The heterogeneity of the fetal hemoglobin of the goat

BIOCHIMICAET BIOPHYSICAACTA

367

BBA 35379 T H E H E T E R O G E N E I T Y OF T H E F E T A L H E M O G L O B I N OF T H E GOAT

J. B. W I I , S O N , H. R. A D A M S AND T. H. J. H U I S M A N

Division of Protein Chemistry, Medical College of Georgia, and Veterans Administration Hospital, Augusta, Ga. 30902 (U.S.A.) (Received J a n u a r y 6th, 1969)

SUMMARY

The heterogeneity of the goat fetal hemoglobin, which has been observed by measurements of the rate of denaturation in an alkaline medium, was reinvestigated. Analyses of the amino acid compositions of various peptides isolated from a tryptic digest of the y chain failed to demonstrate a molecular heterogeneity. Alkali-induced denaturation rates of red cell hemolysates from normal newborn goats, and from newborn goats with a heterozygosity or homozygosity for the HblaB allele, indicated the presence of two distinct components in each of these hemolysates. Similar experiments with isolated fetal hemoglobins containing either lab or II(l polypeptide chains gave distinctly different denaturation curves, which obeyed first order kinetics. It was concluded that the presence of two structurally different a chains, which are the products of non-allelic structural genes, is responsible for the observed phenomenon ; the differences in rate of alkali-induced denaturation apparently result from differences in the stability of the IIct~ and Ia~d dimer subunits.

INTRODUCTION

The possible existence of two fetal hemoglobin components in the domestic goat was reported in I957 (refs. i, 2). The evidence presented was based upon the observation that electrophoretically homogeneous fetal hemoglobin was heterogeneous with respect to its rate of denaturation in an alkaline medium. In this study this heterogeneity has been reinvestigated. First, the possibility of the existence of more than one y chain, similarly as recently observed for human fetal hemoglobin (ref. 3), was studied b y determining the amino acid compositions of the peptides from a tryptic digest of this chain. Secondly, the possibility that the presence of two distinct a chains, the Ia and IIt~ chains, being the products of two closely linked non-allelic a chain structural genes 4-6, was responsible for the observed heterogeneity, was studied b y analyzing the alkali denaturation rates of total red cell hemolysates and of isolated fetal hemoglobins. Biochim. Biophys. Aeta, 181 (1969) 367-372

368

J.B. WILSON et al.

MATERIALS AND METHODS

Animals Blood from one I5-day-old kid (No. 2o3-AA) with an apparent homozygosity for the Hbla and the Hblla genes was used in most analyses. Three new-horns, No. 2oI-AA with a homozygosity for the Hbla and HblIa genes, No. Io3-AB with a heterozygosity for the HblaB allele 4-6 and a homozygosity for the Hblia gene, and No. I25-BB with a homozygosity for the HblaB and the HblIa genes, were also available. Goat lO3 exhibited two fetal hemoglobins, HbF/B or Ia2By2 and HbF/A or a mixture of la2y 2 and Iia2},2, while the two fetal hemoglobins of goat I25-BB contained either lab or IIa chains 7.

Electrophoretic and chromatographic analyses Starch gel electrophoresis of red cell hemolysates was performed as described s. The hemoglobins were also separated by DEAE-Sephadex chromatographyg,l°; a description of this system is presented in previous communicationsT, ~1.

Structural analyses The isolated fetal hemoglobin from goat 2o3-AA was converted into globin by an acid acetone procedure a2. Separation of the y chain and the two a chains was made by a modification of the chromatographic procedure of CLEGG et al. 1~, as described before 7. Quantitative amino acid analyses of the total Y chain were made with a Spinco model 12oB amino acid analyzer as described previously 5. The ~ chain was also aminoethylated with ethylenimine, and next hydrolyzed with trypsin at room temperature for 2 h at pH 9.0. The initial separation of the tryptic peptides was made by Dowex 5o-X2 chromatography, and the separated zones were rechromatographed on columns of Dowex 1-X2 (refs. 14, 15). In one instance repurification of a Dowex I-X2 zone was required; this was achieved by rechromatography on a 0. 9 cm x 50 cm column of Arninex AG5oW-X2 resin with a linear gradient of pyridineacetic acid developer 16. Quantitative amino acid analyses were made after hydrolysis of each purified peptide for 24 h at i i o ° under reduced pressure with 6 M HC1. The presence of tryptophan was established by spot tests on paper 17. The similarity of the total amino acid compositions of the different peptides with those of the y chain of sheep TM was the basis of their arrangement (T-I, T-2, etc.) corresponding to the sequence starting at the N-terminal end.

Alkali denaturation procedures Two procedures were used. The method described by JoNxls AND VISSER19, and referred to as Method I, was employed when total red cell hemolysates were analyzed. The final concentration of NaOH varied between 0.03 and 0.06 M and that of oxyhemoglobin between 0.8 and 1.2 mg/ml. This technique was not suitable for the determination of the rate of denaturation of isolated hemoglobin fractions because of the presence of notable quantities of cyanferrihemoglobin in these preparations. A modification of the technique of BETKE et al. ~°, referred to as Method 2, was developed. The isolated hemoglobin fraction was first converted into the cyanferrihemoglobin derivative by addition of a small amount of solution containing 200 mg KaFe(CN)6 and 200 nag KCN in I 1, and dialyzed for a few hours against distilled

Biochim. Biophys. Acta, 181 (1969) 367-372

369

FETAL GOAT HEMOGLOBIN

water at 4 °. The cyanferrihemoglobin solution was next diluted with distilled water so that the absorbance in a I.o-cm cuvette at 414 m# (Zeiss spectrophotometer) fell around I.ooo i 0-050 (approx. o.15 mg hemoglobin/ml), o.I ml of an 0. 5 or I.O M N a O H solution was added to 1.9 ml of the hemoglobin solution to give a final concentration of 0.025 and 0.o5 M, whereaffer the decrease in absorbance at 414 m/z was recorded at 2 rain intervals. The percentages of undenatured hemoglobin were calculated from these values and from that obtained after heating the sample in a waterb a t h at 37 ° for IO rain in the same way as described in Method I. The absorbance reading of a dilution of 1. 9 ml hemoglobin solution with o.I ml distilled water served as lOO% value. RESULTS AND DISCUSSION

Structural studies of the ~ chain of fetal hemoglobin When data on the amino acid composition of the isolated ~ chain (not presented in detail here) were compared with those of sheep y chain TM, a close similarity between the two proteins was observed; the only differences were the presence of I isoleucyl residue and that of 21 leucyl residues as compared to 2 and 20 in sheep V chain, respectively. Thirteen major zones were obtained by chromatography of the tryptic peptides from the aminoethylated V chain over Dowex 5o-X2; these zones were rechromatographed over Dowex I-X2 for further purification. The results of the amino acid analyses of these peptides are presented in Table I. All peptides were observed with the exception of the T-I2a peptide. The amino acid analyses of the individual peptides yielded amino acid residues in rather distinct molar ratios indicating an acceptable purity; exceptions are the valyl contents of the T- 4 and T-I 4 peptides which were rather low in the 24 h hydrolysates due to the presence of Val-Val peptide bonds. Some of the peptides (T- 3 ; T-5 ; T-9a) were observed in more than one zone of the original Dowex-5o chromatogram; amino acid analyses of these peptides yielded identical compositions. When compared with the amino acid compositions of the tryptic peptides of the sheep V chain TM five distinct differences were observed: peptide T-I contained I seryl residue (threonyl in the sheep ~T-I) ; peptide T-2 contained no isoleucyl residue (one in the sheep yT-2) and I extra leucyl residue; peptide T-3 contained 3 glycyl and I alanyl residue (2 glycyl and 2 alanyl residues in sheep vT-3) ; peptide T- 7 contained I glycyl and I alanyl residue (2 glycyl residues in sheep yT-7) ; and peptide T-9a contained I seryl and I threonyl residue (2 seryl residues in sheep yT-9a ). These differences (Ser-Thr in T-I ; Leu-Ile in T-2 ; Gly Ala in T-3 ; Ala-Gly in T-7; Thr-Ser in T-9a ) correspond remarkably well with the differences observed between the total amino acid compositions of the V chains of goat and sheep. The amino acid compositions of the peptides T-I and T- 7 are identical to those of the corresponding peptides of the bovine y chain 21. The results of these analyses, although not conclusive, do not lend support to the hypothesis of a chemical heterogeneity of the y chain since none of the peptides showed an amino acid composition which indicated the presence of a mixture of two closely related peptides. Consequently, goat fetal hemoglobin probably contains only one type of ), chain. This ), chain apparently closely resembles that of sheep fetal hemoglobin for which also no evidence of a molecular heterogeneity was observed 18. Biochim. Biophys. Acta, 181 (1969) 367-372

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The alkali denaturation rates orfetal hemoglobincomponents Representative data are given in Fig. i. Two distinct denaturation rates were observed when red cell hemolysate from goat 2oI-AA was analyzed in 0.04 and 0.05 M N a O H (Fig. IA). The curves obtained for hemolysates from goats Io3-AB and I25-BB in 0.05 M NaOH are comparable with that for hemolysate from goat 2oI-AA (Fig. IB). This indicates that the replacement of the Ia chain in goat fetal hemoglobin by the lab chain, which differs from the Ia chain by substitution of an aspartyl residue in position 75 by a tyrosyl residue 5, does not change its alkali denaturation properties. Io0

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Fig. i. Alkali-induced d e n a t u r a t i o n curves of the red cell h e m o l y s a t e s of n e w - b o r n goats and of isolated hemoglobin components. Temp., 22 °. A and C. H e m o l y s a t e from goat 2oI-AA. B. H e m o lysates f r o m goats 2oI-AA, Io3-AB and I25-BB. D. The hemoglobins F / B and F / A isolated from the red cell h e m o l y s a t e of goat I25-BB. The Methods i and 2 are described in section MATERIALS .~NO METHODS. Figures indicated at curves: N a O H (M).

A similar type of curve was obtained for fetal cyanferrihemoglobin when the denaturation was allowed to proceed at a lower NaOH normality (Fig. IC). Fig. I D presents the alkali denaturation 'curves of H b F / B (or Ia2B72) and of HbF/A (or Iia272), which were isolated from red cell hemolysate of goat I25-BB. The alkaliinduced denaturation curves in 0.05 and 0.025 M N a O H of both types of fetal hemoglobin obey first order kinetics. A distinct difference in the curves was observed indicating that the rate of denaturation of the IIa chain containing fetal hemoglobin is faster than that of the laB chain containing fetal hemoglobin. The two distinct a chains in the goat, which are considered to be the products of two non-allelic closely linked structural genes, differ in at least four positions, namely 19, 26, 113 and 115; the amino acid residues in these p6sitions are Gly, Ala, Leu, Asn in the Ia chain, and Ser, Thr, His, Ser in the IIa chain, respectively 4-6. The close identity of the alkali-induced denaturation curves of total hemolysates from AA, AB, and BB newborn goats suggests the presence of two distinct components in Biochim. Biophys. Acta, 181 (1969) 367-372

372

j . B . WILSON et al.

each of these w h i c h d e n a t u r e at slightly different rates. The appreciable differences in t h e t r a t e o f d e n a t u r a t i o n o f t h e i s o l a t e d f e t a l h e m o g l o b i n s w i t h e i t h e r IIc~ or I(IB c h a i n s s e e m t o offer a s a t i s f a c t o r y e x p l a n a t i o n f o r t h e m o d e o f d e n a t u r a t i o n o f t h e t o t a l h e m o l y s a t e s . E x p o s u r e o f d i l u t e h e m o g l o b i n s o l u t i o n s t o a p H o f 12 a n d h i g h e r r e s u l t s in a d i s s o c i a t i o n o f t h e d i m e r s u b u n i t s o f t h e m o l e c u l e t o m o n o m e r s 22-~6. R e c e n t s t u d i e s b y GOTTLEB et al. 24 i n d i c a t e t h a t d i f f e r e n c e s in t h e r a t e o f alkalii n d u c e d d e n a t u r a t i o n l i k e l y r e s u l t f r o m d i f f e r e n c e s in t h e alkali s t a b i l i t y o f s y m m e t r i c d i m e r s u b u n i t s . I t s e e m s , t h e r e f o r e , t h a t t h e Ilay d i m e r is less s t a b l e in an a l k a l i n e m e d i u m t h a n t h e l a b 7 ( a n d p o s s i b l y t h e IaT) d i m e r s u b u n i t . A p p a r e n t l y o n e (or p e r h a p s m o r e t h a n one) o f t h e f o u r d i f f e r e n c e s in t h e p r i m a r y s t r u c t u r e s o f t h e s e t w o a c h a i n s is r e s p o n s i b l e f o r t h e o b s e r v e d p h e n o m e n o n . ACKNOWLEDGEMENTS T h i s s t u d y w a s s u p p o r t e d b y U.S. P u b l i c H e a l t h S e r v i c e g r a n t H E - o 5 1 6 8 . T h e r e p o r t c o n t a i n s m a t e r i a l t o b e u t i l i z e d in p a r t i a l f u l f i l l m e n t o f t h e P h . D . r e q u i r e m e n t s for Mr. H. R. A d a m s . REFERENCES i T. H. J. HUISMAN, H. K. A. VISSER AND H. J. VAN DER HELM, Nature, i8o (1957) 758. 2 H. K. A. VISSER, T. H. J. HUISMAN AND M. G. WOLDRING, Blood, 12 (1957) lOO4. 3 v~7" n. SCHROEDER, T . H . J .

HUISMAN, J . R . SHELTON, J . B . SHELTON, E . F . KLEIHAUER

A. M. 4 T. H. 5 T. H. 6 M. D. 7

8 9 IO Ii ~2 13

14 15

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22 23 24 25 26

DozY AND B. ROBBERSON, Proc. Natl. Acad. Sci. U.S., 60 (1968) 537. J. HUISMAN, J. B. WILSON AND H. R. ADAMS, Arch. Biochem. Biophys., 121 (1967) 529. J. HUISMAN, G. BRANDT AND J. B. WILSON, J. Biol. Chem. ,243 (1968) 3675 . GARRICK AND T. H. J. HUISMAN, Biochim. Biophys. Acta, 168 (1968) 585 . H. R. ADAMS. R . N . WRIGHTSTONE A. MILLER AND T . g . J . HUISMAN, Arch. Biochem. Biophys., in the press. T. H. J. HUISMAN, Advan. Clin. Chem., 6 (1963) 231. T. H. J. HUISMAN AND A. M. DOZY, J. Chromatog., 19 (1965) 16o. A. M. DozY, E. F. KLEIHAUER AND T. H. J. HUISMAN, J. Chromatog., 32 (1968) 723 . T. H. J. HUISMAN, J. P. LEWIS, M. H. BLUNT, H. R. ADAMS, A. MILLER, A. M. DozY AND E. M. BOYD, Pediatric Res., in the press. M. L. ANSON AND A. E. MIRSKY, J. Gen. Physiol., 13 (193 o) 469. J. B. CLEGG, M. A. NAUGHTON AND D. J. WEATHERALL, J. ]~Iol. Biol., 19 (1966) 91. \¥. A. SCHROEDER, R. T. JONES, J. CORMICK AND V. MCCALLA, Anal. Chem., 34 (1962) 157°. ~/V. A. SCHROBDER AND B. ROBBERSON, Anal. Chem., 37 (1965) 1583. R. T. JONES, Cold Spring Harbor Symp. Quant. Biol., 29 (1964) 297. R. C. BALDRIDGE AND H. B. LEWIS, dr. Biol. Chem., 202 (1953) 169. J. B. WILSON, W. C. EDWARDS, M. MCDANIEL, M. M. DOBBS AND T. H. J. HUISMAN, Arch. Biochem. Biophys., 115 (1966) 385. J. H. P. JONXIS AND H. K. A. VISSER, A.M.A.J. Diseases Children, 92 (1956) 588. i~. BETKE, H. R. MARTI AND I. SCHICHT, Nature, 184 (1959) 1977. D. R. BABIN, W. A. SCHROI~DER, J. R. SHELTON, J. B. SHELTON AND B. ROBBERSON, Biochemistry, 5 (1966) 1297H. A. ITANO AND S. J. SINGER, Proc. Natl. Acad. Sci. U.S., 44 (1958) 522. S. J. SINGER AND H. A. ITANO, Proc. Natl. Acad. Sci. U.S., 45 (1959) 174. A. J. GOTTLEB, E. A. ROBINSON AND H. A. ITANO, Arch. Biochem. Biophys., 118 (1967) 693. 17. HASSERODT AND J. VINOGRAD, Proc. Natl. Acad. Sci. U.S., 45 (1959) 12. J. VINOGRAD AND W. D. HUTCHINSON, Nature, 187 (196o) 216.

Biochim. Biophys. Acta, 181 (1969) 367-372