Structural characterization of hemoglobin NSeattle:α2Aβ261Lys→Glu

Structural characterization of hemoglobin NSeattle:α2Aβ261Lys→Glu

278 BIOCHIMICAET BIOPHYSICAACTA BBA 35194 S T R U C T U R A L C H A R A C T E R I Z A T I O N OF H E M O G L O B I N Nseattle: a2 Af1261L:cs+Glu RI...

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278

BIOCHIMICAET BIOPHYSICAACTA

BBA 35194 S T R U C T U R A L C H A R A C T E R I Z A T I O N OF H E M O G L O B I N Nseattle: a2 Af1261L:cs+Glu

RICHARD T. JONES, BERNADINE BRIMHALL, ERNST R. HUEHNS AND ARNO G. MOTULSKY Division of Experimental Medicine and Department of Biochemistry, University of Oregon Medical School, Portland, Oreg. (U.S.A.), Medical Research Council Group in Haemolytic Anaemias, University College Hospital Medical School, London (Great Britain) and Division of Medical Genetics, Department of Medicine, School of Medicine, University of Washington, Seattle, Wash.

(U.S.A.)

(Received October 3oth, i967)

SUMMARY

Hemoglobin Nseattle is an abnormal human hemoglobin found in a Seattle man of Negro ancestry. The structure of hemoglobin Nseame was found by chemical analysis to differ from the structure of normal hemoglobin by the substitution of a glutamic acid residue for the lysine residue in position 61 of the fl chain. The structural formula is ~/52 ~ mu

INTRODUCTION

Most abnormal hemoglobins which have been detected differ from normal hmnan hemoglobin by their electrophoretic mobility 1. One group of abnormal hemoglobins, including those designated Hb I and Hb N, have fast mobilities which might result from single amino acid substitutions giving two negative charges per substituted amino acid residue. The purpose of this paper is to describe the chemical characterization of one such hemoglobin which laas been designated as Hb Nseattle. MATERIALS AND METHODS

Isolation of hemoglobin Nsea*tte Hemolysates were prepared from blood obtained from an individual with presence of hemoglobin A, the abnormal hemoglobin was isolated by starch block electrophoresis 2. The purified hemoglobin was converted to globin by removing the heme with cold acid-acetone a.

h e m o g l o b i n Nseattle. Because of the

Starch gel electrophoresis Hemolysates containing Hb Nseattle, Hb JBaltimore (ref. 4), Hb JParis (D. LABIE, personal communication), Hb I (ref. 5), Hb A and Hb F were compared by starch gel electrophoresis at p H 8.6 using a Tris-EDTA buffer system 1. The gels were stained Biochim. Biophys. Acta, 154 (1968) 278-283

279

HEMOGLOBIN NSeattle

with o-tolidine peroxide which detects berne compounds. In the present sample Hb N amounted to 50% of total hemoglobin, while Hb A was 46% and Hb A 2 40/0. Hb F, measured by alkali denaturation, was less than i °/o. The a and/5 chains of Hb Nseattle were separated on a 0. 9 cm x 2o cm column of carboxymethyl cellulose using an ionic gradient from 0.005 to 0.03 M Na2HPO 4 in solutions of 8 M urea and 0.05 M mercaptoethanol at pH 6. 7 according to the procedure of CLEGG, NAUGHTON AND WEATHERALL6. The fl chain was aminoetbylated with ethylenimine to form the S-aminoethyl derivative as previously described6, ~.

Enzymatic hydrolysis The primary enzymatic hydrolysis of the fi chain was carried out with trypsin (Worthington, twice crystallized, salt-free) in a solution buffered with trimethylamine according to the procedure of BAGLIONIs. The abnormal peptide was further hydrolyzed with leucine aminopeptidase for 16 h, at 4 °° using Tris buffer at pH 8- 9.

Fingerprinting Fingerprints were made on a tryptic digest of the globin from Hb Nseatt~e essentially according to the method of BAGLIONI9.

Column chromatography and amino acid analysis The tryptic peptides from the aminoethyl fl chain were separated by automatic column chromatography using the short column (0. 9 cm x 17 cm) Spinco I5A resin of an automatic amino acid analyzer (Spinco Model 12o) and a linear gradient of pyridine-acetic acid developer ~. The peptides were further purified by rechromatography on a column (0. 9 cm X 60 cm) of Aminex AG5oW-X2 (Bio Rad Laboratories, 270-325 mesh). Quantitative amino acid analyses after hydrolysis for 22-70 h at IiO ° in 6 M HC1 were made for each peptide with a Spinco Model 12o amino acid analyzer modified to include 20 mm long path flow cells1°.

Edman degradation Determination of the amino-terminal sequence of the abnormal peptide was carried out by a modification of the Edman phenylthiohydantoin procedure 11. RESULTS AND DISCUSSION

Starch gel electrophoresis (Fig. I) shows that Hb Nseattle migrates close to Hb I, which has two extra negative charges per substituted amino acid residue (Lys--~Glu) as compared to normal Hb A. Hb NSeattle is much faster than either Hb JBaltimore (Gly--~Asp) or Hb JParis (Gln--~Glu), which have only one extra negative charge. The fingerprint tracing (Fig. 2) from a tryptic hydrolysate of the globin from Hb Nseatue indicates the virtual absence of the normal peptides fiT-6 and fiT-7 while two new peptide spots were seen. Identification of the new zone from the fingerprint was not attempted since sufficient material was available to isolate it from the tryptic digest by column chromatography. The tryptic hydrolysate prepared from 15 mg of aminoethyl fl chain from Hb Nseattle, upon chromatography through Spinco I5A resin, gave the peptide pattern shown in Fig. 3, top curve. When compared to the pattern for normal fl chain from Biochim. Biophys. Acta, 154 (1968) 278-283

2~0

R. T. JONES, B. BRIMHALL, E. R. HUEHNS, A. G. MOTULSKY

÷

*

d

~

C~C2>

i

o

iii~A o

'\

3

(~0 o

~2

O

_I+

Fig. I. Starch gel electrophoresis in a T r i s - E D T A borate buffer (pH 8.6). i, H b JBultimore; ii, H b Jparis; iii, H b Nseatt]e; iv, H b I. Fig. 2. Tracing of fingerprint of H b NSeattle. Electrophoresis in p y r i d i n e - w a t e r - a c e t i c acid buffer (pH 6.4), followed b y c h r o m a t o g r a p h y at right angles using isoamyl a l c o h o l - p y r i d i n e w a t e r (35:35 : 27, v/v/v), i and 2 indicate t h e positions of the a b s e n t n o r m a l peptides fiAT- 7 and flAT-6, respectively, while 3 and 4 indicate t h e new a b n o r m a l peptides; 3 is presumably/~NT-6 + 7 + 8, which was only p r e s e n t in low yield, while 4 is/~NT-6 + 7.

Hb A (Fig. 3, bottom curve), it is evident that a new peak has emerged at the position indicated by the cross-hatched zone and that two other zones are absent from their normal positions where peptides/3T-6 and/~T- 7 are usually eluted. The abnormal peptide from this new peak, after rechromatography on Aminex AG5oW-X2 resin and hydrolysis in 6 M HC1, gave the amino acid composition listed for Zone XV of Table I. In the next column of Table I are given the corresponding figures for the normal peptides, fiT-6 plus fiT-7. The two analyses differ only by the presence of one glutamic acid residue and the absence of one lysine residue in the peptide from Hb NseattleSince amino acid analysis on acid-hydrolyzed peptides does not distinguish between glutamic acid and glutamine, a portion of abnormal peptide was hydrolyzed with leucine aminopeptidase, which does not deaminate glutamine. Subsequent amino acid analysis showed that the substituent was indeed glutamic acid.

~.~

~I

HB

it/l

i, ~,, o.t, )v ~ U ~ ~0.0

NSEATTLE

z ~~

VV ~

~

~ ,0

: ~

:~':°

~

I~

~

~

~

~, ~ ~

~

~

~

~u~

~

0.~ 02 0.1 0.0

I

~

50

I00

~

" ~50

---

[

200

0

250 EFFLUENT

~

I

300

350

~

I

400

~

~

~

I

450

(mr)

Fig. 3. A u t o m a t i c c h r o m a t o g r a m of t r y p t i c peptides of a m i n o e t h y ] a t e d ~ chains of H b mse~ti1 e (Lop curve) and H b A ( b o t t o m curve).

Biochim. Bioph3,s. Acta. 154 (1968) 278-283

4

I

0.97

0.76

1.o8

0.95 i.ii

1 0.95

fiT-2 VII

1.21 1.92 0.87

2.19 i.oi 0.07

1.o6

0.86

i.o 3

fiT-; XI**

1-o5

3.02 i.o2 3.02

1.9o

0.97 1.98

fiT- 3 ii

OF TRYPTIC

1.84 o. 8 9

1.89

0.95 o.io 1.16 1.o2 o.io

I .oo

I

fiT- 4 V1

PEPTIDES

2.82

i.io

3.14 i .02 1.95 1.o9 1.94 2.24 i .o6 I.OO o. 7 °

0.99

flI'- 5 I

o.12

1.o 7 i.o 4 0.92

o.99

o.18

o.14

0.95

1.o2

fiT-6, 7 XV

fl CHAIN

OF lib

i i I

o

I

2

i.oo

fiT-6 + flT-8 T- 7 XI

Normal

OF AMINOETHYLATED

i .03

0.97

1.02

1.o2

2.09

o. 13 4.12

0.09 1.o2 o.91 o.15

1.04 1.o3 1.99

2.97

1-85

1.o 5

0.09

1.o2

o.58

IV'***

flT-II flT-I2a XII

0.97

1.o 3 0.98

1.o 3 i .87 0.94 0.98

o.84 0.99

i.oi

flT-zo Xlll

2.12 2.06 0.62

0.96

3.08

i .o6

0.95

fiT- 9 V

NSe~ttle *

0-99

0.23 O.94

1.17 i.oo 0.75

2.11 o.13

I.O 3

I ,O2

I ,OO

1.93 0.08 2.05 0.99

3.00

1.04

0.98

flT-I2b flT-I 3 XVII*** 1i

O,IO I.e6

1.o 7 3.88 2.76

0.93

I.OO

I.O 5

FI

0.94

O.17

o.12

0.0 7

O.12

I.O6

XFI

flT-I4 fiT-IS

* R a t i o o f a m i n o a c i d s r e c o v e r e d follo~ving h y d r o l y s i s in a v a c u u m w i t h 6 M H C 1 f o r 22 h a t 1 1 o °. C y s t e i n e w a s m e a s u r e d a s S - a m i n o e t h y l cysteine which emerges between lysine and histidine on the short column of the amino acid analyzer. ** R o m a n n u m e r a l s d e s i g n a t e z o n e f r o m c h r o m a t o g r a m s b o w n in F i g . 3. ( T h o s e z o n e s w h i c h c o n t a i n e d m o r e t h a n o n e p e p t i d e w e r e r e c h r o m a t o g r a p h e d u n d e r d i f f e r e n t c o n d i t i o n s a s n o t e d in t e x t . } *** T h e a m i n o e t h y l c y s t e i n e r e s i d u e in f l T - I 2 s e r v e s a s a s u b s t r a t e f o r t r y p s i n w h i c h r e s u l t s i n t h e c l e a v a g e o f t h i s t r y p t i c p e p t i d e i n t o t w o p e p t i d e s d e s i g n a t e d f l T - ~ 2 a a n d f l T - 1 2 b a c c o r d i n g t o t h e i r p o s i t i o n r e l a t i v e t o t h e N H e - t e r m i n a l e n d o f t h e fl c h a i n . § Tryptophan detected to be present but not measured quantitatively.

Tryptophan§ Lysine S-Aminoethylcysteine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine YMine Methionine Isoleucine Leucine Tyrosine Phenylalanine

A mino acid

AMINO ACID COMPOSITION

TABLE

o

o

2~2

R . T . JONES, B. BRIMHALL, E. R. HUEHNS, A. G. MOTULSKY

A two-step Edman degradation of the N-terminal end of the abnormal peptide showed the sequence Val-Glu .... , as borne out by amino acid analyses of the peptide remaining after each step: Before Edman: After ist Edman After 2nd Edman

Val o.92 Val o.io

Glu o.99 Glu 0.93

Ala 1.o4 Ala 1.o2

(His, Gly, Lys)

Val o

Glu o.24

Ala 0.98

(His, Gly, Lys)

(His, Gly, Lys)

The remaining fi chain tryptic peptides from Hb Nseatue, which did not deviate from their normal positions on the fingerprint or the column chromatogram, were rechromatographed and subjected to amino acid analyses. The results (Table I) are in agreement with those from normal fl chains. Since approx. 15% of peptide fiT-8, 9 remained with peptide fiT-4 after rechromatography, this amount was subtracted to give the values for the latter reported in the table. Therefore, the lysine in position 61 of the/3 chain of Hb N s e ~ t t l e is replaced by a glutamic acid residue, resulting in an electrophoretic mobility compatible with two extra negative charges over that of Hb A, and making position 61 resistant to cleavage by trypsin. SHIBATA et al. 12 has reported the substitution of the 61 lysine by an asparagine residue in Hb Hikari. The position fi-6I corresponds to position fl-E5, the fifth amino acid of the E helix of the/3 chain of hemoglobin is. Comparison of the amino acid sequences of various normal hemoglobins and myoglobin by these workers shows that this position is consistently occupied by a basic residue 14. It might, therefore, be supposed that the presence of an acidic residue here might impair the stability of function of the molecule. As the sample was obtained from a normal blood donor, clearly there can be no gross deleterious effect. However, before this can be fully excluded detailed hematological studies and examination of the hemoglobin are necessary.

ACKNOWLEDGEMENTS

We thank Miss MARIE DUERST and Mr. RICHARD WRIGHT for their technical assistance. Dr. ROBERT L. HILL kindly supplied the leucine aminopeptidase enzyme. This investigation was supported in part by U.S. Public Health Service Research Grant CA 07941. REFERENCES 1 2 3 4 5 6 7 8

E. [{. ]-{UEHNS AND E. M. SHOOTER, J. 2VIed. Genet., 2 (1965) 1. H. G. •UNKEL, Methods of Biochemical Analysis, Vol. i, I n t e r s c i e n c e , N e w York, 1959, p. 141. 3/I. L. ANSON AND /~. E. MIRSKY, J. Gen. Physiol., 13 (193 o) 469. C. BAGLIONI AND D. J. WEATHERALL, Biochim. Biophys. Acta, 7 (1963) 637. D. BEAL]~ AND H. LEHMANN, Nature, 207 (1965) 259. J. B. CLEGG, ~X¢~.A. NAUGHTON AND D. J. WEATHERALL, Nature, 207 (1965) 945. 1~. T. JONES, Cold Spring Harbor Syrnp. Quant. Biol., 29 (1964) 297. C. BAGLIONI, Biochim. Biophys. Acta, 97 (1965) 37-

Biochim. Bioph3Js. Acta t54 (1968) 278-283

HEMOGLOBIN N s e a t t l e 9 io II 12 13 14

283

C. BAGLIONI, Biochim. Biophys. Acta, 48 (1961) 392. R. T. JoNEs ANn G. WEISS, Anal. Biochem., 9 (1964) 377. W. KONIGSBERG AND R. J. HILL, J. Biol. Chem., 9 (1964) 377. S. SHIBATA, T. MIYAJI, I. UICHI, S. URI)A ANI) I. TAKEDA, Clin. Chim. Acta, io (1964) IOi. M. F. PERUTZ, J. Mol. Biol., 13 (1965) 646. M. F. PERUTZ, J. C. KENDREW AND H. C. WATSON, J. Mol. Biol., 13 (1965) 669.

Biochim. Biophys. Acta, 154 (i968) 278-283