Hemoglobin-A2-coburg or α2δ2116 Arg→His (G18)

Biochimica et Biophysica Acta, 393 (1975) 379-382

© Elsevier Scientific Publishing Company, Amsterdam- Printed in The Netherlands BBA 37076 HEMOGLOBIN-A2-COBURG O R ct2(~2116 Arg-*His (Gt8)

R. S. SHARMAa, L. WILLIAMSa, J. B. WILSONb and T. H. J. HUISMAN b "Department of Hematology, Queen Victoria Memorial Hospital, Melbourne (Australia) and bLaboratory of Protein Chemistry and Comprehensive Sickle Cell Center, Medical College of Georgia, Augusta, Ga. 30902 (U.S.A.)

(Received' January 14th, 1975)

SUMMARY Hemoglobin-A2-Coburg o r Ct2(~2116Arg--*His(GlS) has been found in members of a family of Sicilian origin. The propositus is heterozygous for hemoglobin-A2-Coburg as well as for fl-thalassemia, and family data indicate that the gene for the 6-Coburg chain is in trans of the fl-thalassemia determinant.

During family investigations of a child with homozygous fl-thalassemia(case III-1), the mother's blood examination showed a red cell morphology characteristic of heterozygous fl-thalassemia but a hemoglobin-A2 level which was within normal limits, namely 2.8 7o. However, examination of the hemoglobin of the mother by cellulose acetate electrophoresis at pH 8.6 showed an additional minor fraction (hemoglobin-Xz) which moved slightly faster than hemoglobin-S. The child's father and twin sister (cases 11-2 and 11I-2 of the pedigree in Fig. 1) had findings consistent with a fl-thalassemia heterozygote. Two other members (cases 1-2 and 1I-3) were also fl-thalassemia heterozygotes. Two members (cases I-1 and 11-4) had a normal erythrocyte morphology, but the electrophoretic pattern of their hemoglobins was similar to that of cases II-1 (Fig. 1) except that the quantities of the minor hemoglobin fraction A2 and X2 were 50 ~o of the latter. Hematological data of the members of this family which had migrated to Australia from Vizzini, Sicily, are given in Table I. Approx. 200 ml blood of case II-1 was collected in acid/citrate/dextrose solution and shipped, air mail special delivery, to Augusta, Ga. Red cell hemolysate was prepared by lysis of washed cells with an equal volume of distilled water and 0.2 volume of carbon tetrachloride. Debris was removed by centrifugation. Starch gel electrophoresis at pH 9.0 [2] showed a minor hemoglobin with a mobility like that of hemoglobin-A2-Flatbush [3, 4]. Quantitation by DEAE-Sephadex chromatography [5, 6] gave these data: hemoglobin-A2:2.6 ~ and hemoglobin-X2:2.9 ~o; these values correspond closely to those obtained in Australia. Larger quantities of the X2 component were isolated by DEAE-Sephadex chromatography [5, 6] and the abnormal non-a chain was separated from the a chain by CM-cellulose chromatography [7]. This material was aminoethylated [8, 9], and 52 mg aminoethylated-non-a chain was available for further analysis. Hydrolysis was made with trypsin (Worthington Biochemical Corp., L-(tosylamido-2-phenyl)ethyl

380

-l-

I

2

III (62)

(47)

17 (22)

(20)

(15)

Normol

Trr (I)

CI)

[~

~

A-BThol.

~

(~ Aa-Coburgtrait

Fig. 1. Pedigree of Family C with fl-thalassemia and the 6 chain variant hemoglobin-A2-Coburg. Case III-1 is homozygous fl-thalassemia. The numbers between parentheses are the ages of the persons who participated in this study. TABLE I HEMATOLOGICAL AND HEMOGLOBIN COMPOSITION DATA Case*

Hemoglobin (g/100 ml)

Osmotic** Fragility (g/100 ml)

Hemoglobin-A2*** (%)

Hemoglobin-A2Coburg** * (~)

Hemoglobin-FAD * (%)

I-1 I-2 II-1 II-2 II-3 II-4 II-5 III-1 III-2

13.5 10.8 10.9 I 1.8 11.4 12.8 12.5 5.5 10.5

0.42 0.34 0.35 0.34 0.33 0.43 0.44 0.35 0.36

1.4 5.7 2.8 6.5 6.4 1.4 3.4 3.3 5.4

1.4 0 2.8 0 0 1.4 0 0 0

1.7 4.4 4.4 1.8 4.2 1.7 1.8 67 4.5

* See Pedigree in Fig. 1. ** Median fragility in g/100 ml NaCI, *** By cellulose acetate electrophoresis at pH 8.6. t By alkali denaturation [1].

chloromethyl ketone trypsin) at r o o m temperature for 6 h at p H 9.0 in a pH stat ( R a d i o m e t e r ) ; trypsin (0.5 mg) was added at 0 time and 30 m i n later. The tryptic peptides were separated on a 60 × 0.9 cm c o l u m n of c h r o m o b e a d Dowex 50-X4 resin type P (Technicon I n s t r u m e n t s ) at 37 °C using pyridine/acetic acid developers [10]. Some peptides were r e c h r o m a t o g r a p h e d on c o l u m n s of Dowex l-X2 [11]. A m i n o acid c o m p o s i t i o n of individual peptides was determined with a Spinco Model 121 a u t o m a t e d a m i n o acid analyzer equipped with high sensitivity cuvettes and an info-

381 tronics integrator (Infotronics Inc., Houston, Texas). Hydrolysates were made at 110 °C under reduced pressure with 6 M HC1 for 24 h. Seventeen peptides were isolated from the tryptic digest. Nine of these had amino acid compositions identical to those of the tryptic peptides T-l, T-4, T-6, T-7, T-8, T-9, T-11, T-14, and T-15 found in both the fl chain and the 6 chain [12]. Five peptides corresponded to the T-2, T-3, T-5, T-10, and T-13 tryptic peptides of the normal 6 chain [12] suggesting that the X2 hemoglobin is indeed a 6 chain variant. Peptide 6-T-12 was recovered in three fragments (Table II); the tetrapeptide T-12 a and the octapeptide T-12 a+b had normal amino acid compositions. The octapeptide TABLE II THE A M I N O ACID COMPOSITION OF F R A G M E N T S OF THE 6X-T-12 PEPTIDE In residues per mol of peptide. The numbers between parentheses refer to the numbers of residues in the normal 6 chain. Amino acid

T-12 a

Lysine Aminoethylated Cysteine

T-12 a+b

T-12 ¢ 1.08 (1)

1.O0 (1)

Histidine

0.93 (0)

Arginine Aspartic acid Glycine Alanine Valine Leucine Phenylalanine Yield (/~mol)* Position in 6 chain

2.10 (2)

1.22 1.06 0.17 2.00 2.86

0.3

0.6

0 1.07 1.23 1.05 0.85 1.04 0.86 0.6

105-108

105-112

113-120

0.88 (1) 1.02 (1)

(1) (1) (2) (3)

( 1) (1) (1) (1) (1) (1) (!)

* After Dowex-50 chromatography.

T-12 e had the composition of a combination of two tetrapeptides which can be isolated from a digest of a normal aminoethylated-~ chain (Val-Leu-Ala-Arg in positions 113 through 116, and Asn-Phe-Gly-Lys in positions 117 through 120) except for the replacement of the arginyl residue by a histidyl residue. Sequential degradation of the T-12 e fragment by the phenylthiohydantoin-dansylation procedure [13, 14] placed the amino acids in sequence as follows: Val-Leu-Ala-His(Asn, Phe, Gly)Lys. Thus, the substitution in this 6 chain variant is a Arg -+ His substitution in a position corresponding to position 116 of the 6 chain and to position 18 of the G-helix. The variant is termed hemoglobin-A2-Coburg after the place of residence of the proposita. Hemoglobin-Coburg is the eighth 6 chain variant of which the structural abnormality has been determined. The other seven are listed in a previous communication in this journal [15]. The substitution in the 6 chain of hemoglobin-A2-Coburg is of some special interest because position 116 of the fl chain of hemoglobin-A is also occupied by a histidyl residue. A similar situation exists for the 6 chain of hemoglobin. A2-Flatbush in which position 22 is occupied by a glutamyl residue (as in the fl chain) instead of an alanyl residue (as in the 6 chain) [4]. Identity of sequence between a 6 chain variant and the normal fl chain suggests "that the gene for the 6 chain variant

382 represents either a r e m n a n t o f the evolution change between fl a n d the n o r m a l 6 chain genes or a b a c k - m u t a t i o n o f the 6 chain gene t o w a r d s the fl chain gene" (modified quote f r o m ref. 4). Pedigree d a t a indicate that the gene for the a b n o r m a l 6 chain is inherited ind e p e n d e n t l y o f the gene for fl-thalassemia. Thus, the 6-A2-Coburg locus in the p r o positus is in trans to the fl-thalassemia d e t e r m i n a n t . The n o r m a l level o f h e m o g l o b i n A2 (2.8 ~o) a n d the similar level o f 2.8 ~ for h e m o g l o b i n - A 2 - C o b u r g are again an indication that the 6 chain genes in cis and in trans to the fl-thalassemia d e t e r m i n a n t c o n t r i b u t e (perhaps equally) to the increased p r o d u c t i o n o f the 6 chain in this condition. Other examples have been discussed before [12]. ACKNOWLEDGEMENTS We t h a n k Dr Rae M a t t h e w s for referring this f a m i l y for investigation. This investigation was s u p p o r t e d in p a r t by U. S. Public H e a l t h Service Research G r a n t s HL-05168 and HL-15158. REFERENCES 1 Betke, K., Marti, H. R. and Schlicht, i. L. (1959) Nature 184, 1877-1878 2 Efremov, G. D., Huisman, T. H. J., Smith, L. L., Wilson, J. B., Kitchens, J. L., Wrightstone, R. N. and Adams, H. R. (1969) J. Biol. Chem. 244, 6t05-6116 3 Ranney, H. M., Jacobs, A. S., Bradley, Jr, T. B. and Cordova, F. A. (1963) Nature 197, 164-166 4 Jones, R. T., Brimhall, B. and Huisman, T. H. J. (1967) J. Biol. Chem. 242, 5141-5145 5 Huisman, T. H. J. and Dozy, A. M. (1965) J. Chromatogr. 19, 160-169 6 Dozy, A. M., Kleihauer, E. F. and Huisman, T. H. J. (1968) J. Chromatogr. 32, 723-727 7 Clegg, J. B., Naughton, M. A. and Weatherall, D. J. (1968) Nature 219, 69-70 8 Raftery, M. A. and Cole, R. D. (1966) J. Biol. Chem. 241, 3457-3461 9 Jones, R. T. (1964) Cold Spring Harbor Symp. Quant. Biol. 29, 297-308 10 Huisman, T. H. J., Wrightstone, R. N., Wilson, J. B., Sehroeder, W. A. and Kendall, A. G. (1972) Arch. Biochem. Biophys. 153, 850-853 11 Schroeder, W. A. and Robberson, B. (1965) Anal. Chem. 37, 1583-1587 12 Huisman, T. H. J. (1972) Adv. Clin. Chem. 15, 149-253 13 Gray, W. R. (1967) in Methods in Enzymology (Hits, C. H. W., ed.), Vol. 11, p. 469, Academic Press, New York 14 Woods, K. R. and Wang, K. T. (1967) Biochim. Biophys. Acta 133, 369-370 15 Sharma, R. S., Harding, D. L., Wong, S. C., Wilson, J. B., Gravely, M. E. and Huisman, T. H. J. (1974) Biochim. Biophys. Acta 359, 233-235