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Clinical Chemistry 49: 137-143, 2003; 10.1373/49.1.137
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(Clinical Chemistry. 2003;49:137-143.)
© 2003 American Association for Clinical Chemistry, Inc.

Hemoglobin Görwihl [{alpha}2ß25(A2)Pro->Ala], an Electrophoretically Silent Variant with Impaired Glycation

Emmanuel Bissé1a, Claude Schauber2, Nathalie Zorn2, Thomas Epting1, Antonin Eigel3, Alain Van Dorsselaer2, Heinrich Wieland1, Jean Kister4 and Laurent Kiger4

1 Department of Clinical Chemistry, University Hospital, Hugstetterstrasse 55, D-79106 Freiburg, Germany.
2 Laboratoire de Spectrometrie de Masse Bio-Organique URA31, CNRS Ecole Europeenne de Chimie Polymeres Materiaux, 67087 Strasbourg, France.
3 Institut für Humangenetik, Westfälische Wilhelms-Universität, D-48149 Münster, Germany.
4 INSERM U473, 94276 Le Kremlin Bicêtre, France.

aAuthor for correspondence. Fax 49-761-270-3444; e-mail bisse{at}med1.ukl.uni-freiburg.de.


   Abstract
Top
 Abstract
Introduction
 Patient and Methods
 Results
 Discussion
 References
 
Background: Some of the genetic variants of hemoglobin (Hb) and their chemically modified species are known to affect the measurement of Hb A1c. The purpose of this study was to characterize Hb species in the blood sample of a 74-year-old German male with an exceptionally low Hb A1c value.

Methods: Hemolysates from the propositus and a healthy individual were analyzed by electrophoresis, cation-exchange HPLC, boronate affinity chromatography, and electrospray ionization-mass spectrometry (ESMS). Genomic DNA was amplified by PCR, and the sequencing was performed on an ABI 310 sequencer. Functional properties of Hb were determined by oxygen equilibrium studies and CO recombination kinetics after flash photodissociation. Glycohemoglobin species were synthesized by incubating hemolysates with glucose.

Results: A novel, electrophoretically silent ß chain, ß5(A2)Pro->Ala or Hb Görwihl, was detected by cation-exchange HPLC. It accounted for ~44% of the total Hb and had functional properties similar to those of normal Hb A and a mild degree of heat instability. During incubation with glucose, glycation of the ß chains (assessed by ESMS) in the hemolysate of a healthy volunteer was twice as fast as in hemolysate from the propositus.

Conclusions: The substitution ß5(A2)Pro->Ala seems to affect neither the functional properties nor the heterotropic interactions of Hb, but slows glycation of the N-terminal valine by an unknown mechanism.


   Introduction
Top
 Abstract
 Introduction
Patient and Methods
 Results
 Discussion
 References
 
Hemoglobin A1c (Hb A1c)1 is the result of irreversible posttranslational attachment of glucose to the N-terminal amino acid (valine) of the Hb A0 ß chain (1). Its determination is widely used in routine monitoring of patients with diabetes mellitus (2)(3). Hb A1c is accepted as the most suitable marker for long-term diabetic care. The methods for measuring Hb A1c are based on its physical or chemical properties and on antibodies specific for the glycated N-terminal amino acid of the ß chain. These methods may be affected by pathophysiologic and pharmacologic interferences, such as mutant variants and chemical modifications of Hb (e.g., carbamylation and acetylation), that cause erroneous results for Hb A1c (4)(5). This may lead to inconsistent Hb A1c values for certain patients and indicate a need to search for Hb variants. Here we characterize a new Hb mutant found in a German patient with an exceptionally low Hb A1c value of 1.5% as measured by an automated ion-exchange HPLC analyzer (Tosoh HLC-723 GHb V, A1c3.0). This variant was named Hb Görwihl after the city where the propositus lived.


   Patient and Methods
Top
 Abstract
 Introduction
 Patient and Methods
Results
 Discussion
 References
 
case report
The index patient, a 74-year-old German male, and his sister showed an inappropriate low Hb A1c (1.5% and 2.1%, respectively) values during assessments of glycemic control. They were found to be heterozygotic for a ß-chain variant. The following data concern only the propositus. He was hypertensive with mild type 2 diabetes mellitus. The patient suffered from coronary heart disease and chronic bronchitis attributable to nicotine abuse. He showed neither hepatosplenomegaly nor liver disease. The hematologic data indicated moderate erythrocytosis (Hb, 173 g/L; packed cell volume, 0.529 L/L). The serum bilirubin was 34.2 µmol/L. Serum iron and ferritin were normal. The average value for Hb A1c, determined by two cation-exchange HPLC methods, was 1.4%; it was 4.4% as measured by immunoturbidimetric assay. Glycohemoglobin (GHb), as measured by boronate affinity chromatography, was 9.7%. His fasting blood glucose and serum fructosamine were 8.6 mmol/L and 310 µmol/L, respectively. Concentrations of all three analytes returned to within reference values during treatment with metformin (GHb, 6.8%; fructosamine, 241 µmol/L; blood glucose, 5.3 mmol/L).

samples
Blood samples from the propositus and his sister were collected in EDTA, and standard hematologic procedures were followed. Hemolysates were prepared by lysis of red blood cells (RBCs) with three volumes of water, followed by centrifugation.

HB analysis
Hb analysis was performed by electrophoresis on agarose and by isoelectrofocusing as reported previously (6). RBC lysates were tested for stability, including heat stability and isopropanol stability tests at pH 7 and at 60 °C.

Hb fractions were separated using PolyCAT A cation exchanger as described previously (6) with slight modifications. Buffers A and B were adjusted to final pH values of 6.853 and 6.540, respectively. The column was equilibrated with 88% A and 12% B for 20 min before and after every run. A gradient was developed for 50 min from 12% B to 55% B (88% A to 45% A).

GHB measurement
Hb A1c was measured by five different methods: (a) cation-exchange HPLC using a PolyCAT A column as described previously (6); (b) automated HPLC (Tosoh HLC glycohemoglobin analyzer) with a cation-exchange column; (c) an immunoturbidimetric assay based on the reaction of Hb A1c with a polyclonal antibody against the glycated tetrapeptide fructosyl-Val-His-Leu-Thr (for this assay a Tina-quant Hb A1c reagent set was used as specified by the manufacturer); (d) boronate affinity chromatography to measure total GHb, including Hb A1c and ketoamine structures formed on lysines and N-terminal valine residues of both {alpha} and ß chains (7); and (e) electrospray ionization mass spectrometric (ESMS) analysis of glycated species in the GHb fraction.

ms
A VG BioQ mass spectrometer with an ESI source was used for molecular mass analysis as described previously (8). Crude hemolysate and Hb fractions from affinity chromatography on boronate gel were investigated by ESMS. The globin chain mixture (ßGörwihl + ßA) was digested with endopeptidase (8). The peptide mixture was then analyzed in a liquid chromatography (LC)-MS mode by a BIOQ[1] (Micromass) mass spectrometer, a Waters Model 2690 pump, and a Waters series 996 ultraviolet detector. Peptides were separated on a reversed-phase column [Waters Symmetry 300; 3.4 µm C18; 150 x 2.1 mm (i.d.)] with use of a gradient formed by mixing solvents A (1 g/L trifluoroacetic acid in water) and B (0.8 g/L trifluoroacetic acid in acetonitrile). The flow rate was 250 µL/min. The peptides suspected to be carrying the abnormal residue were sequenced by Edman degradation.

dna analysis
Genomic DNA was isolated from peripheral blood lymphocytes as described previously (9). Exon 1 of the ß-globin gene was amplified by PCR followed by sequencing with the Dye Primer Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems) according to the manufacturer’s recommendations. Sequencing reactions were analyzed on an ABI 310 automated sequencer.

The three-dimensional structure of the deoxygenated Hb A (T state) was obtained using the WebLab ViewerPro 4.0 program (Molecular Simulation Inc.). The Hb A crystallographic coordinates were taken from the file 3HHB (Protein Data Bank, Brookhaven National Laboratory) reported by Fermi et al. (10).

in vitro glycation
Crude hemolysates (0.77 mmol/L Hb) from the patient and a control person were incubated with 55.6 mmol/L D-glucose in the presence of 20 mmol/L NaNBH3 for 6 h at 37 °C (11). The Hb-glucose adduct was separated by affinity chromatography on boronate gel and quantified by ESMS.

functional studies
Removal of anions from hemolysates was performed by ion-exchange chromatography using the ion-retardation resin AG 11-A8 and the mixed bed-resin AG 501-X8 (D), both purchased from Bio-Rad. The procedure was similar to that described by Jelkmann and Bauer (12). The oxygen-binding properties of the stripped hemolysate were measured by a continuous method using the Hemox Analyzer (TCS) at 25 °C in 50 mmol/L bis-Tris buffer, pH 7.2, to which 50 µmol/L sodium EDTA and catalase (20 mg/L) were added to limit methemoglobin formation (13). The Hb concentration was 60–70 µmol/L on a heme basis. The methemoglobin content was calculated from the optical spectrum recorded at the end of the oxygen equilibrium measurements. P50 and cooperativity index (n50) values were calculated by linear regression from the Hill equation for oxygen saturation values between 40% and 60%. For all conditions, the data for the hemolysate were compared with data for stripped Hb A obtained under identical conditions.

The purified Hb Görwihl and Hb A fractions were isolated by PolyCAT A HPLC (using bis-Tris–KCN buffer) and concentrated on 10-kDa membrane, and their functional properties were evaluated by the kinetics of carbon monoxide recombination after photodissociation by 10-ns pulses at 436 nm, as described previously (14). Experiments were performed at 25 °C and pH 7.2 for 5 µmol/L (on a heme basis) samples.


   Results
Top
 Abstract
 Introduction
 Patient and Methods
 Results
Discussion
 References
 
HB analyses
Electrophoretic analysis (at pH 8.6 and 6.2) and isoelectrofocusing showed the presence of only typical Hb variants. Thus, none of the available electrophoretic procedures permitted separation of a possible abnormal Hb variant from Hb A. Globin chain analysis by reversed-phase HPLC failed to resolve the variant ßX-globin from ßA-globin. On cation-exchange HPLC with a PolyCAT A column, the abnormal variant eluted 1.86 min after Hb A and constituted 43.7% of the patient’s total Hb (Fig. 1 ). The Hb A2 concentration was within reference values. The isopropanol test on the hemolysate was negative after 15 min. In heat stability tests on hemolysates, 34% of the patient’s Hb and 18% of the Hb from a healthy individual precipitated after 15 min. For the isolated Hb fractions from the propositus, the precipitation was 88% in the Hb X0 fraction and 68% in the Hb A0 fraction after 15 min; for the hemolysate from a healthy individual, in which the Hb concentration was adjusted to that of the Hb fractions, the precipitation was 61%. These results indicate that the mutant has a mild degree of heat instability.



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  		Figure 1. HPLC chromatogram showing the separation of the Hb components in the whole red cell lysate from the carrier of Hb Görwihl.

Hb components were separated by gradient on a PolyCAT A column as described in Patient and Methods.

ms results
ESMS measurement of the crude hemolysate revealed three major species (Fig. 2A and Table 1 ) accompanied by numerous minor components that were assigned to the adduction of sodium and potassium. Normal ß chain with a molecular mass of 15 866 Da was accompanied by variant species whose molecular mass was 26 Da lower. Most of the structural globin variants result from single-point mutations. Therefore, Pro->Ala, Tyr->His, Ile->Ser, or Leu->Ser substitutions were the single-point mutations conceivable in this case.




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  		Figure 2. MS spectra of the whole hemolysate (A) and GHb fraction (B and C) from the carrier of Hb Görwihl.

(A), electrospray spectrum of the whole hemolysate from the carrier of Hb Görwihl. Transformed data revealed molecular masses of 15 126 ± 1, 15 841 ± 1, and 15 886 ± 1 Da, which match the wild-type {alpha} chains and the wild-type and mutant ß chains, respectively. Shown in panels B and C are the mass spectra of the GHb fraction before (B) and after 6-h incubation (C) of the hemolysate from the patient in glucose-rich medium (for interpretation see Table 2Up ).


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  		Table 1. Expected and measured molecular masses for the free and modified globin chains.

The analysis of the GHb fraction by ESMS (Fig. 2BUp and Table 1Up ) showed 11 peaks representing the {alpha} (15 126 Da), ß (15 866 Da), and mutant ß (15 841 Da) chains, in addition to their glycated forms (species). The proportions of glycated {alpha}, ß, and abnormal ß chains were 16.0%, 8.0%, and 4.0% of total Hb, respectively.

variant structure analysis
During reversed-phase HPLC, both normal and mutant ß chains eluted together. Therefore, the mixture of two ß chains was used for the structure analysis. Proteolysis of the normal and mutant globin ß chains together with LysC enzyme followed by LC-MS analysis (Fig. 3 ) revealed an additional digest peak compared with the LC-MS results obtained for the normal ß chain. This peak eluted within 22.7 min, and its mass was 26 Da lower than the normal ßL1, VHLTPEEK (952.13 Da), which eluted within 23.3 min (Fig. 3 ). This supported the mass difference measured by ESMS. Thus, the ßL1 peptide at 926.13 Da was presumed to be carrying the mutation. These results suggested that the mutation was localized in the N-terminal eight residues within the ß1–8 region. The peptide was then subjected to Edman degradation, revealing a substitution of proline by alanine at position 5 of the peptide L1 (VHLTAEEK).



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  		Figure 3. Separation of LysC digest peptides of modified ß chain by LC-MS.

Signal monitored by ultraviolet absorbance at 214 nm (top) and base peak intensity of the mass spectrum (bottom). The peak eluting at 22.7 min yielded a mass spectrum showing a mass 26 Da lower than that of the wild-type ßL1 peptide, which elutes at 23.3 min.

dna analysis
DNA analysis of the propositus revealed heterozygosity for a C->G transversion at codon 5 of the ß-globin gene, which replaces proline with alanine (CCT->GCT; ß5Pro->Ala).

in vitro glycation
The {alpha} and ß glycoforms were determined by ESMS before and after a 6-h incubation of the Hb samples in glucose-rich medium. The results are shown in Table 2 and Fig. 2CUp . The 6-h incubation of the hemolysate from the patient in glucose-rich medium increased the amount of glycated ß chain (ßA+ ßGörwihl) by 75%. However, a 150% increase in the glycated ßA chain was observed in the hemolysate from a healthy volunteer after incubation with glucose (Table 2 ). In heterozygous RBCs, the amount of glycated ßA was greater than that of the glycated ßGörwihl. These results indicated that in the presence of Hb Görwihl, the ß N-terminal residue was less glycated.


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  		Table 2. Percentages of glycated {alpha} and ß chains in hemolysates from a healthy control (N) and from the carrier for Hb Görwihl (P) before and after 6-h incubation with glucose.1

functional studies
The oxygen-binding properties of the stripped hemolysate (containing ~43.7% of Hb Görwihl) were determined as described previously (9). Under standard conditions (50 mmol/L bis-Tris, pH 7.2, 0.1 mol/L NaCl; 25 °C) the P50 was identical to that of Hb A with the same n50 value as for normal Hb (Table 3 ). The oxygen equilibrium curves of the hemolysate in the presence of 1 mmol/L diphosphoglycerate (DPG) were also identical to those of Hb A in the same experimental conditions. These data indicate that Hb Görwihl and Hb A display nearly identical oxygen-binding properties, including the DPG effect (Table 3 ).


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  		Table 3. Oxygen binding properties of purified Hb A/Hb Görwihl hemolysate.

As shown in Fig. 4 both kinetics exhibit typical fast (R-state) and slow (T-state) CO-rebinding rates. The amplitude of the slow phase, proportional to the species that have made a R->T transition after flash photolysis of CO, was also similar to the amplitude for the mutant and the Hb A control purified by the same experimental procedure (Fig. 4 ). Consequently, the ß5 Pro->Ala mutation does not affect the allosteric properties of Hb with regard to the CO binding in agreement with the oxygen-binding experiments.



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  		Figure 4. Kinetics for CO bimolecular recombination of the Hb Görwihl (—-) and Hb A (•) purified by PolyCAT A HPLC.

Experimental conditions: buffer, 0.05 mol/L bis-Tris, pH 7.2, 0.1 mol/L NaCl, 0.5 mmol/L sodium dithionite; CO concentration, 0.1 mmol/L; heme concentration, 5 µmol/L; temperature, 25 °C; detection at 436 nm. {Delta}AN, normalized absorbance at 436 nm.


   Discussion
Top
 Abstract
 Introduction
 Patient and Methods
 Results
 Discussion
References
 
The first human Hb variant detected containing a substitution at residue ß5 was Hb Warwickshire, or {alpha}2ß25(A2)Pro->Arg (15). It has been described as having normal functional properties in spite of additional positive charges close to the central cavity along the dyad axis of the Hb molecule. In addition, it showed a decrease in oxygen affinity in the presence of phosphates. The functional properties of Hb Warwickshire awakened interest in determining the functional implication of proline ß5. Accordingly, Baudin et al. (16) studied the functional properties of a recombinant Hb in which proline ß5 had been replaced by alanine. They found an increased oxygen affinity with a normal Bohr effect. The DPG and inositol hexaphosphate effects were normal, but the sensitivity to chloride had increased by 30%. These findings suggest that the absence of proline ß5 slightly affects the spatial positions of Val at NA1 and His at NA2 and indirectly affects the off-loading of 2,3-DPG as observed in Hb Warwickshire.

Hb Görwihl, or {alpha}2ß25 (A2)Pro->Ala, is the third occurrence of a mutation at the ß5 residue and the first natural human Hb in which proline ß5 has been replaced by alanine. It exhibits normal oxygen affinity with normal cooperativity, Bohr effect, and 2,3-DPG interactions. The heat stability test was weakly positive. The electrophoretic behavior of Hb Görwihl was similar to that of Hb A. It comprised 43.7% of the total Hb and was not associated with clinical symptoms or hematologic abnormalities.

Our findings are inconsistent with the data from the artificial Hb [rHbß5(A2)Pro->Ala] obtained by directed mutagenesis (16). This discrepancy could be attributed to the occurrence of some impurities in the recombinant Hb. Indeed, one of the crucial problems in recombinant protein technology is the persistent occurrence of a small quantity of proteins closely related in structure to the main component (17). In addition, some minor misrefoldings of the protein affecting the oxygen affinity may occur during the recombination process.

In light of our data, it seems probable that the ß5 Pro->Ala substitution does not induce functional alterations, and as shown in Fig. 5 , the external location of this residue indicates that the T-state structure is not destabilized by the absence of the proline at this position. This could be also postulated to explain the normal functional properties of Hb Warwickshire [ß5(A2)Pro->Arg] (15). Therefore, the change in off-loading of 2,3-DPG in the case of this Hb variant could be attributable to the introduction of the positively charged arginine; although according to Wilson et al. (15), its distance from the DPG is too far to permit a direct interaction.



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  		Figure 5. Three-dimensional structure of the ß5Pro site and the Hb A ß chain in T-state obtained by the WebLab ViewerPro 4.0 program.

Surprisingly, Hb Görwihl demonstrated impaired glycation of the ß chains. This observation was substantiated by the comparison of wild-type vs heterozygous hemolysate, as well as by in vitro incubation with glucose (Table 2Up ). The glycation rate of the ß chains (ßA +ßGörwihl) for the heterozygote was two times lower than that for the wild-type. In contrast to Hb Rambam (18), there was a substantial difference in the glycation of both normal and mutated ß chains. RBCs from diabetic and nondiabetic carriers of Hb Tyne, or {alpha}2ß25(A2)Pro->Ser (19), have been shown to have an inappropriately low percentage of GHb, a finding consistent with our observations for Hb Görwihl. We do not have a picture of the conformation that occurs because it has not been possible to isolate enough material to produce a crystal. However, Perutz et al. (20) indicated that the substitution ß5(A2)Pro->Ala confers more flexibility to the first turn of helix A and may allow the two N-terminal residues to stretch out toward the dyad axis. This situation may perhaps lead to an impairment of the reaction of glucose at these residues. The ß N-terminal amino group is a critical site for 2,3-DPG binding. The fact that Hb Görwihl exhibits a normal DPG effect makes it difficult to explain the slower rate of nonenzymatic glycation observed in the heterozygous RBCs. Consequently, the available information does not permit formulation of the molecular mechanism by which the replacements ß5(A2)Pro->Ala and ß5(A2)Pro->Ser lead to decreased glycation of the ß chain. Additional studies of the crystal structure will be necessary to address this question.


   Acknowledgments
 
We thank R. Scholl and G. Kögel (Department of Clinical Chemistry, University Hospital, Freiburg, Germany), G. Caron (INSERM U473), and U. Antowiak (Institut für Humangenetik, Univerisität, Münster, Germany) for skillful technical assistance.


   Footnotes
 
1 Nonstandard abbreviations: Hb hemoglobin; GHb, glycohemoglobin; RBC; red blood cell; ESMS, electrospray mass spectrometry; LC, liquid chromatography; and DPG, diphosphoglycerate.


   References
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 Abstract
 Introduction
 Patient and Methods
 Results
 Discussion
 References
 

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