Haemoglobin J Guantanamo (α2β2(H6) AlaåAsp ) A new fast unstable haemoglobin found in a Cuban family

Biochimica et Biophysica Acta, 491 (1977) 1-6

© Elsevier/North-Holland Biomedical Press BBA 37588 H A E M O G L O B I N J G U A N T A N A M O (a2f12128(H6) Ala -+ Asp) A NEW FAST UNSTABLE H A E M O G L O B I N F O U N D IN A CUBAN FAMILY

G. MARTINEZ, F. LIMA and B. COLOMBO Instituto de Hematologia e Inmunolog~a, Apartado 8070, La Habana 8 (Cuba)

(Received August 3rd, 1976)

SUMMARY Haemoglobin J Guantanamo (azf12128(H6)Ala-~ Asp) was found during a screening for abnormal haemoglobins in three members of a Cuban family, of negro ancestry. The substitution in this variant is located at the alfl~ contact. This explains the slight instability and the mild haemolytic anaemia and morphological abnormalities found in the carriers of this variant. The in stability of haemoglobin J Guantanamo indicates that the presence of Asp at the position fl-128(H6) weakens the ctlfl 1 contact.

INTRODUCTION During a screening for haemoglobin variants by electrophoresis, carried out in pregnant women attending the E. Cabrera Hospital in Havana, one fast haemoglobin was found out of 650 blood samples analyzed. The variant was also found in two other members of the family. We report here the chemical characterization of this new haemoglobin. MATERIALS AND METHODS Routine haematological data were obtained by standard methods [1]. Blood red cells were counted in an automatic blood cell counter (TOA Microcell Counter, Mod. CC-1002). The thermolability test was performed by the methods of Dacie et al. [2], and isopropanol stability test by the method of Carrell and Kay [3]. Foetal haemoglobin was evaluated according to the method of Betke et al. [4], and haemoglobin A 2 by the method of Bernini [5]. Starch gel electrophoresis was carried out at pH 8.6 in a Tris/EDTA/borate buffer [6]. Glucose-6-phosphate activity was determined as recommended by the WHO [7]. The haemoglobin variant was separated from haemoglobin A by column chromatography on DEAE-Sephadex [8]. Globin chains were separated from a total haemolysate according to the method of Clegg et al. [9]. The purified abnormal chain was digested with trypsin (TPCK-treated, Merck) and fingerprinted according to

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Fig. 1. Fingerprint of the fl-chain of haemoglobin J Guantanamo. The arrow indicates the abnormal fl~T-13 peptides. Baglioni [10]. Purification of the abnormal peptide was obtained by the method of Jones [11], slightly modified. The chromatography was run on a 0.9 x 16 cm. Aminex A-5 (Biorad) column, using a linear gradient of pyridine/acetic acid developers from p H 2.5 (0.05 M) to pH 3.5 (0.67 M). The purified abnormal peptide was

partially hydrolysed with 0.03 N HCI at 110 °C for 6 h [12], and fingerprinted by the same peptide mapping procedure described above. A Carlo Erba Model 3A27 Amino Acid Analyzer was used for quantitative amino acid analysis of each peptide after hydrolysis for 18 h at 110 °C in vacuo with 5.7 N HC1 containing 9 mg/100 ml of phenol. RESULTS A N D DISCUSSION

Starch-gel electrophoresis at alkaline pH of a specimen of the subject and of two other members of the family revealed a fast moving haemoglobin with the mobility of the haemoglobins J. In all cases, mild haemolytic anaemia (10-11 g/100 ml Hb), morphological abnormalities, mild reticulocytosis (3-4~), high number of target cells in the peripheral blood, and reduced osmotic fragility, were noticed. Haemoglobin At ranged between 3.2 and 3.6 ~o. All the other routine haematological examinations were within the normal range. The thermostability test, performed according to the technique of Dacie at 50 °C was negative; however, the same test carried out at 60 °C showed a flocculent precipitate after 30 min ; at this temperature a normal control remained negative. By the Carrell test, a slight precipitate was observed after 20 min. Blood samples showed the formation of multiple intraerythrocytic inclusion bodies, after 2 h incubation with brilliant cresyl blue. It was thus concluded that the abnormal haemoglobin is unstable. The amount of the abnormal variant was estimated to be between 36 and 38 of the total by means of the separation of haemoglobins on DEAE-Sephadex and globin chain separation on CM-cellulose. Fingerprinting of the abnormal fl-chain showed that the normal fl-13 peptide was absent and a new peptide shifted anodally was present (Fig. 1). The composition of this peptide indicated a substitution of an Ala for an aspartyl or an aspariginyl residue (Table I). Since the abnormal haemoglobin and the abnormal peptide showed a change in electrophoretic mobility it was concluded that the new amino acid was Asp. Moreover, according to the genetic code, the substitution for Ash is not possible by a single base change. To determine which of the two alanyl residues present in the normal fl-13 peptide, at the positions fl-128 and fl-129, was substituted, the peptide purified by column chromatography (see Materials and Methods) was partially hydrolysed with G l u - Phe - T h r - P r o - Pro - V a t - G i n - A l a - A l a - T y r - Gin - Lys 121

125

130

132

pT 13 i

I

Gtu - Phe - T h r - Pro - P r o - V a t - G t n ~ Asp'= Ala - Tyr - Gin - Lys

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I

~JT 13 a

~JT 13 b I

Glu - Phe - T h r - P r o - P r o - V a l -

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G i n - A l a ~ A s p at T y r - G i n - L y s

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J~JT13~

J~JT13 ~"Fig. 2. Comparison of the amino acid sequence of the fiST- 13 normal peptide and the expected peptides when alanine fl-128 or fl-129 is substituted for aspartic acid.

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Fig. 3. Fingerprint of the HCI partially hydrolised flq'-13 peptide. 0.03 N HCI, under conditions in which peptide bonds between Asp and the adjacent amino acids are preferentially cleaved [11]. The fragments expected in the two cases are shown in Fig. 2; together with the normal fl-13 peptide sequence. The fingerprint of the HCI partially hydrolysed abnormal peptide is shown in Fig. 3 and the composition of the main peptides are shown in Table I. Peptides 1, 4, 5 and 6 were recovered in high yield and correspond to the fragments fl-13a and fl-13b (Fig. 2), indicating that the substituted Ala is in fl-128. The presence of the more acidic peptides 1

and 5 is most likely explained by the formation of Glu from the Gin fl-127 and Gin fl-131 respectively [13]. Moreover, peptides and free amino acids produced by unspecific cleavage were found. The analysis of the peptides obtained by partial HC1 hydrolysis clearly indicated that the location of the abnormal aspartyl residue is at the position fl-128. In fact, if the replacement were at the position fl-129 the tripeptide containing Tyr, Gln and Lys (fl-Tp 13 b', fig. 2) should be produced; instead, a positively charged tetrapeptide (fl-Tp 13 b, Fig. 2), containing also Ala, was released. Moreover, the composition of peptides 1 and 4 (Fig. 2, Table I), further confirmed the substitution of Ala fl-128. It was then concluded that haemoglobin J Guantanamo has the structural formula a2f12128 Ala -~ Asp. TABLE I Composition of fiT-13 Guantanamo tryptic peptide

Composition of the derivative peptides obtained by partial HC1 hydrolysis of fiT-13 Guantanamo (molar ratio)

Amino a c i d residue

1

2

3

4

5

6

0 0.98 1.88 1.98 0 0.99 0 1.05 0

0 0 1.14 0 0.54 0 0.94 0 0

0 0 1.07 1.59 0 1.00 0 0 0

0 0.97 2.09 1.96 0 1.02 0 1.18 0

0 0 1.18 0 0.58 0 0.96 0 1.07

0 0 1.02 0 0.87 0 0.99 0 1.18

Asp Thr Glu Pro Ala Val Tyr Phe Lys

Molar ratio Found 0.93 0.97 3.20 1.95 1.18 1.03 0.86 1.16 1.10

Expected 0 1 3 2 2 1 1 1 1

The substitution of the adjacent Ala fl-129 by Asp hzs been reported for haemoglobin Taichung, found in a Chinese subject in Taiwan [14]. No detailed clinical or haematological examination was made, but it has been reported that this haemoglobin does not produce anaemia [14]. Haemoglobin J Guantanamo, on the contrary, is slightly unstable, and produces mild anaemia, high number of target cells in the peripheral blood, and reduced osmotic fragility. However, while Ala fl-129 lies at the surface crevice, Ala fl-128 is an alfll contact [15]; it contacts Leu residue 34 and Gly residue 35 of the a-chain. Many abnormal haemoglobins showing substitutions at different sites of the alfll contact have been found [16, 17]; most of these variants are benign, but some are unstable. This occurs when the a~fl~ contact is weakened, as in the cases of haemoglobin Philly [18] and haemoglobin Tacoma [19]. A substitution of Ala fl-128 has not previously been reported in human haemoglobins. The characteristics of haemoglobin J Guantanamo indicate that the presence of an Asp in this position, i.e. the introduction of a charged group in a site normally occupied by a hydrophobic residue, weakens the stability of the alfl I contact. Since this is known to be the more extensive of the contacts between a and fl-chains [15], only a slight instability of the molecule results and a mild haemolytic anaemia.

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