Structural studies on the major and minor haemoglobin of the monkey Macaca irus

45 °

BIOCHIMICAET BIOPHYSICAACTA

BBA 35682 S T R U C T U R A L S T U D I E S ON T H E MAJOR AND MINOR H A E M O G L O B I N OF T H E MONKEY M A C A C A I R U S

PATRICIA T. WADE*, N. A. BARNICOT AND E. R. HUEHNS*"

Department of Anthropology, University College London, Gower St. London W.C. I, and **Department of Clinical Haematology, University College Hospital Medical School, University Street, London WC J: (Great Britain) (Received June 8th, 197 o)

SUMMARY

The amino acid compositions of the tryptic peptides of both chains (a Ami and f3TM) of the normal major haemoglobin of Macaca irus (Hb-A mi) have been analysed (excluding aT I2B and aT 13). The most probable sequences of these chains have been constructed by comparison with the a- and//-chain sequences of human Hb-A and Macaca mulatta Hb-A. A minor component (a2Xmifl2Ami) is found as a polymorphism in M. irus. Amino acid analysis of the soluble tryptic peptides of the axmi-chain has elucidated the positions of four amino acid differences between this chain and the aAmi-chain.

INTRODUCTION

The Old World monkey Macaca irus (fascicularis) has a major haemoglobin with similar electrophoretic mobility to human Hb-A. This haemoglobin has been designated Hb-A mi (ref. I). About 20% of the monkeys also have a minor haemoglobin comparable in concentration and electrophoretic mobility to human Hb-A 2 (ref. I). Two types of minor haemoglobin have been detected. One (Hb-X mi) has a slightly more cathodal mobility than human Hb-Ae on starch gel electrophoresis at pH 8.6 (see Fig. i). The other has the same mobility as Hb-A 2 but is usually in lower concentration than H b - X mi. We have demonstrated previously that H b - X mi has a different a-chain (a TM) from Hb-A mi and also that the a xnli- and aAmi-chains are the products of non-allelic genes1, 2. I t was therefore suggested that the minor component in M. irus arose by duplication of the a-locus in a manner analogous to the//-locus duplication proposed for the origin of the human &chain a. In order to obtain further information about the nature and origin of the a-locus duplication further structural data on the (/Ami_, //Ami_, and axmi-chains has been obtained and is presented here. * Present address: Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, Wash. 981o5, U.S.A.

Biochim. Biophys. Acta, 221 (197 o) 450-466

HAEMOGLOBINS OF ~ r . ires

451

Fig. I. Comparison of M . irus haemoglobins with h u m a n haemoglobins. Starch gel electrophoresis in Tris E D T A - b o r a t e (pH 8.6)6; h a e m stain. (a) H u m a n haemolysate, H b - A and Hb-A 2. (b) M . irus haemolysate, Hb-A mi and H b - X mi.

METHODS

The major haemoglobin (A mi) and tile minor haemoglobin (X mi) were purified from two M. irus individuals in which the minor haemoglobin comprised 4 % of the total haemoglobin. Haemolysates from these animals were freed of carbonic anhydrase by chromatography on CM-Sephadex 4 and H b - X mi was separated from Hb-A mi by further chromatography on DEAE-Sephadex 5. Each component was pure as judged by starch gel electrophoresis p H 8.6 (ref. 6). Some of the Hb-A mi used for structural analyses was prepared from animals having both Hb-A mj and the major haemoglobin variant Hb-Q mi (ref. I). The Hb-A mi was purified by starch block electrophoresis first in 0.05 M barbiturate buffer (pH 8.6), and then in 0.04 M phosphate buffer (pH 7.0) 7. Pooling of material from more than one individual was avoided throughout the course of this work. Globin was prepared from the purified haemoglobins by precipitation in acid acetone at --20 ° (ref. 8). The a- and fl-chains of the major and minor globins were separated by chromatography on CM-cellulose in 8 M urea and then aminoethylated 9. Separated chains and whole globin were digested with trypsin and fingerprinted 6. Fingerprints were stained for tryptophan, histidine, arginine and tyrosine 1°. Peptides for amino acid analysis were purified by one of two different fingerprinting methods6, 9. The tryptic peptides were eluted from the paper with 6. 7 M HC1 and hydrolysed under reduced pressure at lO7 ° for 17 h unless otherwise stated. The hydrolysates were analysed on an amino acid analyser using a single column system 1~,42. The analyser was built in University College Hospital Medical School and incorporated 2o-mm lightpath cells. Asparagine and glutamine were estimated as aspartic acid and glutamic acid, respectively. The peptides which contained methionine were seen Biochim. Biophys. Acta, 221 (197 o) 450-466

ox

%

o

4

-b

(a)

d~b

s -e

- electrophoresis

origin

- etectrOp horesi

8,9

4

o

o

b

g> O

O

@

(d)

@

Oo×Y

~ Ami ~ c h a i n

o( x m ' - c h a i n

(b)

C3

I origin

origin _ etetrophoresis

d?®

@

+

o

L_ r~ 0 +0

2

O@

- electrophoresis

8+9

Fig. 2. F i n g e r p r i n t maps of M . i r u s a m i n o e t h y l a t c d a- and fl-chains. (a) /~Ami-chain. (b) and (c) aami-chain. (d)aXmi-chain. The fingerprinting s y s t e m for (a) and (b) was electrophoresis in p y r i d i n e - a c e t i c acid (pH 6.4) followed b y c h r o m a t o g r a p h y in p y r i d i n e - i s o a m y l alcohol w a t e r (35:35:27, b y vol.), and for (c) and (d) electrophorcsis in pyridine acetic acid (pH 4.7) followed by c h r o m a t o g r a p h y in n - b u t a n o l - a c e t i c a c i d - w a t e r pyridine (15:3:12:1o, b y vol.). The t r y p t i c peptides were labelled b y comparison with hunlan a- a n d fl-chain peptides6, 9. Additional p e p t i d e s are labelled alphabetically.

~)oxy

/~ Am' -chain

O

(C) 0 ~ A m J - c h a i n

O

o

12a

o L u

>

~J

4a tO

I

0

~a

.~

~"

~ ~.~

ACID

Asp Thr Set Glu Pro Gly Ala Val Leu Tyr Phe His Lys Arg Met Cys Trp

Amino acid

but the amount

was not estimated.

i. i 0.9

0.9 i .o

2.3 I. I

0.8

+

I,O

i.i I.O I.I 0.9

I.O 1.8

0.9

3.0 1.2 2. 7 0.9

2.2

2.I

+

I.O

2.1 1.8 0.7

I.i I.O

0.9

+

I,O

2.8

3.0 1.2 2.2 2.0 o. 9 o. 7 i.o

3.I

I.O

i.o

I.O 1,6

i.o I.O

I.O I.O 1.8

2.1 1.2 i.o 3.8

0.9

4.2

i.o I.O I.I

2.1 1.2 I.i 3.8

0.8

3.8

+

0. 9 I.O I,O

2.0

0. 9 I.I

I.I 0.9 0.8 2.I

+

0. 9 I.O

2.2

I.O I.I

0.9 0.9 2.1

2.1 1.8 1.2 0.9 1. 9 o, 9 1, 9

5.2 2.1

2.0 I.I

+

1.8 3.2

1.2

0. 9

2.o

i.i

0.8

Y = f i t i i -- f i T i 2 a Z = f i T i o a 1:3 [ST I 2 a i

fl-CltAIN TRYPTIC P E P T I D E S OF 1"~I. i~'Us H b - A

fiT I f i t 2 f i t 3 fiT 4 fiT 5 fiT 6 f i t 7 + a fiT a + 9 f i t 9 f i t zo

Peptide :

OF THE

acid was present

COMPOSITIONS

indicates the amino

AMINO

+

I

TABLE

+

1.9 i.i

i.o 1.8 I.I

1.2 1.2 0.8 0.8

I.i

0. 7 0. 9

1. 7 1,2

4.0 1.2

0.9

I.O I.O

I.i 3.8 3.0 i.o

I.O

1.2

0.8

X = f i t i2b f i t 1 3 fiT 1 4 fiT 1 5 fit z2aii

~0

42-

0

X

t~ 0

0

m >

4a

?

&

II

Asp Thr Ser Glu Pro Gly Ala Val Leu Zyr Phe His Lys Arg Met Cys Trp

Amino acid

found

between

3l.irus

and

man

iii III

III

III

ii1 iii

iio 221 ooi

iii 112 tli Zli

222 III

III

iii

333 iii 333 iii

222

222

Peptide." flT ± f i T 2 fiT 3 species." I M H I34H I 3 f H

i n f i T 4.

Differences

found

iii

ii1

III

232

212

III iti

tli

III

III

333

333 ooi 332 III 222 222 iii iii tli

III

ii1

Iii III

iit iii

fiT 7 I3IH

iii

fiT8 I3IH

i n f i T 2, f i T 5, f i T 9 ,

fiT 4 f i T 5 f i T 6 I3IH 1MH I3IH

were

ioa,

O F .l/l. fiT ii

(I),

fiT 13. One

iYlgS H b - A and

iii iii III

444

222 112 Iii

IIi

443

III

III III

222

iii Iii

I12 III 22I

III

III

Iii Iii iio ooi

iii itt

Iii IiZ

222

fiT 9 fiT ioa flT rob f i T , , 13IH I3IH IMH IMH

fiT

COMPARISON OF THE AMINO ACID COMPOSITIONS OF THE fl-CHAIN TRYPTIC PEPTIDES

TABLE

3,l.ir*ts

and

(H)

3l.mulatta

(M) AND HUMAN Hb-A

between

Hb-A

was

222

iit

iii

iii

222 iii

iit 222 III

iii iii itt tli

III

Iii Iii

222 iii

443 I12

Iii

II1 III

444 333 iIi

IIl

III

III

iii

fiT z2ai fiT r2aii fil"I2b fiT1:3 r i T z 4 f i T 1 5 I3IH 13IH I3IH I3IH IMH I3IH

difference

5I. mulatta

>

4~ %n

HAEMOGLOBINSOF M. irus

455

mainly as the oxidised peptide on the fingerprints. Methionine was therefore detected by the presence of methionine oxides on amino acid analysis. Cysteine was detected by the presence of cysteic acid and aminoethyl cysteine, and tryptophan was demonstrated by specific staining of the fingerprint. RESULTS

Analysis of the fAtal-chain of the major Hb-Am* A tracing of an aminoethyl fAmi-chain fingerprint is shown in Fig. 2a, and the amino acid composition of tile tryptic peptides is given in Table I. The analyses of f T i, f T 4 and f T 14 shown in Table I were carried out after an 48-h hydrolysis at 112 °, since low values of valine and/or leucine were observed after I7-h hydrolysis at lO7 °. f T II and f T 13 were analysed together as they overlapped one another on the fingerprint, f T 13 was then obtained in pure form from a fingerprint of non-aminoethylated fAmi-ehain in which the core peptides (fT IO, f T i i and f 12) were absent. f T 7 and f T 7 + 8 (fT 7 + lysine) also overlapped one another and were analysed together. Lysine from f T 8 is thus present in non-integral amounts. Analysis of peptide Y (Fig. 2a) showed that it corresponded to f T Ioa, from the N-terminus of f T IO up to and including the aminoethyl cysteine residue. The yield of f T ioa was much less than f T IO. It has been reported 1~ that the corresponding human peptide is not susceptible to tryptic digestion. In contrast trypsin completely splits both the M. irus and human f-chains after aminoethyl cysteine in f T 12, yielding BIT I2a a n d f T I2b. In addition, analysis ofpeptides X and Z ofM. irusf-chain (Fig. 2a) showed that together they comprised f T i2a. Peptide Z consisted of the first four residues of this peptide and peptide X the last four. The chain had presumably been cleaved after asparagine lO8. Chymotryptic activity such as this has been observed previously 13. In Table II, the fAmi-chain analyses are compared with the compositions of the tryptic peptides of the human fA-chain and also the M. mulatta E-chain. The latter is taken from the sequence of M. mulatta Hb-A determined by MATSUDA el al. ~4-18. In f T 2 there are two differences between fAtal_ and the human E-chain. Aspartic acid and threonine were found in M. irus in place of serine and alanine in man. Since the two peptides have the same electrophoretic mobility the aspartic acid found in M. irus f T 2 must be present as asparagine, f T 2 was analysed from two individuals of M. irus of different haemoglobin phenotypes and the results were the same. The composition o f f T 2 in M. irus and M. mulatta is the same. M. irus and man do not differ in f T 4 whereas M. mulatta has one less valine and one more leucine than man. In the case of M. irus, f T 4, the same result was obtained from two individuals of different haemoglobin phenotypes, and it may be that this is a constant difference between these two closely related species. In each of the peptides f T 5, f T 9, f T Ioa, f T II and f T 13 ofM. irus, one amino acid difference from the human E-chain was found, but there were no differences from M. mulat~a. All the tryptic peptides of M. irus have the same electrophoretic mobility as the corresponding human peptides and it is therefore likely that asparagine and glutamine are present in homologous positions. The amino acid compositions of the tryptic peptides of M. irus f A m i - c h a i n Biochim. Biophys. Acta, 22I (197o) 450-466

Leu, Ala, His, Lys, Tyr, His

Arg Lys Glu, Asn, Phe, Lys, Leu, Leu, Gly, Asn, Val, Leu, Val, Cys, Val, Leu, Ala, His, His, Phe, Gly, Lys, 125 Pro Gln Glu, Phe, Thr, Pro, Gin, Val, Gln, Ala, Ala, Tyr, Gln, Lys, Val, Val, Ala, Gly, Val, Ala, Asn, Ala,

zo 4

Leu, Lys, Gly, Thr,

Lys, Ala, His, Gly,

Phe, Phe, Glu, Ser,

Asp, Gtu, Val, Gly,

13 Thr Thr Thr, Leu, Trp, Gly, Lys, Val, Asn, Val, 33 Val Leu Gly, Glu, Ala, Leu, Gly, Arg, Leu, Leu, Val, Val, Tyr, Pro, Trp, Thr, Gln, Arg, 5° Thr Set Phe, G l y , Asp, Leu, Ser, Ser, Pro, Asp, Ala, Val, Met, Gly, Asn, Pro, Lys, Val, 76 Ata Asn Lys, Lys, Val, Leu, Gly, Ala, Phe, Ser, Asp, Gly, Leu, Asn, His, Leu, Asp, Asn, 87 Thr Gln Phe, Ala, Gin, Leu, Ser, Glu, Leu, His, Cys, Asp, Lys, Leu, His, Val, Asp, Pro,

9 Ser Asn Val, His, Leu, Thr, Pro, Glu, Glu, Lys, Asn, Ala, Val, Thr,

Fig. 3- Comparison of 31. i r u s , 31. m u l a t t a and human/J-chain sequences.

Amino acid number Human Hb-A 3 I . m u l a t t a Hb-A M . i r u s Hb-A ~" Amino acid number Human Hb-A M . m u l a t t a Hb-A -~ M . i r u s Hb-A "L" Amino acid number ~D --~ Human Hb-A M . m u l a t t a Hb-A M . i r u s Hb-A Amino acid number Human Hb-A 3,I. m u l a t t a Hb-A M . i r u s Hb-A Amino acid number Human Hb-A ]~I. m u l a t t a Hb-A M . i r u s Hb-A Amino acid nmnber Human Hb-A ~ l . m u l a t t a Hb-A M . i r u s Hb-A Amino acid number Human Hb-A 3 I . m u l a t t a Hb-A M . i r u s Hb-A Human Hb-A 3I. m u l a t t a Hb-A 31. i r u s Hb-A

£

>

¢dl

~n

indicates

Met Cys Trp

Tyr Phe His Lys Arg

Leu

Val

Ala

Thr Ser Glu Pro Gly

Asp

Amino acid

+

0.9

i.i I.o i.I

0.9

0.9

i,i

aT •

Peptide :

i.o

I.O

o. 4 o.4

i,o

•

i.o

I.O

0. 5 0.3

i.o

2

but

+

I.I

1. 9

i.o

aT 3

acid was present

aT 2

the amino

i .o

i.o

0.9

3.i I.O I.O

3.8

3.2

aT 4

the amount

+

i.i

2.0

i.I

i.i

1. 7 0.8

aT 5

0.9 2.0 1.9 i.i

i.o I.I i.i

o.9 1.6 1.2 I.O 1.2

i.o

aT 6

was not estimated.

I.O 2.0

2.0

aT 7 + 8

AMINO ACID COMPOSITIONS OF T H E CL-CHAIN T R Y P T I C P E P T I D E S OF 1~/. iYUS H b - A

TABLE III

+

3.0 1.9

4-9

1.3 6.3 2.9

1.7 1.6 O. 9

I.O

3.9

aT8 9

+

I.O

i.o

aT •o

I.O

0.9

2.0

I.O

2.I

aT •z

+

I.O

0.9

0.8

2.0

I.O

2.2

+

1. 3

1.9

o.8

a T zo + a T I 2 a z•

I. O

+

aT •4

1.9 0. 7

2.I

2.0

I.I

1.2

~I

"-o

©

:Z

0

0

>

458

P.T. WADE et al.

show seven differences from those of the human /SA-chain. However, the M. irus peptides that differ from human have the same amino acid compositions as those in M. mulalta 14 18. Since the fl-chain sequence of M. mulatla Hb-A has been determined 1~-18, it is possible to construct a likely sequence for M. irus if we assume that peptides with the same composition in the two species have the same sequences. M. irus and M. mulatta fl-chains would then differ at only one site, namely fl 33, which is leucine in M. mulatta and valine in M. irus (and man). This proposed sequence of M. irus flAmi-chain is given in Fig. 3. Analysis of the aaron-chain of the major Hb-A m*

Two fingerprinting systems (Figs. 2b and 2c) were used to obtain the a T M peptides. Amino acid analyses of the tryptie peptides are given in Table I I I . The analyses of aT 2 from both fingerprinting systems (Analyses I and 2, Table III) gave half values for threonine and serine. Impurities in the first analysis corresponded to less than o.17 of an amino acid residue, and in the second analysis impurities were less than 0.06 of an amino acid residue. Since the M. irus individual from which tile aT 2 peptide was obtained, had only the one major haemoglobin Hb-A mi, we believe the most likely explanation is that there are two alleles at the locus determining the aAmi-chain, one giving rise to serine in aT 2 and the other giving rise to threonine. On this hyp(~thesis, the animal analysed was heterozygous for these two alleles. An analysis of Hb-A mi aT 2 from an animal which also has the major haemoglobin variant Hb Pmi (ref. I) should yield only serine or threonine if this explanation is correct. The composition of peptide aT 8 + 9 from the pH 6. 4 fingerprint is shown in Table I I I . aT 9 could not be obtained pure from this fingerprint. No peptides corresponding to aT 9 or aT 8 + 9 were found with the pH 4.7 system. Peptide M, Fig. 2c (and peptide B, Fig. 2b) was a low yield of the last part of aT 9, arising from the hydrolysis of the peptide at serine 81. The extreme lability of bonds formed by the amino groups of threonine and serine has been noted previously la. No peptides corresponding to aT I2b or aT 13 were obtained in sufficient quantity for analysis from either the pH 4.7 or the pH 6. 4 fingerprints. In the latter fingerprint (Fig. 2b) peptide A was a small quantity of an oxidised derivative of aT 12a. Peptide L in the pH 4.7 fingerprint (Fig. 2c) contained only serine and glycine. Table IV compares the a-chain peptides of M. irus Hb-A mi, M. mulatta Hb-A and human Hb-A (excluding aT I2b and aT 13). The difference in aT 2 between the three species has already been mentioned. There is one difference in aT 4 between the two monkeys and man, glycine in man being replaced by alanine in the macaques. The composition of M. irus aT 9 given in Table IV is the most probable from the aT 8 + 9 analysis (Table III). The validity of taking glutamic acid as one and not two residues is supported by the analysis of the homologous region of the aXmi-chain of the minor haemoglobin in M. irus (see below). The results show M. irus aT 9 differs from man in two residues (Ash Leu, Ala Gly) which correspond to the changes observed in M. mulatta, and assuming homology of these two changes with those in M. mulatta, there is a third difference (Asp Glu) between M. irus and man in this peptide. This third change is therefore also a difference between the two Macaca species. F(mr of tile five differences between the M. irus aAmi-chain and human a-chain can be assigned to specific positions if it is assumed that peptides with the same amino Biochim. ldiophys. Acta, 221 (197o) 45o-466

o

2

IV

Asp Thr Ser Glu Pro Gly Ala Val Leu Zyr Phe His Lys Arg Met Cys Trp

Amino acid

O F T H E a - C H A I N TRYPTIC P E P T I D E S OF

Peptide: Species:

iii

iii

Iii

iii IiO ooi

aT 2 IMH

iii iii iii

iii

iii

iii

aT i IMH

iii

iii

III 222

aT 3 IHM

III

III

333 444 iii III iii

333

aT 4 IMH

III

Xli

222

iii

iii

222 III

aT 5 IMH

III III iii iii III III iii 222 222 iii iii III

222

III

Iii

333 III

ioo iii IiO 667 333 554

222

aT 9 IMI-I

222

aT 8 IMH 456 iii

aT 7 IMH

III

III

aT zo IMH

iii

III

222

III

222

aT ii IMH

s p e c i e s i n a T 2 a n d a T 9-

iii iii

aT 6 IMH

the two macaque

Iii

iii

222

III

aT i2a IMH

iii

iii

a T 14 IMH

M . i r u s H b - A (I), M . m u l a t t a H b - A (M), AND HUMAN H b - A ( H )

M . i r u s a n d m a n w e r e f o u n d i n a T 2, a T 4 a n d a T 9, a n d b e t w e e n

OF THE AMINO ACID COMPOSITIONS

Differences between

COMPARISON

TABLE

©

Z

0

0

m

Ser,

[Thr/

8 Thr Ser / Ser / Lys, Val, Lys, Ala, Ala, Leu, Gly, Lys, Val, Gly, Arg, His,

14

19 Ala Gly Asn, Val, Lys, Ala, Ala, Trp, Gly, Lys, Val, Gly, Gly, His,

9

Ser, His, Gly, Ser, His, Gly, 71 Ala G1y Ala, Val, Gly, Ala, Val, Arg,

Ser, Ala, Gln, Val, Lys, Gly, His, Gly, Lys, Ser, Ala, Gln, Val, Lys, Gly, His, Gly, Lys, 74 75 78 Asn . A sn His, Val, Asp, Asp, Met, Pro, Gln, Ala, Leu, His, Val, Asp, Asp, Met, Pro, Gln, Ala, Leu,

Tyr, Arg. Tyr, Arg.

l:ig. 4. Comparison of 3I. irus, 31. mulatta and h u m a n a-chain sequences.

* (;111 is tentatively assigned to position a78 in 5I. irus Hb-A and Hb-X. However it is possible t h a t a78 is Asn and position a74 or a75 is Ghl.

Leu, Ser, His, Cys, Leu, Ser, His, Cys,

Ser, Ala, Leu, Ser, Asp, Leu, His, Ala, His, Lys, Leu, Arg, Val, Asp, Pro, Val, Asn, Phe, Lys, Leu, Ser, Ala, Leu, Ser, Asp, Leu, His, Ala, His, Lys, Leu, Arg, Val, Asp, Pro, Val, Ash, Phe, Lys, Leu, lO4 14o

Thr, Tyr, Phe, Pro, His, Phe, Asp, Leu, Thr, Tyr, Phe, Pro, His, Phe, Asp, Leu, 64 68 Asn Le u Lys, Val, Ala, Asp, Ala, Leu, Thr, Leu, Lys, Val, Ala, Asp, Ata, Leu, Thr, Leu, 85

Ala, Gly, Glu, Tyr, Gly, Ala, Glu, AIa, Leu, Glu, Arg. Met, Phe, Leu, Scr, Phe, Pro, Thr, Thr, Lys, Ala, Gly, Glu, Tyr, Glv Ala, Glu, Ala, Leu, Glu, Arg, Met, Phe, Leu, Ser, Phe, Pro, Thr, Thr, Lys,

Val, Leu, Ser, Pro, Ala, Asp, Lys,

M. trus Hb-X

Amino acid number Human Hb-A M . mulatla Hb-A M . irus Hb-A ,© 3~I. irus Hb-X Amino acid number Human Hb-A M . mulatta Hb-A da M . irus Hb-A M . irus H b - X Amino acid number H u m a n Hb-A M . mulatta Hb-A M. irus Hb-A M. irus Hb-X Amino acid number H u m a n Hb-A M . mulatta Hb-A M. irus Hb-A 3I, irus H b - X Amino acid number Human Hb-A M. mulatta Hb-A M. irus Hb-A M. irus Hb-X

Val, Leu, Ser, Pro, Ala, Asp, Lys,

Amino acid number Human Hb-A M . mulatta Hb-A M . lrus Hb-A

H

>

-ix ©

HAEMOGLOBINSOF M. irus

461

acid compositions in M. irus and M. mulatta have the same sequence. The fifth difference, glutamic acid for aspartic acid, or glutamine for asparagine, may be at any one of the five positions : a64, a74 , a75, a78 or a85 in peptide aT 9. Analysis of peptide M (Table III), which comprised residues a82-9o, showed that residue a85 was aspartic acid. Investigation of the axmi-chain of the minor haemoglobin (see below) showed that in this haemoglobin a64 was aspartic acid, although the same Asp-Glu change was present in aT 9. Therefore, by homology, a64 is probably aspartic acid in a T M . We tentatively assign glutamine at position a78 since this site is much more variable than a74 or a75 in other mammalian haemoglobins 19. Glutamine is chosen rather than glutamic acid, since human aT 9 and M. irus aT 9 have the same charge. The probable sequence of M. irus aAmi-chain is shown in Fig. 4Analysis of M. irus minor H b - X m* Fingerprinting of the aminoethyl/~-chain of Hb-X mi, with subsequent staining ff)r tryptophan and histidine revealed no differences from the /~-chain of Hb-A mi, confirming previous results with whole globin ~. The compositions of fiT 6, fiT 7 + 8 and/~T 9 from Hb-X mi were the same as in Hb-A mi. A fingerprint of the aminoethyl axmi-chain at pH 4-7 is shown in Fig. 2d. Peptide aT 2 is absent and peptides aT 3 and aT 4 are in different positions from the corresponding peptides in a T M (Fig. 2c). This confirmed the previous results 2. Table V shows the amino acid compositions obtained for the a Xmi peptides of the minor haemoglobin. The peptides aT I and aT II of the minor component were analysed together since they did not completely separate from one another. Analysis of the two peptides, T and S (Fig. 2d) obtained in good yield showed that the former had the composition serine, lysine and the latter consisted of valine and lysine. Val-Lys is found in the/%chain but no trace of any other/Lehain peptide was seen. This suggests that T and S together account for the region of the a xmichain corresponding to aT 2 (Thr or Set, Asn, Val, Lys) of the major component, the substitution of lysine for asparagine occurring at position a 9. In addition, only serine was present at position a8 of the minor component a-chain. The peptides T and S have therefore been designated aT 2a and aT 2b. Analysis of peptide aT 3 which had a lower chromatographic mobility in a T M fingerprint (Fig. 2d) than in the comparable a Ami fingerprint (Fig. 2c), showed that the tryptophan at aI 4 in the latter was replaced by leueine in a Xmi. The absence of tryptophan in aT 3 was in accordance with the very low 28o-nm absorbance of the aXmi-chain noted during the globin chain separation of the minor component. Analysis of peptide aT of the minor component showed that the single valine residue and two glycine residues were absent, when compared with aT 4 of the major haemoglobin. We had previously showed 2 that aT 4 of the minor component had a greater electrophoretic mobility towards the anode, again consistent with it being a shorter peptide. In addition, a new peptide staining for arginine was seen in fingerprints of the minor component (peptide N, Fig. 2d, and peptide 3, ref. 2), and it was in such a position that its composition could have been valine, glycine, arginine. However, this peptide was difficult to locate with the 0.02% ninhydrin used prior to the elution of the peptides and was not analysed. We believe, however, that the change Gly-Arg at position a i 9 accounts for the altered mobility and composition of aT 4 and the appearance of a new arginine-positive peptide (aT 4a). Biochim. Biophys. Mcta, 221 (197 o) 45o-466

?

"5"

h,

4

V

indicates

Asp Thr Ser Glu Pro Gly Ala Val Leu Tyr Phe His Lys Arg Met Cys Trp

A mine acid

+

2.0

0.9

1.2 2.8 i.i

2.o

i.i

2.9

i.i

o.9

(IT 2a

i.o

I.O

(IT 2b

+

i.o

i.I

i. i 1. 9

aT 3

but the amount

+

0. 7

1.2 0. 7

1.9 3.1

3.2

aT 4a

+

i.o

1.8

I.i

o.9

1.9 i.i

aT 5

was not estimated.

1.2 o-7 1.8 1.2 I. i I.O i.o I. i i.i 0.9 1.8 1. 9 i.I

aT 6

(I-CHAIN T R Y P T I C P E P T I D E S OF M . i r u s H b - X

acid was present

(IT i -w I I

Peptide :

the amino

A M I N O A C I D C O M P O S I T I O N S OF T H E

TABLE

I.I I.I

1.8

aT 7

0.7

3.0 1.9 2.1

1.2 1.2

aT 9a

+

2.6 I.O

3.0 I.O 3.0

1.8 1. 3 1.2

3.o

a T 9b

I.O

I.O

a T ~o

+

1.2

1.8

I.O

aT I2a

I.I

o.9

(IT 14

~4

t~

%

o

~o

H

t~

Cys Trp

Arg Met

Asp Thr Set Glu Pro Gly Ala Val Leu Tyr Phe His Lys

Amino acid

Peptide: Haemoglobin."

I

I

i

i

I I I

I

I

I I I

i

i

aT I X I

a and

i

I

I

I

I

o o i o o ½ i o ½

ab

aT2 X I

and

o

i

I

I 2

I

i

0

I 2

aT3 X I

I

i

I i

2 3

3

ab

i

I

I

i

i

4 3 I I i

3

aT4 X I

M . irus H b - A

termed

Hb b.

Differences between M. the sum of two peptides

irus s l o w m i n o r

OF THE a-CHAIN

VI

COMPARISON OF THE AMINO ACID COMPOSITIONS

TABLE

found

i

I

2

2 I

I

i

2 i

I

i

2 i

aT5 X I

i

were

i I 2 i i I I I I i 2 2 i

i I 2 i i I I I I i 2 2 i

3, aT

I i

2

I I

2

aT

i

aT8 X I

4 and

2, aT

I 0

I

I

I

I

I

I

o

I

3 3

336 213 235

0

134 i i 2 2 i i

ab

aT 9 X I

9. aT

m . irus S L O W M I N O R H b

aT 7 X I

OF

2, a T

aT6 X I

in aT

TRYPTIC PEPTIDES

I

I

I

I

aT Io X I

aT

AND

4 and

(X)

I

I

2

I

2

I

I

2

I

2

aT11 X 1

9 in the

Hb

(I)

I

I

2

i

I

I

2

i

aT I2a X I

minor

M . irus H b - A

each

I

I

I

I

aT i 4 X 1

are

4~

~o 0

0 0

0

464

P.T. WADE el al.

Peptide Q (Fig. 2d) comprised the first half of aT 9 (residues a62 71) with a7 I glycine of the major haemoglobin substituted for arginine in the minor component. Peptide P, Fig. 2d, corresponded to the second half of aT 9, namely residues (~72 9 o. The two peptides, Q and P of the minor haemoglobin, will therefore be called aT 9 a and aT 9 b. Peptide R (Fig. 2d) was a small portion of the end of aT 9 b, arising from the hydrolysis of the labile peptide bond at serine a8I (cf. major component). The insoluble peptides aT I2b and aT 13 of the **xmi-chain have not yet been analysed. The results, summarised in Table VI, show that the slow minor haemoglobin of M. irus differs from the major Hb-A mi (containing serine at position a8) in at least four sites; a 9 Asn-Lys, a I 4 Trp-Leu, ~I 9 Gly Arg and a7I Gly Arg. Assuming homology of M . irus aam*- and aXrai-chains, the likely sequence of the latter is shown in Fig. 4. The differences from the aAmi-ehain are underlined. DISCUSSION

Our analyses of the major haemoglobin component Hb-A mi show that its achain differs from the human aA-chain in at least five sites (if we include the a8 Thr-Ser change) and the fl-chain differs from the human in at least seven, implying minimal mutational distances 2° of six and nine, respectively. The two macaque species, M . mulatta and M . irus differ at a78 and also a8 if the presence of threonine in non-integral values at the latter site is counted. They differ only at position 33 in the fi-chain. The tryptic peptide composition of the fl-ehain of the Japanese macaque, M . fuscata 21, is the same as that of M. irus, while its a-chain 22 appears to be the same as that of M . mulalta. All the sites at which M. irus differs from man are variable in other mammals. Leucine at a68 has been reported in the horse and the macaques, f19 Ash and fiI3 Thr have only been found in the macaques. Glutamine tentatively assigned to a78 in M . irus has not been reported previously. At some sites, such as a8 and f15o Thr-Ser, a I 9 and f171 Ala Gly, a78 Asn-Gln and fllO4 Arg Lys, man and M. irus differ in residues with similar physico-chemical properties. I f the substitutions in M. irus are considered in relation to the tertiary structure of horse haemoglobin 2a all but two are seen to lie at the surface or in surface crevices. The exceptions include fli25 (H3 Pro Gln) which takes part in the sift 1 contact; but about one-third of the sites here are known to vary in mammals and flI25 is one of the most variable. The other exception is fllO 4 (G6 Arg-Lys) which lies in the internal cavity where several basic residues occur and Arg Lys substitutions in this region are not uncommon in vertebrates. The fact that most of these substitutions would not be expected to affect function significantly is consistent with the view of KIMURA24 and KI.~G AND J U K E S 25 that most substitutions in proteins are selectively neutral and are fixed by random processes. Polymorphic variation at some of these sites would be expected, though not necessarily at very high frequency levels, and some evidence that this is so at position ~8 has been presented here. The minor component of M . irus, H b - X mi, has been shown to differ from the normal a-chain in at least four sites, namely a9 Asn-Lys, a i 4 Trp-Leu, aI 9 Gly Arg and a7I Gly Arg, Fingerprint patterns of two different individuals suggested that the same four changes were present in both. These sites all lie at the surface of the Biochim. Biophys. Aria, 22i (197 o) 450-466

HAEMOGLOBINS OF M. irus

465

molecule. The substitution aI 4 T r p - L e u is interesting since tryptophan is said to act as a spacer between the A and E helices 23. It is difficult to say what effects this change m a y have, if any, since the physiological functions of both Hb-A2 and the macaque minor component are unknown. Since the minor haemoglobin is found together with the major Hb-A ml and a major haemoglobin a-chain variant H b - P mi in some M. irus individuals (and frequency data are consistent with H b - P m1 and Hb-A m1 being allelic), it was concluded that the distinctive a-chain of the H b - X mi probably arose by duplication of the a-locus1, 2. The present work showing that there are at least four differences from the normal a-chain strengthens this view. Misreading of a codon has been suggested to account for certain cases where more than one type of a-chain is regularly observed, for example in the rabbit 26, but other workers have shown that it m a y be due to allelic variation in this species27, es. The misreading hypothesis is not tenable for M. irus since three of the differences between a xmi and a T M involve charge changes and more than one electrophoretically distinct minor component should regularly be present, a-locus duplication m a y not be uncommon since there is evidence of it in the goat 29-32, the m o u s e 3 3 , 34 and the horse ~5. In M. irus, a minor component is present only in some individuals in contrast to man, the chimpanzee, gorilla, orang and gibbon in which the great majority of individuals appear to have one 36. The ape minor haemoglobins differ from the normal major haemoglobins in their non-a-chains and probably have a homologue of the h u m a n 6-chain 43. We have also found that all individuals in samples from seven New World genera have a minor haemoglobin which also differs from the major component in the non-a-chain. The distinctive chain of the ceboid minor component has recently been analysed in three New World monkey genera3L It is difficult to tell from the sequences available at present whether it is a homologue of the &chain or whether the two chains originated b y separate duplications in the hominoid and ceboid lineages; it m a y be significant that both the human &chain and the ceboid min(~r component chains differ from known anthropoid fl-chain sequences in having 116 arginine and 117 asparagine instead of two histidines which are found in most mammals. It is curious that a minor component has only been found in M. irus among Old World monkeys. During the last few years we have examined haemolysates from

Macaca cyclopis, Macaca radiata, Macaca sinica, Cynopithecus (Macaca) niger, Mandrillus, Theropithecus, Cercopithecus mitis, Cerc@ithecus ascanius, Cercopithecus neglectus, Cercocebus albigena, Presbytis entellus and Presbytis obscurus and no minor haemoglobins were found. This m a y be an artifact of small sample size but considerable numbers of Macaca mulatta, Papio anubis, Papio cynocephalus, Cercopithecus aethiops and Erythrocebus patas, ranging from one hundred to several hundred, have also been screened in our laboratory and in others as-4°. It is possible that a minor component that cannot be separated from the major component by electrophoresis is present in some of these species. I f the minor component arose since M. irus became specifically distinct, if indeed it is 4~, this would imply the accumulation of at least four differences from the normal a-chain in a period of perhaps one or two million years, which is quite a rapid rate of change. I f it arose earlier in the cercopithecoid lineage we might expect to find it in other macaque species or even in other genera. Biochim. Biophys. Acta, 221 (197 o) 45 ° 466

466

P.T. WADE et al.

ACKNOWLEDGEMENTS

We would like to thank Dr. Anne F. Skinner for preparation of some of the ~Alni_ and flArni-chains while she was working in the Department of Anthropology with financial support from the Medical Research Council, London. We are also grateful to Dr. J. Moor-Jankowsky and Mr. J. Davis, Lemsip Primate Laboratory, New York, U.S.A. and Dr. A. C. Lawsen, Glaxo Laboratories, Greenford, Middlesex, England for supplying M. irus blood specimens. REFERENCES I N. A. BARNICOT, E. R. HUEHNS AND C. J. JOLLY, Proc. Roy. Soc. London, Ser. B, 165 (1966) 224 . 2 P. T. WADE, N. A. BARNICOT AND E. R. HUEHNS, Nature, 215 (1967) 1485. 3 V. M. INGRAM, Nature, 189 (1961) 704 . 4 E. R. HUEHNS AND E. M. SHOOTER, J. Mol. Biol., 3 (1961) 257. 5 T. H. J. HUISMAN AND A. M. DOZY, J. Chromatog., 19 (1965) 16o. 6 E. R. I-IUEHNS, in I. SMITH, Chromatographic and Electrophoretic Techniques, P a r t II, Heinem a n n , London, 1968, p. 291. 7 H. G. KUNKEL, in D. GLICK, Methods of Biochemical Analysis, Interscience, New York, 1954, p. 141. 8 A. L. ANSON AND A. E. MIRSKY, J. Gen. Physiol., 13 (193 o) 469. 9 J. B. CLEGG, M. A. NAUGHTON AND D. J. WEATHERALL, J. Mol. Biol., 19 (1966) 91. i o I. SMITH, in I. SMITH, Chromatographic and Electrophoretic Techniques, P a r t I, H e i n e m a n n , London, 1969, p. 119. I I D. H. SPACKMAN, W. H. STEIN AND S. MOORE, Anal. Chem., 3 ° (1958) 119o. 12 R. T. JONES, Cold Spring Harbor Syrup. Quant. Biol., 29 (1964) 297. 13 R. L. HILL, Advan. Protein Chem., 20 (1965) 37. 14 G. MATSUDA, Z. MAITA, H. TAKEI, H. OTA, M. YAMAGUCHI, T. MIYAUCHI AND M. MIGITA, J. Biochem. Tokyo, 64 (1968) 279. 15 G. MATSUDA, T. MAITA AND H. TAKEI, Intern. J. Protein Res,, 2 (197 o) i. 16 G. MATSUDA, Z. MAITA, N. IGAWA, H. OTA AND T. MIYAUCHI, Intern. J. Protein Res., 2 (197 ° ) 13. 17 G. MATSUDA, T. MAITA, M. YAMAGUCHI AND M. MIGITA, Intern. J. Protein Res., 2 (197 o) 83. 18 G. MATSUDA, T. MAITA, H. OTA AND H. TAKEI, Intern. J. Protein Res., 2 (197 o) 99. 19 M. O. DAYHOFF, Atlas of Protein Sequence and Structure, National Biomedical Research F o u n dation, Silver Springs, Md., 1969. 2o W. M. FITCH AND E. MARGOLIASH, Science, 155 (1967) 279. 21 I. TACHIKAWA, Acta Med. Nagasaki, 13 (1969) 157. 22 Y. TEREO, Acta Med. Nagasaki, 13 (1969) 17o. 23 M. F. PERUTZ, H. MUIRHEAD, J. M. Cox AND L. C. G. GOAMAN, Nature, 219 (1968) 131. 24 M. KIMURA, Nature, 217 (1968) 624. 25 J. L. KING AND T. H. JUKES, Science, 164 (1969) 788. 26 G. VON EHRENSTEIN, Cold Spring Harbor Symp. Quant. Biol., 31 (1966) 705 . 27 T. HUNTER AND A, MUNRO, Nature, 223 (1969) 127o. 28 G. SHAPIRA, M. BENRUBI, N. MALEKNIA AND L. RIEBEL, Biochim. Biophys. Acta, 181 (1969) 216. 29 T. H. J. HUISMAN, J. B. \VILSON AND H. R. ADAMS, Arch. Biochem. Biophys., 121 (1967) 528. 3 ° T. H. J. HUISMAN, G. BRANDT AND J. B. WILSON, J. Biol. Chem., 243 (1968) 3675. 31 M. D. GARRICK AND T. H. J. LIUISMAN, Biochim. Biophys. Acta, 168 (1968) 585 . 32 M. D. GARRICK AND J. P. CHARLTON, Biochem. Genet., 3 (1969) 393. 33 K. HILSE AND R. A. PoPP, Proc. Natl. Acad. Sci. U.S., 61 (1968) 93 o. 34 R. A. PopP, J. Heredity, 60 (1969) 126. 35 J. v . KILMARTIN AND J. B. CLEGG, in M. O. DAYHOFF, Atlas of Protein Sequence and Structure, P a r t 4, D 43, N a t i o n a l Biomedical Research F o u n d a t i o n , Silver Springs, Md., 1969. 36 N. A. BARNICOT, C. J. JOLLY AND P. T. WADE, Am. J. Phys. Anthropol., 27 (1967) 343. 37 S. H. BOYER, E . F . CROSBY, T. F. THURMON, A. 1N-.NOYES, G . F . FULLER, S . E . LESLIE, M. K. SHt~PARD AND C. N. HERNDON, Science, 166 (1969) 1428. 38 J. BUETTNER-JANUSCH AND V. BUETTNER-JANUSCH, in J. BUETTNER-JANUSCH, Evolutionary and Genetic Biology of Primates, Vol. I I , Academic Press, New York, 1964, p. 75. .39 N. A. BARNICOT, C. J. JOLLY, E. R. HUEHNS AND N. DANCE, in H. VAGTBORG, The Baboon in Medical Research, U n i v e r s i t y of T e x a s Press, Austin, 1965, p. 322. 4 ° J. BUETTNER-JANUSCH AND V. BUETTNER-JANUSCH, Nature, 197 (1963) lO18. 41 J. FOODEN, Science, 143 (1964) 363 . 42 K. A. PIEZ AND L. MORRIS, Anal. Biochem., I (196o) 187. 43 P- T. \¥ADE AND N. A. BARNICOT, Comp. Biochem. Physiol., in the press.

Biochim. Biophys. Acta, 221 (197o) 45o-466