Abnormal human haemoglobins VI. The chemical difference between haemoglobins A and E

520

BIOCHIMICA ET BIOPHYSICA ACTA

ABNORMAL HUMAN HAEMOGLOBINS VI. T H E CHEMICAL D I F F E R E N C E B E T W E E N HAEMOGLOBINS A AND E J. A. H U N T * AND V. M. I N G R A M

Medical Research Council Unit/or Molecular Biology, Cavendish Laboratory, Cambridge (Great Britain) and Division o[ Biochemistry, Department o[ Biology, Massachusetts Institute o] Technology, Cambridge, Mass. (U.S.A.) (Received N o v e m b e r I 4 t h , 196o )

SUMMARY

The primary structures of normal human haemoglobin and of haemoglobin E appear to differ only in the sequence of a single tryptic peptide (peptide 26). These peptides have been degraded and their amino acid sequences have been determined in the two cases. The sole difference is the replacement of a glutamic acid residue in normal haemoglobin by lysine in haemoglobin E.

INTRODUCTION

In previous papers of this series 1-4 the chemical differences between normal human haemoglobin (A) and haemoglobins S and C were shown to be localised in a single peptide found in tryptic digests of the particular haemoglobin; the differences consisted of the replacement of a glutamic acid residue in haemoglobin A for valine and lysine in each of the two identical half molecules of haemoglobins S and C, respectively. Haemoglobin E (see ref. 5) is another frequently occurring inherited abnormality of haemoglobin, which is thought to be allelic or linked with the haemoglobin S abnormality ~. However, unlike haemoglobins S and C which are found mainly in Africa, haemoglobin E is largely localised in Southeast Asia 7. Previous work on the distinguishing properties of haemoglobin E have been summarised elsewhereS; it is sufficient to state, that virtually the only difference detected hitherto between the haemoglobins was one of electrophoretic mobility. Although at pH 8.6 haemoglobin E moves near to haemoglobin C, at pH 6.5 it behaves more like haemoglobin S. The pH-mobility curve of haemoglobin E is not parallel with that of haemoglobin A, in contrast to the curves of haemoglobins S and C, which are parallel with each other and with that of haemoglobin A. Abbreviations: PTH, phenylthiohydantoyl; DNP, dinitrophenyl. * P r e s e n t a d d r e s s : N a t i o n a l I n s t i t u t e for Medical R e s e a r c h , L o n d o n (Great Britain).

Biochim. Biophys. Acta, 49 (I961) 52o-536

HUMAN HAEMOGLOBINS A AND E

521

As in haemoglobin C (see ref. 4), it has been shown 8 in tryptic digests of haemoglobin E that one of the peptides normally found in tryptic digests of haemoglobin A has been replaced by two new peptides s. The present paper describes in detail the experiments which led to these conclusions and also those experiments which were made to determine the amino acid sequences of the peptides involved. Once again it would appear that the genetic mutation causing the production of haemoglobin E has altered only one amino acid in the total sequence of about 280 amino acids which made up each of the haemoglobin half molecules. The implications of these findings have been discussed elsewheres. MATERIALS AND METHODS

Haemoglobin solutions were prepared in the same way as previously described 1. Haemoglobin E from two different individuals who were homozygous for the abnormality and haemoglobin E from a person who was heterozygous were used for the fingerprinting and for the preparation of the differing peptides. In the heterozygous case, the haemoglobin E occurred mixed with A (60%), but it was not found necessary to isolate it. Tryptic digestions of the heat denatured haemoglobins and chylnotryptic digestion of the trypsin resistant cores of haemoglobins were performed as previously described1, 2. Elastase digestion of peptide A-26 was performed in a pH-stat at pH 9 and 37 °. 0.025 mg of purified elastase ° (gift of Dr. M. A. NAUGHTON,Cambridge) to 1-2 t,moles of peptide in 1.5 ml was used; digestion time was 2.5 h. Alternatively, where the micro vessel for the pH-stat was not available, the peptide was dissolved in I ml 2% NH4HCO3 (pH 7.8) and incubated with the elastase at 37 ° for the same length of time. The NH4HCO~ was sublimed by evaporating the solution to dryness and then treating the residue with acetic acid; the ammonium acetate so formed was readily removed in vacuo. Electrophoresis and "fingerprinting" were performed as previously described 1. In addition the technique of paper electrophoresis at pH 6.4 was used, followed by descending chromatography of eluted bands on a fresh paper with n-butanol-acetic acid-water (4:1:5, v/v). Use was made of combinations of paper electrophoresis with the two pyridine acetate buffers at p H 3.6 and pH 6. 4 (see refs. I, 4)N-terminal end group analysis was performed with the GANGER fluoro dinitrobenzene method 1°, detecting the N-terminal amino acid by two dimensional paper chromatography with tert.-amyl alcohol and 1.5 M phosphate solvents (see ref. 4). The SJOQUISTmodification 11 of EDMAN'S phenylisothiocyanate stepwise degradation was also employed, in which case the liberated PTH-amino acids were identified after hydrolysis in H I and chromatography of the liberated free amino acids in the butanol-acetic acid (Redfield 2) solvent system, as previously described 4. For the quantitative analysis, standard solutions were used in the range 0.02 to 0.I0 tzmole for each paper 4. They contained either arginine, aspartic acid, glutamic acid, lysine, glycine, alanine, valine and leucine; or: arginine, aspartic acid, glutamic acid, lysine, glycine, histidine, serine, alanine, tyrosine, valine, methionine, phenylalanine, leucine and threonine. Partial acid hydrolysis of the peptide samples was carried out by incubation Biochim. Biophys. Acta, 49 (1961) 520 536

522

J. A. HUNT, V. M. INGRAM

of the peptide in concentrated HC1 (M.A.R.) at 37 ° for 4o-6o h or by refluxing the DNP-peptide in 5.7 N HC1 in a test tube for the time indicated, using a cold finger for condenser. The DNP-peptides so formed were extracted with ether and ethyl acetate and the aqueous residue was used for fractionation of the non-DNP-peptides. Tests for tryptophan and cysteine were performed directly on the paper chromatograms as previously described4; the SAKAGUCHI reaction was used in the test for arginine 1.. Peptide A-26 (i.e. tryptic peptide number T-26 from haemoglobin A (see ref. I)) was prepared from the tryptic digest of haemoglobin A by paper electrophoresis at pH 6.4, where it was the most acidic peptide (Fig. 2); further purification was not usually necessary, but a repetition of the paper electrophoresis at pH 6, 4 was occasionally used. A sample of the peptide, which had been prepared by column chromatography by Dr. G. BRAUNITZER 1~, w a s also subjected to paper electrophoresis at p H 6. 4 before use. Peptide E-26a (i.e. peptide 26a from haemoglobin E) was prepared by electrophoresis of the tryptic digest of haemoglobin E at pH 6.4 followed by re-electrophoresis at the same pFI (Fig. 2). Peptide E-26b was prepared by electrophoresis of the tryptic digest of haemoglobin E at pH 6. 4, followed by elution and descending chromatography on Whatman No. i paper in the n-butanol-acetic acid-water (4:1:5) system (Fig. 2). Final purification was achieved by further paper electrophoresis at pH 6.4. The chromatography step could be replaced by paper electrophoresis at pH 3.6. RESULTS AND DISCUSSION

Comparison of the tryptic and chymotryptic digests of haemoglobins A and E Photographs of the tryptic digests of haemoglobins A and E are reproduced in Fig. I. They show that the most acidic, faintly ninhydrin reacting, peptide (A-26) of haemoglobin A has been replaced in the tryptic digest of haemoglobin E by the new basic peptide E-26a. The disappearance of peptide A-26 is better shown by the use of the arginine stain either on a fingerprint or on a one-dimensional electrophoretogram of the digests at pH 6. 4. The peptide marked E-26b on the tracing was not properly identified until a more thorough examination of the tryptic digests was instituted. For this, paper electrophoresis of all the peptides at pH 6.4 was followed by descending chromatography with n-butanol-acetic acid-water ( 4 : i :5) (Fig. 2) of groups of peptides; it was shown that the negative band I contained an additional slow moving peptide in haemoglobin E digests. Qualitative amino acid analysis on paper of all the peptides separated in the two-step procedure failed to reveal any further differences. Likewise, a similar study of the chymotryptic digests 2 of the trypsin resistant "cores" of the two haemoglobins showed no further differences to be present, the pattern of peptides being essentially the same as that published for haemoglobin C (ref. 4). As far as can be judged by the methods used, the only differences between haemoglobins A and E reside in the peptides A-26 and E-26a and b. The determination of the amino acid sequence of peptides A-26, E-26a and E-26b are reported in the following section. Biochim. Biophys. Acta, 49 (196I) 520-536

HUMAN HAEMOGLOBINS A AND E

523

The amino acid composition of peptides A-26 and E-26a and b All of the amino acid compositions were determined using the paper chromatographic method 4 with less than o.I /zmole of each peptide. The result for peptide A-26 has been confirmed b y quantitative column chromatography on a MOORE

(a)

(b)

©

oO

O• ~(~ peptide ":'"'

--

Hb A

+

Hb E

-

+

Fig. I. (a) Photographs of the fingerprints of haemoglobins A and E. (b) Tracing of the fingerprints showing the position of the changed peptides. Biochim. Biophys. Acta, 49 (1961) 520-536

524

J. A. H U N T , V. M. I N G R A M

AND STEIN automatic amino acid analyser, model SpincoS,la, a4. From these results the amino acid composition of each peptide is found to be: Peptide

A-26,

Peptide

E-z6a, Arg, Gly, Ala, Leu

A r g , A s p v G l u z , G l y z, A l a , V a l 3, L e u

Peptide

E - 2 6 b , L y s , A s p 2 , G l u , G l y 2, V a l 3

The amino acid sequence of the haemoglobin A peptide A-26 End group analysis using the fluoro dinitrobenzene method 1° showed that valine was the sole amino acid in this position. Some information of the N-terminal sequence TABLE QUANTITATIVE

AMINO

ACID

I OF PEPTIDES A - 2 6 , E - 2 6 a ,

ANALYSES

E-26b

M o l a r r a t i o s of e a c h a m i n o a c i d . Amino acid

P eptide A-26

Arginine Lysine Aspartic acid Glutamic acid Glycine Alanine Valine

Leucine Amount of peptide in/~moles

0.9 -i. 8 2. i 2.9 I. I 3.0 I. i

I. I ---0.9 i .o -I.O

-0.9 1.9 i. i 2. I

o.057

0.035

0.039

0.054

TABLE

DNP-amino acid

4 5 6 7 8 9 Io

Val ? Val Val Val? Glu? Val Val

E- 26b

o.9 -2. i 2. I 2.9 lost 2.8 i. i

A N A L Y S I S OF D N P - P E P T I D E S

DNP-peptide*

E- 26a

--

2.8 --

II

DERIVED

FROM P E P T I D E

A-26

A queous residue

Asp . + + + ± + + +

Glu .

. -. -+ + --

.

Gly . -. . -+ ---

¥al + -+(+) + +

* See Fig. 3a.

T-26 HbA

il

+ Hb____EE

T-26a Fig.

2a.

Biochim. Biophys. Acta,

49 (1961) 5 2 0 - 5 3 6

C~

I

O

IA

i

A

)

"IA

i

: ' .....

i E

A__~

Bond 2

I~HIc. 4.1.5.

I

Fig, 2b. i.

I

•

"

/ I

I E

I'

T-26a

i

~,,..~1

IE

Band 4 i AI

Itu~e. 4,1.5

1

I

Fig. 2b. z

00

QO

"1 A

A

Band 3 I

,I E

I

i AI

I E I

Band 6

BuHAc. 4.1.5.

U27C2

Fig. zb. 3

C22~ C - ~

I A

Band S

Fig. z. (a) Separation of t h e t r y p t i c digests of haemoglobins A and E by p a p e r electrophoresis at p H 6. 4, (b) Separation of t h e p e p t i d e s from t h e b a n d s marked in (a) b:y" descending 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 (4 : ~ : 5),

IIZ

Bond I

T-26b ~

,

t~

>

~z

0

0

526

J. A. HUNT, V. M. INGRAM

of the peptide was obtained by refluxing the DNP-peptide in 5.7 N HC1 for 12 min, extracting with ethyl acetate and separating the extracted DNP-peptides by the two-dimensional paper chromatographic system used for DNP-amino acids 4. The DNP-peptides were eluted from the paper with i N HC1 and extracted with ether and ethyl acetate; complete hydrolysis with 5-7 N HC1 was used to determine the terminal DNP-amino acid and the other amino acid residues in the DNP-peptides. The results obtained are shown in Fig. 3a and Table II. The N-terminal sequence is seen to be Val-Asp; peptides 5 and IO indicate that it is most probably Val.Asp-Val, although the difference in chromatographic mobility of these two peptides might suggest that they contain different amounts of aspartic acid and valine. In order to clarify this point the EDMAN stepwise degradation was used. It is also possible that DNP-peptides 5 and IO differ by an amide group on the aspartic acid side chain. The first two steps of the EDMAN degradation 11 yielded valine and aspartic acid, respectively; some indication could be obtained that valine is also present in the third position (Table III). However, after two steps of the EDMAN degradation, both the unpurified and the purified residues (Fig. 7) yielded after dinitrophenylation DNP-valine as the sole DNP-amino acid. The N-terminal sequence of peptide A-26 is then Val. Asp .Val.

(Do

:}':

',

t-AmOH

i

t /

tAmOH

il

;" 206 (K:)minute hyclrolysate)

-

176 DNP AT-26

Q2minutehydrolysate)

DNP E T-26b

Fig. 3a.

Fig. 3 b.

Fig. 3. (a) Separation b y t w o dimensional c h r o m a t o g r a p h y of the D N P - p e p t i d e s derived from peptide A-26. (b) Separation b y t w o dimensional c h r o m a t o g r a p h y of the D N P - p e p t i d e s from peptide E-26b. TABLE III EDMAN STEPWISE DEGRADATION OF PEPTIDE A-26 A rg

I s t step 2rid step 3rdstep

----

A sp

± + + + + ++

Glu

± 4+

Gly

A la

Val

Leu

+ + +

-t+ ++

+ + + + + + +++

:~ + +

Biochim. Biophys. Acta, 49 (1961) 520-536

527

HUMAN HAEMOGLOBINS A AND E

Partial acid hydrolysis of peptide A-26 The peptide fragments obtained by partial acid hydrolysis were fractionated either by fingerprinting (Fig. 4a), or by a combination of paper electrophoresis at

["

(

, i~

@

@-e

(,'G ,~

s v

-.+ ---

A T--26 peptides E T - 2 6 b ~pti~s

Fig. 4 a. 150

A T-26

pH 6.4 2

3 4

5

789

6

-4-

\

Band I

///~Ban(

2 t +

i+

~b (qlu}~.)

b

oO

+91#,J (arq) I s/ t I BuHAc ~y ~ 3:1:1 Amino ocids I .I

1 3.9

I

3.6

3.6

P

]

Fig. 4 b. Fig. 4. (a) Separation b y fingerprinting of the peptides obtained b y partial acid hydrolysis of peptides A-26 and E-26b. (b) Separation by paper electrophoresis a t pH 6.4 and 3.6 of the peptides obtained from peptide A-26.

Biochim. Biophys. Acta, 49 (I96I) 5"'0-536

528

J. A. HUNT, V. M. INGRAM

pH 6.4 and 3.6 (Fig. 4b). The compositions of the peptides and their N-terminal amino acids are shown in Tables IV and V. In a repeat experiment, fingerprinting was used to separate the peptides from a i2-min hydrolysate of the peptide A-26 in 5.7 N HC1 at IOO°. Peptide 7 (see Table IV) contained Gly + + , Ala ± , Leu + + ; peptide io after electrophoresis at pH 3.6 contained Glu + , Ala + , Leu + ; and peptide 13 purified further by electrophoresis at pH 3.6 had Glu + , Gly + , Val + : peptide 15 was clearly Glu + , Gly + , Ala + . TABLE IV PARTIAL

HYDROLYSIS

PRODUCTS

FROM

PEPTIDE

A-26,

HYDROLYSED

IN

CONe.

He1

AT

37 °

See Fig. 4a. Peptide

2 3 4 5 6 7 8 9 Io I1 12 13 14 I5 16 17 18 19

A rg

A sp

Glu

Gly

++++

+++ +++ + +++ + ++++ + ++ ++ ++ ? ±

+++ ++ +++ +++ ~ + + + + + +++

± ++ ++ ++ + ±

A la

Val

N-terminal amino acid

Leu

Gly +++

++ +++ +

+++ ++ ++++ ++

+++ ++++ +++ +++ +

++ ~ +

Val ++++ ++ ++++ ++

Ala, L e u Ala Glu Val Val Glu, Val

++

+ +

Glu Gly

+ +

++

TABLE V PARTIAL

HYDROLYSIS

PRODUCTS

FROM

PEPTIDE

A-26

BY CONe.

He1

AT

37 °

See Fig. 4b. Peptide

Asp

Glu

Gly

A la

I-C

I-d

+++

++

+

+

+

?

+ ++

I-e

l-f* 2

4* 5-a 5-b 6-a 6-b

Leu

Remarks

++

l-a, I-b

3

Val

--

++++ +

+ ++ ++

+ +

+++ + ++

End group : Glu

++++ + ++ ++

* A t t e m p t e d d i n i t r o p h e n y l a t i o n , w i t h n o result. ** T h e a m i n o acids are f r o m t h e a q u e o u s p o r t i o n a f t e r d i n i t r o p h e n y l a t i o n of p e p t i d e 3.

Biochim. Biophys. A~ta, 49 (1961) 520-536

HUMAN HAEMOGLOBINS A AND E

529

From the structures of the di- and tri-peptides and from the end group studies the following partial sequence may be formulated: Amino acid composition: Arg, Asp2, Glu2, Gly3, Ala, Val 3, Leu

End group determination : Val. Asp.Vat

Partial acid hydrolysis: Table GIy" Arg (Leu, Gly) Ala. Leu (Ala, Leu, Gly) Glu. (Ala, Leu) Glu' Ala Gly. Glu (Gly, Olu, Ala) Val. Gly (Glu, Val, Gly) (Val, Asp)

Peptide number 2 7 (purif) 9 I-e 3 16 17 15 (purif) 5 13 (purif) 5a

IV IV V V IV IV IV V

The partial sequence for peptide A-26 is: Val. Asp.Val. (Asp, Glu, Val. Gly) Gly. Glu. Ala. Leu. Gly. Arg

In order to complete the sequence studies it was necessary to resort to elastase digestion of the peptide A-26. About I/,mole of the peptide was digested and divided into two portions, which were both fractionated by paper electrophoresis at pH 6.4 and 3.6, (Fig. 5). The peptides obtained from the first portion were used for quantitative amino acid analysis and those from the other portion for end group analysis, where necessary (Table VI). A T-2b elastase

I 4

2

I

pH b.4

+

band 4

band 8

7 3

I

h

I

,}

2

3 4

pH 3.6

--

,

pH

3.5

--

,

3.sI

pN

-

-

Fig. 5. Separation by paper electrophoresis at pH 6-4 and 3.6 of the peptides from the elastase digestion of peptide A-26.

Biochim. Biophys. Acta, 49 ~I96I) 520-536

530

J . A. H U N T , V. M. I N G R A M

Further confirmation of the structure of the peptide A-26 was obtained by heating in 0.25 M acetic acid; under these conditions the peptide bonds on both sides of aspartic acid residues are expected to be cleaved is. The peptide was heated in the acetic acid for I i h at 12o ° and the mixture of peptide fragments separated b y paper electrophoresis at pH 6. 4 and 3.6 (Fig. 6). The quantitative amino acid analysis of the peptides (Table VII) showed that two molecules each of valine and aspartic acid had been cleaved from the N-terminus of the molecule leaving the rest intact. No DNP-amino acid could be obtained by the fluoro dinitrobenzene procedure 13 from the remaining portion of A-26, perhaps due to pyrrolidone formation of the now N-terminal glutamic acid residue (see complete structure). k T-26 (oceticacid) pH 6.4

- [4-

I

Iol Ii

F i g . 6. S e p a r a t i o n

1

2

3

b y p a p e r e l e c t r o p h o r e s i s a t p H 6 . 4 of t h e p e p t i d e f r a g m e n t d i g e s t i o n of p e p t i d e A - 2 6 . TABLE

AMINO

ACID

ANALYSIS

OF

PEPTIDES

FROM

from acetic acid

VI

THE

ELASTASE

DIGESTION

OF

PEPTIDE

A-26

S e e F i g . 5Molar ratios o] amino acids Peptide

ArE

2* 3 4-1 4-2 5-I 7-2 8-1 9-1 9-2

Asp

Glu

Gly

I.I 0.9

Ala

Val

1.2 i.I

I.O

i.I Unchanged I.O I.I 1.9 2.1

1.8 1.6 1.8

Yield in lanole

Leu

0. 7 I I.i

2.8 i.I peptide A-26 2.2 0. 9 2.2 2.1

0.027 o.124 o.145 0.080 0.070 O.Ol 5 o.074 o.o14 0.058

3.1 2.7 2.9

0.9 I.O

End group

Val

None** Val Val Val

* P e p t i d e 2 is p r o b a b l y c o n t a m i n a t e d b y s o m e p e p t i d e 3, t h u s a c c o u n t i n g f o r t h e h i g h v a l u e s of Arg and Gly. ** D N P - p e p t i d e 7-2 w a s h y d r o l y s e d i n 5 . 7 N H C 1 u n d e r c o n d i t i o n s w h e r e D N P - G l y w o u l d b e destroyed. TABLE

VII

ANALYSIS OF PEPTIDES OBTAINED BY HEATING PEPTIDE A - 2 6 WITH DILUTE ACETIC ACID S e e F i g . 6. Peplide

x (Unhydrolysed) 2 3 2 (After dinitrophenylation and hydrolysis)

Arg

i.o i.i

Asp

-i --

Glu

G/y

Ala

Val

Leu

i.i 0.9

2.2

2.7

i.o

i 0. 9

2.o

2.9

I.I

o. 9

Yield itt l,moles

o.1io 0.o34 0.o90 o.o49

Biochim. Biophys. Acla, 4 9 (1961) 5 2 o - 5 3 6

HUMAN HAEMOGLOBINS A AND E

531

It is evident that the N-terminal tetrapeptide sequence A-26 has been split off b y the action of acetic acid and that, moreover, the sequence of this portion must be Val.Asp.Val.Asp. This result is in excellent agreement with PARTRIDGE AND DAVIES15 concerning the proposed mechanism of action of acetic acid on proteins, namely, that the peptide bonds on both sides of the aspartyl residues are cleaved. The complete sequence of peptide A-26 may now be formulated using the previous results, which include the dipeptide sequence Val.Gly and the results of acetic acid and elastase hydrolysis: Previous results : Val- Asp" Val(Asp, Glu, Val, Gly) Gly. Glu- Ala. Leu. Gly. Arg. Val- Gly

Acetic acid hydrolysis: (Val, Asp, Val, Asp) (Glu, Val,Gly, Gly, Glu, Ala, Leu, Gly, Arg)

Elastase digestion : Val(Asp, Val, Asp, Glu, Val) Val(Asp, Val, Asp, Glu, Val, Gly, Qly, Glu, Ala) (Gly, Gly, Glu, Ala) (Gly, Gly, Glu, Ala, Leu, Gly, Arg) (Leu, Gly, Arg) (Gly, Arg)

Hence : Val. Asp .Val. Asp. Glu .Val. Gly. Gly. Glu. Ala. Leu. Gly. Arg

The data from the elastase digestion enable us to say that the glutamyl residue in peptide 4-2, Table VI, must have a flee carboxyl group to neutralize the guanidino group of the arginyl residue, since this peptide is neutral at pH 6.4. Of the charged groups, the other glutamyl residue must also have a free carboxyl group, since peptide 2 from the acetic acid digest is negatively charged at pH 6. 4. There is a little evidence for the presence of an amide group on the aspartyl residue in position 2, since after two EDMAN degradations the residual peptide has a greater net negative charge than the original peptide (see Fig. 7). Apparently, the aspartyl residue in position 2 was uncharged, giving the residual peptide a greater net negative charge by the removal of two neutral amino acids.

The amino acid sequence of peptide E-26b This peptide presented similar difficulties with regard to the N-terminal sequence as peptide A-26; moreover these difficulties were made more acute by the very small amounts of peptide available. The N-terminal sequence was determined using partial acid hydrolysis of the DNP-peptide, and by the EDMAN stepwise degradation procedure.

T A T-26after 2 Edman$

~

.,~. I

2

Expt. 195 Fig. 7- Purification by paper electrophoresis at pH 6.4 of the residual peptide A-26 after two steps of the ~DMAN degradation. Biochim. Biophys. Acta, 49 (1961) 520-536

532

J. A. HUNT, V. M. INGRAM

Partial acid hydrolysis of the peptide E-26b in concentrated HC1 at 380 followed by fingerprinting (Fig. 4a), gave the results shown in Table IXa. The DNP-peptide E-26b was refluxed in 5.7 N HC1 and the water soluble fraction was fractionated by paper electrophoresis at pH 6.4 (Fig. 8), giving the results in Table IXb. We may again summarise the most clearly defined peptides as follows: Table I X-5 I X-6 IX-I

Val- Gly Glu (Val, Gly) Gly. Lys

and the N-terminal sequence: Val-Asp.Val. (Asp, Glu, Val, Gly 2, Lys)

By hydrolysis of a small amount of the peptide (approx. o.oo4/,mole) in o.25 N acetic acid a small amount of a peptide having the composition Glu, Val, Gly=, Lys, determined by quantitative analysis, was found after paper electrophoresis at pH 3.6, indicating again that the N-terminal sequence is Val.Asp.Val.Asp. TABLE X A N A L Y S I S OF

PEPTIDES

PRODUCED

BY

ELASTASE

FROM

PEPTIDE

]~-26b

See Fig. 9.

Peptide

Asp

2 4 6

Glu

1.9

I.O

--

ye!low at

Lys

Gly

Ala

I.I I. 9 Undigested peptide E-26b (o.I)

I

pH

(o.1)

6.4

I "al

Yield in f~m)les

3.1

0.033 O.OLO 0.028

+

first with r~nhydrin

171 peptide T-26b (elastase)

Fig. 9. Separation by paper electrophoresis at pH 6. 4 of the peptides from the elastase digestion of peptide E-26b.

Elastase digestion in buffer of a slightly impure preparation of the peptide E-26b yielded only two peptides (Table X) in high yield which could have been derived from peptide E-26b. They were detected by paper electrophoresis at pH 6.4 (Fig. 9). The sequence of the whole peptide E-26b may now he formulated as follows : Partial acid hydrolysis and end group studies: Val. Asp.Val(Asp, Glu, Val, Gly)Gly. Lys Glu (Val, Gly) Val. Oly

Acetic acid hydrolysis: (Glu, Val, Gly, Gly, Lys)

Elastase digestion : (Val, Asp, Val, Asp, Glu, Val) (Gly, Gly, Lys)

Hence : Val. Asp. Val. Asp. Glu" Val. Gly' Gly. Lys

Biochim. Biophys. Acta, 49 (196I) 520-536

533

HUMAN HAEMOGLOBINS A AND E

Fig. 3b shows the pattern of the DNP-peptide obtained by partial hydrolysis of DNP-E-26, The results of the hydrolysis of these peptides are shown in Table VIII. Hence the N-terminal sequence is seen to be Val. Asp.Val. The EDMAN stepwise degradation n confirmed the above N-terminal sequence; after two steps of the degradation, dinitrophenylation lo of the residual peptide revealed only DNP-valine. TABLE VIII ANALYSIS OF D N P - P E P T I D E S

Peptide

Residue ol peptide

DNP-amino acid

Asp

Val

1

Val

+ nk

+

2 3

Val Val

+ 4+ 4-

+

188

_

FROM PEPTIDE E-26b

flow 2 3

E T-2bb

(12 min h,/drol,/sate, ~ueous)

4

7 8

5 6

9

I0

-Jr-

II

Fig. 8. Separation b y paper electrophoresis at pH 6.4 of the peptides obtained b y partial acid hydrolysis from DNP-peptide E-26b. TABLE

IX

PARTIAL ACID HYDROLYSIS PRODUCTS FROM PEPTIDE E-26b

a. Hydrolysis in conc. HC1 (see Fig. 4a). Peptide

Lys

A sp

GIu

+++ 3 5 6 io Ii I3 i8

++ + + :k

+(+) 4++ ±

Gly

+++ +++ +++ 44+ +(+)

Val

End group

Gly 4-++ q++(+) 4-+ ++

VaI Val Val and Glu

b. Hydrolysis of DNP-peptide E-26b in 5-7 N HC1 (see Fig. 8). Peptide

A sp

Glu

Gly

Val

End group

++ +

Glu

4

+

+

+

6*

--

4-

+

7

4-

4-

IO Ii

+

+

4-

" T h e amino acids in peptide 6 are from the aqueous portion after dinitrophenylation a n d hydrolysis.

B i o c h i m . B i o p h y s . A c t a , 49 (1961) 52o-536

534

j.A.

HUNT, V. M. INGRAM

No data is available on the presence of arnide groups, except that in order for the peptide to have its negative charge at pH 6.4, at least two of the three carboxyl groups must be free, leaving the possibility of only one amide group. T h e a m i n o acid sequence of peptide E - 2 6 a

End group determination by the fluoro dinitrobenzene method 1° revealed alanine as the only DNP-amino acid; the residue contained only arginine, glycine and leucine. Partial acid hydrolysis in concentrated HC1 at 38o of the peptide E-26a from 2 Fmoles of haemoglobin, followed by fractionation by paper electrophoresis at p H 6.4 and at pH 3.6 gave the results shown in Figs. Ioa and b and Table Xl. From the dipeptides thus found the sequence of peptide E-26a may be formulated thus : End group: Ala (Leu, Gly, Arg).

ET-2ba +

pH b.4

l Band1

pH3.6

b

_[_ 3

--

2

-~

I

An 71 Fig. Io. S e p a r a t i o n b y p a p e r electrophoresis a t p H 6. 4 (a) a n d 3.6 (b) of t h e p e p t i d e s from t h e p a r t i a l acid h y d r o l y s a t e of p e p t i d e E-26a. TABLE XI ANALYSIS

OP

PEPTIDES

DERIVED

BY

PARTIAL

ACID

HYDROLYSIS

PROM

PEPTIDE

E-26a

See Fig. IOa. Peptide

A rg

G/y

Ala

Leu

++

++

--

--

--

Gly

A la

Leu

Remarks

+ + -k ++++

~ + ++++

2

++

++

3

++++

+++

End group

See Fig. lob. Peptide

Gly

I 2 3

+ + +(+)

Free a m i n o acids ?

B i o c h i m . B i o p h y s . A c t a , 49 (I96I) 520-536

HUMAN HAEMOGLOBINS A AND E

535

Partial acid hydrolysis: (Ala, Leu) (Leu, Gly) G l y . Arg

Hence: Ala. Leu- Gly. A r g EDMAN stepwise degradation n confirmed this sequence in its entirety. By assuming that peptide E-26a is attached to the C-terminal end of the peptide E-26b, we are able to account for a peptide having as m a n y amino acids as the peptide A-26 which is missing in haemoglobin E; moreover, these two peptides are identical in their sequences except that the second glutamic acid has been replaced b y a lysine as shown below: peptide A-26:

Val. Asp. Val. Asp . Glu . Val. Gly. Gly. Glu . Ala. Leu. Gly. Arg

peptides E-26b and a : Val. A s p . Val. A s p . Glu. Val. Gly. Gly. Lys. Ala. L e u . Gly. Arg.

Again, as in haemoglobins S and C, the only detectable difference between a m u t a n t form (E) of haemoglobin and its normal form (A) is the replacement of one amino acid in one of its chemically distinct chains. The change in haemoglobin E has been shown b y INGRAMTM and b y BRAUNITZER et a l W to occur in the fl-chain; in the whole molecule of haemoglobin it will occur twice, thus giving a net charge difference of four units, the same as the charge differences found in haemoglobin C. HUNT AND INGRAMTM and BRAUNITZER et al. ~7 have shown that the haemoglobin S and C change occurs at residue 6 and the haemoglobin Gsan Jose ( = Gselawartz) changO 9 at residue 7. The haemoglobin E difference is at residue 26 from the Nterminus of the fl-chain 17. I t is difficult to explain why haemoglobin E does not manifest its charge change fully as does haemoglobin C. I t m a y be that the changed amino acid is buried deeper in the molecule and so has a different charge environment. ACKNOWLEDGEMENTS

We are grateful to Drs. H. LEI-IMANNand E. R. HUEHNS (London) for samples of haemoglobin E, to Dr. G. BRAUNITZER (Munich) for a gift of peptide A-26 and to Dr. M. A. NAUGHTON (Cambridge) for a gift of purified elastase. We wish to thank Drs. F. H. C. CRICK and M. F. PERUTZ (Cambridge) and Dr. H. LEHMANN (London) for m a n y stimulating discussions. One of us (J.A.H.) is grateful to the M.R.C. for a Scholarship. Part of this work was performed b y .1. A. H. while holding a Carlsberg-Wellcome Travelling Fellowship at the Carlsberg Laboratorium, Copenhagen. Some analyses were performed on equipment provided b y a grant to V. M. I. from the National Institute for Arthritis and Metabolic Diseases, U. S. Public Health Service. REFERENCES I V. M. INGRAM,Biochim. Biophys. Acta, 28 (1958) 539. J. A. HUNT AND V. M. INGRAM, Biochim. Biophys. Acta, 28 (1958) 546. a V. M. INGRAM, Biochim. Biophys. Acta, 36 (1959) 402. 4 j . A. HUNT AND V. M. INGRAM, Biochim. Biophys. Acta, 42 (196o) 409 . s H. A. ITANO, W. R. BERGREN AND P. STURGEON,J. Am. Chem. Soc., 76 (1954) 2278.

Biochim. Biophys. Acta, 49 ~I96I) 52o-536

536

j.A.

HUNT, V. M. INGRAM

M. AKSOY AND H. LEHMANN, Nature, 179 (1957) 1248. 7 H. LEHMANN AND R. BHAGXVAN SINGH, Nature, 178 (1956) 695. s j . A. HUNT AND V. M. INGRAM, Nature, 184 (1959) 870. 9 M. A. NAUGHTON AND F. SANGER, Biochem. J., 7 ° (1959) 4 p10 F. SANGER AND H. TuPPY, Biochem. J., 49 (1951) 465 • 11 J. SJ~QUIST, Arkiv Kemi, i i (1957) 129. 12 j . B. JEPSON AND I. SMITH, Nature, 172 (1953) ilOO. 13 K. HILSE AND G. BRAUNITZER, Z. Naturforsch., I 4 b (1959) 6o 4. 14 D. H. SPACKMAN, S. MOORE AND V~r. H. STEIN, Anal. Chem., 3 ° (1958) 119o. 15 S. M. PARTRIDGE AND H. F. DAVIES, Nature, 165 (I95O) 62. 16 V. M. INORAM, Nature, 183 (1959) 1795. 1: G. ]3RAUNITZER, N. HILSCHMANN AND R. MULLER, Z. physiol. Chem. Hoppe Seyler's, 318 (I96o) 284 . 18 j . A. ]7IUNT AND V. M. INGRAM, Nature, 184 (1959) 64 o. lU R. L. HrLL AND H. C. SCHWARTZ, Nature, 184 (1959) 642.

Biochim. Biophys. Acta, 49 (1961) 52o-536

T H R O M B O S T H E N I N - - A CONTRACTILE PROTEIN FROM THROMBOCYTES. ITS EXTRACTION FROM HUMAN BLOOD PLATELETS AND SOME OF ITS PROPERTIES M. B E T T E X - G A L L A N D AND E. F. L U S C H E R

Theodor Kocher Institute, University o] Berne, and Blood Trans]usion Service o] the Swiss Red Cross, Berne (Switzerland) ( R e c e i v e d J a n u a r y 2oth, 1961)

SUMMARY

On extraction of human blood platelets with a buffered o.6 M KC1 solution, a protein fraction is obtained, which becomes insoluble at ionic strengths below 0.2. If ATP is present, this material will show the phenomenon of superprecipitation or active contraction. Because of its role in thrombocyte function, we have named this contractile protein thrombosthenin. Like actomyosin from muscle, thrombosthenin acts as an ATP-ase. The dependence of this enzymic activity on the ionic strength, on the concentrations of Ca e+ and Mg 2+ ions, and of ATP has been investigated. The sensitivity of solutions of thrombosthenin towards ATP has been determined. Although the obtained results suggest t h a t thrombosthenin belongs to the actomyosin group, a quantitative comparison with the data on muscle actomyosin leads to the conclusion, that thrombosthenin is distinct from the contractile protein of muscle. The significance of the presence of thrombosthenin in blood platelets in relation to viscous metamorphosis and clot retraction is discussed. A b b r e v i a t i o n s : VM, viscous m e t a m o r p h o s i s , TCA, t r i c h l o r o a c e t i c acid.

Biochim. Biophys. Acta, 49 (1961) 536-547