Hb Puttelange [beta 140(H18)Ala-->Val] was found as a de novo mutation in two siblings of a French family suffering from polycythemia. Both parents were phenotypically normal and exclusion of paternity has been ruled out by the study of several polymorphic markers located on different chromosomes. The structural modification of Hb Puttelange was established by reversed-phase HPLC analysis of the tryptic digest of the abnormal chain. The amino acid composition of an abnormal beta T14 peptide revealed that one of the four residues of Ala was replaced by a Val. Tandem mass spectrometry demonstrated that the substitution concerned position beta 140 (H18). This hemoglobin displays an increased oxygen affinity that is responsible for the polycythemia. De novo mutations, as demonstrated again in the case of this variant, have the highest probabilities of detection when they lead to pathological manifestations. They may result either from a somatic mutation in a very early stage of the embryological development of the propositus or may have a parental origin with occurrence of a germline mosaicism. The study of the beta-globin gene indicated that this case of Hb Puttelange probably arose from a mutation affecting a part of the germline of the father, therefore leading to a true recurrence risk.

Adult↗