HEMOGLOBIN, 20(2), 141-146 (1996)

WORT COMUUNlCATlON

HB TSURUMAI [f382(EFG)LYS+GLN]: A NEW HB VARIANT

WITH HIGH OXYGEN AFFINITY AND ERYTHROCYTOSIS

High oxygen affinity hemoglobin (Hb) variants constitute an estab- lished cause of erythremia (1). Their clinical expression is characterized by a chronic, benign, often familial erythremia without accompanying thrombo- cythemia or leukocytosis. A 46-year-old Japanese male with plethora and erythrocytosis was examined at the Hospital of Nagoya University School of Medicine. Blood samples were delivered to Yamaguchi University and Osaka University for structural and functional studies, respectively. A new abnormal Hb, Hb Tsurumai, was identified in the patient and his son in 1987 (2). Red cell indices of the proband and his three children were as shown in Table I. Platelet and leukocyte counts were normal in all members. Total and direct serum bilirubin concentrations in the proband were 1.5 and 0.2 mg/dl, respectively, and uric acid was 8.4 mg/dl. Arterial blood oxygen saturation, as calculated from PO,, was 97.7%.

TABLE I

Red Cell Indices

Proband 11-1 11-2 11-3 M-46 F-19 M-17 F-14

RBC ( l O l 2 / l ) 6.47 4.51 6.22 4.69 PCV (l/l) 0.583 0.425 0.560 0.420 Hb (g/dl) 20.0 13.3 18.3 13.1 MCV (fl) 90.0 94.0 90.0 90.0 MCH (PY) 30.9 29.5 29.4 27.9 MCHC (g/dl) 34.3 31.3 32.7 31.2

The proband and 11-2 carry the Hb Tsurumai variant.

Received: Sep te rn6~ 1 2 , 1995 . Accepted: Muhch 2 t i , 1 9 9 6 .

141

Copyright 0 1996 by Marcel Dekker, Inc.

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142 OHBA ET AL.

0 50 100 P a r t i a l oxygen pressure (rnrnHg)

FIGURE 1

OEC of fresh red cell suspensions at 37OC. Open circles: Normal control, pH 7.4. Closed circles: Proband, pH 7.4. Open triangles: Normal control, pH 7.9. Closed triangles: Proband, pH 7.9.

Oxygen equilibrium curves (OEC) (3,4) of the proband's fresh red cell suspension were shifted toward the left (Fig. 1). The P50 values at pH 7.4 and pH 7.9 were 19.7 and 12.4 mmHg, respectively (control: 26.2 and 16.0); the n values of the Hill plot were 2.8 and 2.9 (control: 3.0 and 2.8). The concentration of 2,3-diphosphoglycerate (2,3-DPG) was 4.62 mmol/L RBC (control: 4.25). These results suggested that the cause of erythremia was related to the high oxygen affinity of Hb Tsurumai.

lsoelectrofocusing (IEF) (5) revealed an abnormal Hb with a low isoelectric point between Hb A and Hb J (Fig. 2). Results of the glycerol lysis time, heat precipitation at !&'C, and the isopropanol tests were normal. Values for Hb A2 and Hb F were also normal. The abnormal Hb comprising 49.4% of total Hb emerged after Hb A (47.3%) in anion exchange high performance liquid chromatography (HPLC) on a TSK gel DEAE-5PW column (Tosoh Company, Tokyo, Japan) (6).

The abnormal p chain was prepared by urea CM-cellulose column chromatography (7). It emerged earlier than normal, and showed a retention time similar to that of the glycated p chain. It comprised 52% of the total p chains. The normal and abnormal p chains were digested with 20% (w/w)

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HB TSURUMAI 143

A2

FIGURE 2

IEF of hemolysate. This was specially run in order to compare Hb Tsurumai (lane 1) with Hb Providence (lane 2). A normal control is placed on both sides. The anode is to the top. The minor uppermost "aging bands" in lane 1 used to be invisible in the fresh hemolysate. Q, N, D: Substitution of glutamine, asparagine, and aspartic acid, respectively, for lysine at P82 (EF6).

HCI for 24 hours at 105OC and subjected to amino acid analysis. Molar ratio of [glutamic acid + glutamine] was 0.9 higher and that of lysine was 0.9 lower in the abnormal chain. Differences in other amino acids were within k0.5.

Reversed phase HPLC of the tryptic digest of the S-aminoethylated abnormal P chain (8) showed that the PT-9, PT-10, and PT-10,I 1 peptides were missing. There were several abnormal peptides that emerged much later than any of the normal tryptic peptides (Fig. 3). Two major abnormal peptides were recovered and purified by rechromatography. Amino acid analyses disclosed that the most abundant peptide was the abnormal PT- 9,lO and the other PT-9,10,11, with substitution of either a glutamine or a glutamic acid for a lysine. Results of amino acid analysis of the abnormal PT-9,lO were as follows (molar ratios; the expected normal values in paren- theses): Asp + Asn 3.9(4), Thr 1.8(2), Ser 1.9(2) Glu + Gln 2.2(1), Gly 3.0(3), Ala 3.0(3), Val 1.3(1), S-aminoethyl Cys 0.9(1), Leu 6.1(6), Phe 2.0(2), Lys 1.0(2), and His 1.9(2).

Results of gas phase Edman degradation (470A Protein Sequencer, Applied BioSystems Japan, Inc., Osaka, Japan) of the abnormal PT-9,10 assigned glutamine at the 16th position, which is equivalent to position 82 in the p chain. These results established an amino acid sustitution of

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144

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OHBA ET AL.

14

1-L

4

9-10KO

Retention t ime (min)

FIGURE 3

Peptide map of the abnormal p chain. Tryptic digest (about 0.1 mg) was applied to a pBondapack C,, column (0.39 x 30 cm), that was developed with a gradient of acetonitrile (from 0 to 32% in 120 minutes) in 0.05% trifluoroacetic acid at a flow-rate of 1.5 mL/min. Arrows: The normal posi- tions of the missing peptides. KQ: With substitution Lys+Gln.

P82(EF)Lys+Gln. We named the variant Hb Tsurumai. The result of urea CM-cellulose column chromatography was consistent with a basic-to-neutral amino acid substitution, while this charge difference was only partially ex- pressed in IEF.

The lysine at p82(EF6) provides one of the important basic residues in the DPG pocket, a slit-like space which is formed by the two partner p subunits in the heterotetramer molecule (9,lO). The binding of 2,3-DPG in this pocket stabilizes deoxy T conformation, thus lowering the oxygen affinity of Hb. The a-amino group of valine at p l (NAl) , imidazol groups of histidine at P2(NA2) and p143(H21) engage in electrostatic attraction with the phos- phate groups, and the &-amino group of the lysine at p82 (EF6) of one p chain is in contact with the carboxyl group of 2,3-DPG (1 1). In Hb Tsurumai loss of the p82 Lys will disrupt the DPG binding site so that the T and R equilibrium is shifted in favor of the oxy R state and high oxygen affinity.

Substitution of the p82 Lys by other amino acids have been reported in Hb Providence (12-16), Hb Rahere (+Thr) (17), and Hb Helsinki (+Met)

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HB TSURUMAI 145

(18). Their functional properties have been published in detail. The stripped, purified variants showed normal or even slightly low oxygen affinity, normal inter-subunit cooperation, and slightly decreased alkaline Bohr effect. Actually, in the presence of physiological concentration of 2,3-DPG, these variants show a higher oxygen affinity relative to Hb A because of the inability to bind the effector. This is the basis for the high oxygen affinity of the whole blood. The OEC of Hb Helsinki was little affected even by inositol hexaphosphate (IHP), which is a more potent effector than 2,3-DPG.

Because of these common properties all carriers of these variants showed a similar left shift of the OEC (increased oxygen affinity) of fresh blood or red cell suspension. Carriers of Hb Rahere and Hb Helsinki had relative or absolute erythrocytosis. Some carriers of Hb Providence also ap- peared to have it to a lesser degree. Although oxygen equilibrium studies have never been done on the stripped Hb Tsurumai, the mechanism for the high oxygen affinity of fresh red cell suspension would be the same: the de- creased effects of 2,3-DPG due to loss of the positively charged lysyl resi- due and to the steric hindrance exerted by the new residue.

The asparaginyl residue of Hb Providence undergoes non-enzymatic deamidation, and the post-transcriptionally modified Hb Providence (+Asp) constitutes the major component in the peripheral blood. Hb Tsurumai shows the same isoelectric point as the unmodified Hb Providence (+Am) (Y. Ohba, unpublished data), but its glutaminyl residue is not deamidated in vivo.

The authors thank Ms. M. Ami for screening tests and Ms. M. Sadamyo for drawing the figures.

Department of Clinical Laboratory Science Yamaguchi University School of Medicine 1144 Kogushi, Ube 755, Japan

Department of Internal Medicine Chubu National Hospital Obu, Aichi 474, Japan

Department of Blood Transfusion Nagoya University School of Medicine 65 Tsurumai, Nagoya 466, Japan

Department of Physiology Osaka University Medical School Suita, Osaka 565, Japan

Y. Ohba

H. Yamada

S. Takamatsu

K. lmai

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146 OHBA ET AL.

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