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Abnormal human haemoglobins V. Chemical investigation of haemoglobins A, G, C, X from one individual
BIOCHIMICA ET BIOPHYSICA ACTA
253
ABNORMAL HUMAN HAEMOGLOBINS V. CHEMICAL INVESTIGATION OF HAEMOGLOBINS A, G, C, X FROM ONE I N D I V I D U A L C. B A G L I O N I AND V. M
INGRAM
D~ws~on o/B~ochem~stry, Department o / B w l o g y , ~Iassachusetts Instztute of Technology Cambr,dge, 3'Iass (U S A ) (Recetved O c t o b e r 6th, 196o)
SUMMARY
The four adult haemoglobins, A, G p h t l a , C, X present in the blood of D.K.P. have been isolated and fingerprinted. Their constitution is shown to be Hb-A = %A8~, Hb-Gphtla G A A C G C -- -- % B 2, Hb-C = a 2 B 2 and Hb-X = a 2 ~ 2 , a doubly abnormal haemoglobin. The amino acid substitution in Hb-Gphna is a lysine for asparagine change in a tryptic peptide from the a-chain.
INTRODUCTION
The normal human haemoglobin molecule--a2BA--is known to be made up of four polypeptide chains, two a- and two B-chains 1. In many abnormal haemoglobins amino acid substitution has been shown to take place in the a- (haemoglobin I = Hb-I) or in the B-chain 2, 8 (Hb-S, Hb-C, Hb-E, Hb-Gschwartz). All these abnormal haemoglobins are inherited In a strictly Mendelian manner. It is thought that the a- and B-chains are controlled by two sets of allelic genes 2. Recently a pedigree of a colored family has been reported 4, which does not leave any ambiguity about the existence of two distinct a and B genes and about the fact that they are inherited independently. The presence of four electrophoretically different haemoglobins in the blood of the propositus (D.K.P) has been observed 4. On the basis of their electrophoretlc mobility, three of the four haemoglobins were assumed to be respectively Hb-A, Hb-C and Hb-G. Since this haemoglobin G was discovered in Philadelphia, we propose to call it Hb-Gphlla. The name Hb-X was assigned to the slowest component, whose mobility was much less than that of any of the known haemoglobins. Hb-C is known to result from a mutation of the B gene, since a lysine has been reported to substitute for a glutamlc acid in the B-chain 5 The Hb-G described by SCHWARTZ et al. ~ was shown to result also from a mutation of the B gene. ATWATER et al. 4 thought that D.K.P.'S haemoglobin Gl,hlla IS identical with the Hb-G reported by EDINGTON AND LEHMANN6. Hb-Gl,~lla has now been shown to be the product of a Abbreviations: DNP, dmltrophenyl, PTH, phenyl thlohydantoyl. B~och,m Bzophys Acta, 48 (1961) 253-265
254
c. BAGLIONI, V. M. INGRAM
mutated c¢ gene, as previously suggested 4. Since a normal ~ a allele 1, aNo present, D.K.P. was supposed to be a doubly heterozvgote whose genotype can be written as follows in the terminology suggested by INGRAMANI) STRETIO~,: xGphtla
tic
0~-k
flA
The presence of the slow moving component called Hb-X was rather surpnsang, it was supposed to result from the combination of ~Cph~la-chalns with /~C-chalnb The occurrence of a haemoglobin molecule entirely composed of abnormal chains ~s of considerable interest, although the possibihty that such a molecule could be encountered was strongly suggested by the m vztro reassociatmn experlment~ of ITANO
et al. s. The present paper is concerned with the identification of the chemical abnormahty ~)f the Hb-Gphna and with the demonstration that Hb-X is a doubly altered haemoglobin, resulting from the comblnatmn of the products of two independent mutated genes. The genetical implications of these findings have already been discussed elsewhere2L MATERIALS AND METHODS
Haemoglobin A generous supply of D.K.P.'s blood was made available through the courtesy of Miss J. ATWATER, Philadelphia The haemoglobin solution was prepared as previously described by INGRAM9. After dialysis agamst 0.05 3I veronal buffer at pH 8 6, the haemoglobin solution was centrifuged for 3 ° rain at IOO,OOO g m a Spinco preparatlve ultracentrifuge and the supernatant used Immediately for starch block electrophoresis.
Starch block electrophoresis The electrophoresis on starch block was carried out following the method of KUNKEL10,11. 5 ml of haemoglobin solution (47 mg/ml) were applied to each block. o.05 M veronal buffer pH 8.6 was used to wash the starch and to fill the electrode vessels. A voltage of 350 V across the starch block (30 cm) and the paper wicks (2 ', 5 cm) was applied for 48 h in the cold (2°). After that time, four bands were clearly separated, three migrating toward the anode and one toward the cathode. The bands were eluted from the starch with distilled water, discarding the zones where overlapping was observed. Three starch blocks were run and the homologous fraction pooled together.
Concentration of the haemoglobins The method described by INGRAMAND STRETTON12 was applied. The starch block eluates were dialysed against o.oi M phosphate buffer pH 6.8 and concentrated by adsorption on columns of carboxymethylcellulose whose dimensions were I × 5 cm. The columns were washed with the same phosphate buffer and then eluted with small volumes (4-5 ml) of 0.05 M veronal buffer pH 8.6. 129 mg of Hb-A, 98 mg of Hb-Gl, hna, 85 mg of Hb-C and 52 mg of Hb-X were obtained. The total yield was about 50 °o. The purity and the electrophoretic mobility of each fraction were determined by starch gel electrophoresls, according to SMITHIESla B~och~m.B~ophys Acta, 48 (t96I) 253-265
CHEMISTRY OF HAEMOGLOBINS A, G, C, X
255
Digestion Tryptic digestion of the heat denatured haemoglobins was performed in a thermostated automatic titrator as previously described 9.
F~ngerprinting The peptlde mixtures obtained by tryptic digestion were analyzed by the original fingerprinting technique 9. In order to get a better resolution of the peptides, the digests were also examined by a modified fingerprinting technique 14, in which a pyridineisoamyl alcohol-water (35:35:30) so Ivent15 was used for chromatography.
Pept~de preparation For quantitative preparation of peptides, the tryptlc digests from 2- 5 mg of haemoglobin were applied per inch of a line on a sheet of W h a t m a n 3MM paper. Votatile buffers p H 6.4 (see ref. 9) or 4.7 were used. The buffer of pH 4.7 was prepared with pyridine-acetic acid-water-butanol (25 : 25 : 19oo : 50, v/v). Electrophoresis was carried out as described by INGRAM9, but applying 3o V/cm to the paper on a watercooled brass plate. The papers were dried, a guide strip was cut and the peptides located with o.2 °'o nmhydrin in acetone. The bands corresponding to peptides were cut out from the paper and eluted with I N acetic acid.
End group analysis of peptides Dinitrophenylation of peptides was performed as described by SANDER AND THOMPSON1~, dissolving the peptides in I ° o aqueous trimethylamine and adding a 5°0 solution in alcohol of I-fluoro-2,4-dinitrobenzene. The identity of the DNPamino acids was determined in a two dimensional paper chromatographic system, using for the first dimension the tert-amyl alcohol solvent of BLACKBURN AND LOWTHER17 and for the second the 1. 5 M phosphate solvent of LEVY18. EOMAN stepw~sedegradation of peptides The method of SJOQUIST 19 w a s followed with minor modifications. At each step the peptides were reacted twice with phenylisothiocyanate in triethylamine buffer. Identification of the PTH-amino acids was achieved using the chromatographic solvent system described by Sj/3QUIST2°.
Quantitative arn,no acid analys~s The peptides to be analyzed were hydrolyzed in 5.7 N HC1 at i i o ° for 16 h. Amino acid composition was determined by means of a Spinco model MOORE ANn STEIN automatic amino acid analyzer. RESULTS AND DISCUSSION
Fingerprints The haemoglobins separated from D.K.P.'S blood were subjected to the fingerprinting analysis. The photographs of the four fingerprints, obtained with the modified technique 1~ are shown in Figs. I and 2. The fingerprint of Hb-A shows a pattern of peptldes similar to that described by INGRAM9. The fingerprint of D.K.P.'s haemoglobin component characterized by an electrophoretic m o b l h t y equal to that of Hb-C shows a pattern of peptides equal to B~och~m B,ophys .4cta, 48 (I961) 253-265
d
~o
k_
e0 Fxg l Photograph of f i n g e r p r i n t s q of h a e m o globins \ a n d Gphlla Electrophoresls was m the usual buffer, but on a (ooled bras~ plate at about 3° V/cm for 2 h -Xscendlng chlomatogr a p h v la is in p y n d m e lsoainyl alcohol water (35 35 3 " , v / v )
>
t~
>
CHEMISTRY OF HAEMOC~LOBI~IS A, G, C, X
257
e" O ~ 0
~r5
B~och~m. Bwphys. Acta, 48 (I96r) 253-265
258
C. BAGLIONI, V. M. INGRAM
c~ ®
C32e +
(-)
0 ~, ~
Hb-A
(+)
e
~,
3b (: ~ )
¢b
t.,0,
=
:
÷
(-)
Hb-G
(+)
Fig. 3. Tracing of f i n g e r p n n t s of haemoglobins A and Gphtla Peptldes A- 3, A-9, G-3b, G-3b ox and C--9a are marked m black. Peptlde A- 4 is shaded.
~0
.- . 3b(:gb) ......
--~'
3 b o×
4a :-x..
90
+
(--)
1.4b -X
(-)
Hb-¢
(+)
(+)
Ftg. 4- Tracing of fingerplants of haemoglobins X and C Peptldes G-3 b, G-3b ox a n d G-9a are marked m black, peptldes C-4a and C-4b are shaded
Bzo~htt~z Btoph>s 4c/a, 48 (1961) 253-265
CHEMISTRY OF HAEMOGLOBINS A, G, C, X
259
t h a t described b y HUNT AND INGRAM21 for Hb-C. One can see (Figs. I and 2) the absence of peptide A- 4 and the appearance of two new peptides, C-4a and C-4b, resulting from the cleavage b y trypsin of the peptide analogous to A- 4 where In haemoglobin C a lysine substitutes for a glutamic acid residue. In the Figs. 3 and 4 are shown the tracings of the fingerprints, the peptide A- 4 in Hb-A and the peptides C-4 a and C-4b in Hb-C are indicated by shading. In the fingerprint of the Hb-Gpuna, peptide A- 3 is missing from the band of uncharged peptldes; instead three positively charged peptldes, called G-3b, G-3b ox, and G-9a, which are not present in fingerprints of Hb-A, are evident. The reasons for indicating these peptides in this way will be made clear below. Later reexamination by ionophoresls at p H 4.7 of the area of the fingerprint which has peptides G-3b and G-3b ox showed the absence of peptide A- 9. Therefore in haemoglobin G peptldes A- 3 and A- 9 have disappeared to be replaced by G-3b ,G-3b ox and G-9a (see Figs. I and 3 )and G-3a (see later). Peptide 3 is known to contain histidlne and methlonine, since it gives the reactions on paper which are characteristic for these amino acids 22. B y staining Hb-G fingerprints with sulphanilic acid 22 for histidine and with platlnic iodide 22 for methionine or cystelne, it was shown that peptide G-3b gives a positive reaction while peptide G-9a gives no reaction for those amino acids. Amino acid analysis (see below) showed the a m m o acid reacting positively with platinic iodide to be methionine. The meth~onine contained In peptide G-3b undergoes partial oxidation during the tryptic digestion and the fingerprinting, a proportion of it being transformed into methionine sulphoxide. The resulting peptlde has an electrophoretic mobility equal to the peptide containing methionlne, but behaves differently in chromatography, having a lower RF. This phenomenon is known to occur in other peptides, for example, peptides A-25 (containing methionlne) and A-2 4 (containing the sulphoxide), which have the same electrophoretic mobility but a lower RF in chromatography. In addition peptide A- 9 shows sometimes a partial separation into two components, which contain respectively the oxidized and the reduced form of methionine. It is easyto differentiate the two peptides, because the one containing methionlne sulphoxlde, while having all the properties in common with the peptide containing methionine, gives no reaction with the platlnic iodide reagent 22. The peptide G-3b containing methionlne sulphoxlde is mdlcated as G-3b ox (ox for oxidized). The fingerprint of H b - X shows the abnormalities characteristic of both Hb-C and Hb-G Both peptides A- 3 and A- 4 are absent. In addition to a normal pattern of peptides four new positively charged peptides are present, namely the two described b y HUNT .a~ND INGRAM21 in Hb-C and the two found in the Hb-Gl, htia fingerprint (Fig i). In the Figs 3 and 4 are shown the tracings of the fingerprints of haemoglobin Gl~hlla and X; peptides A-3 in Hb-A fingerprints, G-3b and G-9a in Hb-G and H b - X fingerprints are marked in black. The peptldes C-4a and C-4b In the H b - X fingerprint are shaded H b - X shows two abnormalities, being a combination of the pattern characteristic of Hb-C and that of Hb-G. However, these findings do not show whether the chemical change, which takes place in Hb-G, is situated on the a- or on the B-chain of haemoglobin. The fingerprint of the isolated a-chain has indeed shown that a peptlde with the properties of peptlde 3 belongs to the a-chain, while no peptide with similar properties was found in the B-chain fingerprint 23. Btoch~m B~ophys Acta, 48 (I961) 253-265
260
C BAGLION1, V. M INGRAM
Two sets of analyses of the amino acid composition of all the tryptic peptldes from the a-chain of haemoglobin A have so far been reported by \:ON HILSE AND BRAUNITZER 24 and by HILL AND I{ONIGSBERG ~a. The latter authors also determined the N-terminal amino acid of each peptide. Therefore, we decided to analyze peptide A-3 and peptides G-3b and G-9a, to ascertain whether peptide A- 3 IS indeed an ~-cham peptide and to discover the ammo acid substitution which had occurred in our HbGPwla and in Hb-X.
Peptides C-4a and C-4b Preparation and analysis ofpepSdes. The peptldes C-4a and C-4b were prepared by paper electrophoresis accordmg to HUNT AND INGRAM21. Amino acid analysis of peptide C-4a showed the presence of glutamic acid and lysine only. Amino acid analysis of peptide C-4b gave lysine, histidine, threonme, proline, valine and leucine in equal amount These compositions agree with the compositions reported by HUNT AND INGRAM21 and imply a lysine for glutamic acid substitutmn in the expected peptide of the fl-cham It was concluded that the haemoglobin present in D.K.P.'s blood is authentic Hb-C, since it has the same electrophoretlc mobility of Hb-C and shows the same amino acid substitution.
Peptides A-3 and A-9 Peptide A-3 was prepared by electrophoresls at pH 6.4 of a Hb-A dlgestg; the band of uncharged peptides (see Fig. 5) was cut from the paper and eluted. The eluate was then separated by electrophoresis at pH 4.7, applying 30 V/cm for 4 h. The separation at pH 4-7 IS shown in Fig. 6. After the second electrophoretlc separation, peptide A- 3 was obtamed almost
,
O0 @000¢ 09 00 000
22
21
20
t9
18
17-15
14-)0
9
~ band
23
;~4-25
26
Fig 5- Comparison by one-dimensional paper electrophoresls9 at pH 6 4 of the tryptlc digests of haemoglobins A, C, Gphtta and X Peptides are marked as in Fig 3 a n d Flg. 4
O0 <+>
<_>
0 0 N@ t
2
5-6
3
@ 4
Fig. 6. Paper electrophoresm a t p H 4.7 of the trypelc peptides isolated from the neutral bands (see Fig. 5) of haemoglobms A and Gpwt~. Peptides containing hlstldine (posture reaction 2~ with P a u l y ' s reagent) are shaded
B*och~n Blophys. Acta, 48 II96I) 253 265
CHEMISTRY
O F H A E M O G L O B I N S A, G, C, X
261
pure. As a check of the purity the N-terminal amino acid was determined by SANGER'S methodiC; only DNP-valine was obtained. The amino acid composition of peptide A- 3 was determined (see Table I) and was found to be equal to the amino acid composition given by HILL AND KONIGSBERG25 for the a-chain peptide called by them Ta6, except for our finding of 2 histldme reslI
TABLE AMINO
ACID COMPOSITION
OF
THE
PEPTIDES
Peptutes A rrllno a c i d
Lys His Asp Thr Ser Pro Ala Val Met Leu Total residues
A-3
A "9
lb ( 2G-G-9b)
G_.9a
i.ii 2.13 5 7° 0.90 i 84 i.o9 6.78 3.22 (I) 4 oI
1.98 (2.59) 5 72 i 28 i 94 1.3 6.56 3.17 0.84 4.28
o 91 2 28 4oi -i 83 i.o 4 4.92 2.03 (I) 3.oo
1.9I -i 09 0.87 --2.I3 0.96 -i oo
21
8
28
29
dues compared with 3 reported by these authors. The N-terminal amino acid was reported to be valine 25, in agreement with our findings for peptide A-3. The amino acid composition of peptide A-3 is slightly different from the composition of a peptide found by HILSE AND BRAUNITZER24 in the tryptic digest of the a-chain and indicated by them as peptide I I ; our peptide A- 3 has 2 histidines instead of 3 and one more aspartic acid residue. Peptide A-3 is the largest peptide which is present in tryptic digests of haemoglobin apart from the "core." Its 28 amino acids constitute a large unit, which has no equivalent m the B-chain of haemoglobin, nor can it be confused with any other peptide. The absence of peptide A- 9 from the tryptic digests of Hb-G and Hb-X was rather surprising, since it could indicate that two peptldes are in some way changed by the same mutation. Peptide A- 9 is known to be present in variable amount in different tryptic dlgests of the same haemoglobin preparation; it is usually observed as a rather weak spot, which gives positive reactions for hlstidine and methionine. By fingerprinting the tryptic digest of the isolated a-chain, HUNT23has shown that peptide A- 9 is an a-chain peptide On the other hand the amino acid composition of the a-chain of Hb-A gives only two methionine residues per chain 24,25HUNT2Zhas instead found three methionme containing peptides in the fingerprint of the a-chain. These facts led us to suppose that peptide A- 9 has a definite relationship with peptide A- 3, since both give positive reactions for methionine and histidine and both disappear in Hb-Gl,hila. The peptide A- 9 was prepared by electrophoresis at pH 6.4 of the tryptic digest of Hb-A (see Fig. 5) and subjected to quantitative amino acid analysis (see Table I) Bzochtm. B*ophys
,4cta, 4 8 ( I 9 6 I ) 2 5 3 - 2 6 5
262
c. BAGLIONI, V. M. INGRAM
The amino acid composition of A- 9 was found to be almost equal to the composition of peptide A-3, except for the lvsine content, which is double in peptlde A- 9. When subjected to end group analysis by SANGER'S method '~, peptide A- 9 gives di-DNPlysine. With the exception of the higher value for hlstidlne, the analysis agrees well with the idea that peptlde A- 9 ---- Lys-(A-3). TABLE II N-TERMINAL AMINO ACID BY SANGER'S METHOD18
Peptzde
A- 3 A-9 G--3a G-9a G-3b ( = G-9b)
N-termmal
Valme Lyslne Vahne Lvsme Aianlne
Peptides G-3b and G-9a The peptldes G-3b and G-9a were prepared by electrophoresis at pH 6. 4 of the tryptic digest of Hb-G and Hb-X (see Fig. 5). Further check of purity by electrophoresis at pH 4.7 was considered to be necessary for peptide G-3b, since it overlaps in electrophoresis at pH 6.4 with the peptide A- 9. The eluate of the band containing the peptide G-3b was run side by side with a sample of peptide A- 9 isolated from the tryptic digest of Hb-A by electrophoresls at pH 6.4. No trace of peptide A- 9 was found to contaminate our peptide G-3b. The peptides G-3b and G-9a were consequently used without further purification. Analysed by SANGER'S end group method le, peptide G-3b gives DNP-alanine and peptide G-9a gives di-DNP-lysine.
Peptide G-3a The neutral band isolated by electrophoresls at pH 6. 4 of the tryptic digest of Hb-G (see Fig. 5) was eluted and subjected to the electrophoretic separation at pH 4.7. The neutral band from Hb-A was run along side (see Fig. 6). As expected, no trace of peptide A-3 was observed in Hb-G digest; a new faint peptide, not present in Hb-A, was instead observed. This peptide gives no reaction for histidine and methionine. It is also present in Hb-X. The yield of this peptide G-3a is rather poor and it was found to be not sufficiently pure in several preparations to give an accurate amino acid composition. The analysis of this peptide G-3a is rather similar to that of peptide G-9a, but showed only one lysine residue. Subjected tg end group analysis by SANGER'S method :e, peptide G-3a gives DNP-vahne. CONCLUSIONS
In order to prove unequivocally that peptides A-3 and A- 9 and that peptides G-3a and G-9a are the same peptides, except for the additional N-terminal lysine in A-9 and G-9a, the EDMAN analysis of the N-terminal sequence was undertaken according to SJOQUIST1~,so. The results are reported in Table III. The peptides A-3 and G-3a show the same N-terminal amino acid, valine ; peptides A- 9 and G-9a also show Bzoch~m Bzophys Acta, 48 (196:) 253-265
CHEMISTRY OF HAEMOGLOBINS A, G, C, X
263
the same amino acid, lysine. Except for the N-terminal lysine of peptides A- 9 and G-9a, all these peptides have the common N-terminal sequence Val. Ala.Asp. The presence of lysine in N-termmal position is rather unexpected, since trypsin is supposed to split the peptide bond between the carboxyl group of lysine and the amino group of the next amino acid in the peptide. We would expect, however, that TABLE I I I STEPWISE
DEGRA.DATION OF
Pept*de
~st step
2nd step
A-3 A-9 G-9a G-3 a
Valme Lysme Lysme Vahne
Alanme Vahne Vahne Alamne
P E P T I D E S 19
~rd step
Aspartlc Alanme Alanme Aspartic
4th step
-Aspartic Asparttc --
an N-terminal lysine residue is split at a very slow rate, if at all, by trypsin 26. Since the lysine of peptides A- 9 and G-9a is not in N-terminal position in the whole a-chain (the N-terminal amino acid of the a-chain is valine), we have to suppose that somewhere along the polypeptide chain there is a sequence such as L y s . L y s or Arg. Lys. The trypsin will alternatively split the peptide bond between the two basic amino acids or between the lysine and the next amino acid. When the first split occurs between the two basic amino acids, the lysine will occur in the N-terminal position of the following peptlde where it will be removed only slowly by the trypsin. This explanation allows us to describe the relationship between the peptides A-3 and A- 9, G-3 a, G-9a and G-3b (see Table IV). The amino acids suffering the mutational change in Hb-Gphna and Hb-X are underlined. In this figure amino acids whose sequence is unknown are placed inside brackets. The provisional assignment of amide groups to two of the four aspartlc acid residues in peptide G-3b is based on the electrophoretlc behaviour of this peptide and of peptides A-3 and G-3a at pH 6. 4. Peptide G-3b is basic at this pH. Since it contains 2 histidine and I lysine residue TABLE IX"
Pept,de A -3 Val. Ala. Asp(Leu,Ala,Thr)Asp[NH,] • Ala(HlsvAspi, Asp[NH2]~,Ser2,Alaa,Pro,Val2,Met,Leu3) Lys
Pept~de G-3a Val. Ala. Asp(Leu, Ala,Thr) Lys
Pept,de G-3b Ala(Hls2,Asp~,Asp[NH~]2,Ser2,Ala2,Pro,Val2,Met,Leu3) Lys Pept~de A-9 Lys. Val. Ala- Asp(Leu,Ala,Thr) Asp [NH~] • Ala(Hls z,Aspz,Asp [NH~]z,Ser vAla4,Pro,Val2,Met,Leua) Lys
Pept,de G-9a Lys- Val. Ala. Asp(Leu, Ala,Thr) Lys (Peptlde G-9b ~ G-3 b)
B,och,m. Biophys Acta, 48 (I96I) 253-265
264
C. BAGLIONI, V. M. INGRAM
not more than 2 aspartlc acid residues can be free. Again, peptlde G-3a IS uncharged; hence the first aspartic acid residue in A- 3 must be free The second aspartlc acid residue in A- 3 on the other hand is hkely to be covered by an amide group, since it~ substitution by lysine in the whole haemoglobin G molecule gives rise to a change in electrophoretic mobility corresponding to one charge unit per a-chain Therefore, and also because peptlde A- 3 is uncharged, we would expect two free and two covered aspartlc acid residues in peptlde G-3b These arguments wait to be supported by the determination of the complete sequence of peptide A- 3. The amino acid compositions of peptides G-3a and G-3b together give the composition of peptide A-3, except for one aspartic acid residue missing and one addlt,onal lvsme residue present in G-3a G-3b Similarly, the sum of G-9a and G-3b has the same relation to A- 9" A-3 = G-3a + G-3 b + L y s - - A s p [NH2] A-9 = G-9a + G-3b + L y s - - A s p [NH2~
The extra lysine present in Hb-G provides the additional cleavage site for the action of trypsin and therefore peptides A- 3 and A- 9 are split into two peptides, G-3a and G-3b, G-9a and G-9b ( = G-3b) respectively. The new lysine in Hb-G probably substitutes an asparagine residue. The evidence for an asparagine and not an aspartlc acid being substituted by lysine rests on the consideration of the charge difference between Hb-Gputl~ and Hb-A. Double the observed charge difference would be expected from an aspartic acid to lysine substitution, by analogy with the behaviour of Hb-C NOTE ADDED IN PROOF
Since this paper went to press additional information has become available which together with the results in the present paper, shows that the asparagme residue of peptide A- 3 and A-9, which undergoes the mutational change, occupies position number 68 from the N-terminus of the peptide chain (BRAUNITZER e~ al.2S; CRAIG et al.*9). RAPER e~ al. 8° have reported an analogous situation in which four haemog l o b i n s - A , G, C and G / C - - a r e found in one person, a double heterozygote for the G and C abnormalities. Their characterisation of the four haemoglobins rests on experiments involving dissociation and reassociation of the various component haemoglobins. Received January 28th, 1961 ACKNOWLEDGEMENTS
This work has been supported by grants from the National Science Foundation and from the National Institute for Arthritis and Metabolic Diseases, U.S. Public Health Service. REFERENCES
1 H S. RHINESMITH,"~V A. SCIIROEDER AND L PAULING, J A m . Chem Soc , 79 (1957) 4682. 2 j A HUNT AND V. M INORAM, C I B A Foundation Sympos,um Biochemistry o / H u m a n Genet*cs, I959, P 114 3 H. C. SCHWARZ, T. H. SPAET, ~¥. ~V ZUELZER, J X~. NEEL, A. R ROBINSON AND S. F. KAUFMANN, Blood, 12 (1957) 238. 4 j ATVV'ATER, I R SCHWARTZ AND L M TOCANTINS, Blood, 15 (196o) 9ol
B~ochlm. Bzophys..4~t~, 48 (19611 -'53-205
CHEMISTRY OF HAEMOGLOBINS A, G, C, X
265
HUNT AND V. IV[. IN,RAM, Nature, 181 (1958) lO62 EDINGTON AND H. LEHMANN, Lancet, 2 (1954) 173. INGRAM AND A O. W. STRgTTON, Nature, 184 (1959) 19o3. ITANO, S J. SINGER AND E ROBINSON, GIBA Foundat,on Symposium B,ochemzstry o/ Human Genet,cs, 1959, p 96 9 V. M INGRAM, B*och*m. B*ophys Acta, 28 (I958) 539. 10 H . G. KUNKEL, R CEPPELLINI, O MULLER-EBERHARD AND J. V~rOLF,J. Chn Invest, 36 (1957) 1615 11 H G HUNKEL, ~Iethods o/B~ochem~ca1.4nalys~s, Vol. I, Interscxence Publishers, 1959, P. 141. 12 V. 1~I INGRAM AND A O. W. STRETTON, 111 t h e press. 13 O SMITHIES, B*ochem J., (1955) 629. 14 C. BAGLIONI, Bzochim. Bzophys Acta, in the press 15 H G. ~,VITTMAN AND G BRAUNITZER, Virology, 9 (1959) 726 le F. SANG~R AND E O. P. THOMPSON, B*ochem. J., 53 (1953) 353. 17 S BLACKBURN AND A. C- LOW'THOR, B~ochem. J , 48 (1951) 126. ts A L LevY, Nature, 174 (1954) 126. 18 j . SjoQUIST, Ark,v. Kemp, i i (1957) 129, 151. 20 j . SJOQUIST, B~och~m. B~ophys Acta, 41 (196o) 2o 21 j A HUNT AND V M INGRAM, Bzochzm. Bzophys. Acta, 42 (I96O) 409 ~2 R J BLOCK, E L. DURRUM AND G ZWEIG, Paper Chromatography and Paper Electrophoresis, Academic Press, 1958, p. 128. z3 j . A HUNT, Nature, 183 (1959) 1373 24 K VON HILSE AND G BRAUNITZER, Z Natur/orsch , 146 (I959) 6o3. 2s R J. HILL AND W. KONIGSBERG, J Biol Chem., 235 (196o) PC2I 2s C. H . W. HIRS, S MOORE AND W. H STEIN, J. B,ol. Chem., 219 (1956) 623. 27 C BAGLIONI AND V M INGRAM, Nature, 189 (1961) 465 2s G BRAUNITZER, V. RUDLOFF, K HILSE, B LIEBOLD AND R MULLER, Z. physzol. Chem , HoppeSeyler's, 32o (196o) 283 29 L C CRAIG, R L HILL AND W KONIGSBERG, u n p u b l i s h e d observations. 3o A. B RAPER, D B. GAMMACK, Ca. R. HUEHNS AND E M. SHOOTER, Brat Meal. J., (I96o) I257. J. G. 7 V. 8 H.
A. M. M. A
Bzochzm. B~ophys. Acta° 48 (I96I) 253-265
253
ABNORMAL HUMAN HAEMOGLOBINS V. CHEMICAL INVESTIGATION OF HAEMOGLOBINS A, G, C, X FROM ONE I N D I V I D U A L C. B A G L I O N I AND V. M
INGRAM
D~ws~on o/B~ochem~stry, Department o / B w l o g y , ~Iassachusetts Instztute of Technology Cambr,dge, 3'Iass (U S A ) (Recetved O c t o b e r 6th, 196o)
SUMMARY
The four adult haemoglobins, A, G p h t l a , C, X present in the blood of D.K.P. have been isolated and fingerprinted. Their constitution is shown to be Hb-A = %A8~, Hb-Gphtla G A A C G C -- -- % B 2, Hb-C = a 2 B 2 and Hb-X = a 2 ~ 2 , a doubly abnormal haemoglobin. The amino acid substitution in Hb-Gphna is a lysine for asparagine change in a tryptic peptide from the a-chain.
INTRODUCTION
The normal human haemoglobin molecule--a2BA--is known to be made up of four polypeptide chains, two a- and two B-chains 1. In many abnormal haemoglobins amino acid substitution has been shown to take place in the a- (haemoglobin I = Hb-I) or in the B-chain 2, 8 (Hb-S, Hb-C, Hb-E, Hb-Gschwartz). All these abnormal haemoglobins are inherited In a strictly Mendelian manner. It is thought that the a- and B-chains are controlled by two sets of allelic genes 2. Recently a pedigree of a colored family has been reported 4, which does not leave any ambiguity about the existence of two distinct a and B genes and about the fact that they are inherited independently. The presence of four electrophoretically different haemoglobins in the blood of the propositus (D.K.P) has been observed 4. On the basis of their electrophoretlc mobility, three of the four haemoglobins were assumed to be respectively Hb-A, Hb-C and Hb-G. Since this haemoglobin G was discovered in Philadelphia, we propose to call it Hb-Gphlla. The name Hb-X was assigned to the slowest component, whose mobility was much less than that of any of the known haemoglobins. Hb-C is known to result from a mutation of the B gene, since a lysine has been reported to substitute for a glutamlc acid in the B-chain 5 The Hb-G described by SCHWARTZ et al. ~ was shown to result also from a mutation of the B gene. ATWATER et al. 4 thought that D.K.P.'S haemoglobin Gl,hlla IS identical with the Hb-G reported by EDINGTON AND LEHMANN6. Hb-Gl,~lla has now been shown to be the product of a Abbreviations: DNP, dmltrophenyl, PTH, phenyl thlohydantoyl. B~och,m Bzophys Acta, 48 (1961) 253-265
254
c. BAGLIONI, V. M. INGRAM
mutated c¢ gene, as previously suggested 4. Since a normal ~ a allele 1, aNo present, D.K.P. was supposed to be a doubly heterozvgote whose genotype can be written as follows in the terminology suggested by INGRAMANI) STRETIO~,: xGphtla
tic
0~-k
flA
The presence of the slow moving component called Hb-X was rather surpnsang, it was supposed to result from the combination of ~Cph~la-chalns with /~C-chalnb The occurrence of a haemoglobin molecule entirely composed of abnormal chains ~s of considerable interest, although the possibihty that such a molecule could be encountered was strongly suggested by the m vztro reassociatmn experlment~ of ITANO
et al. s. The present paper is concerned with the identification of the chemical abnormahty ~)f the Hb-Gphna and with the demonstration that Hb-X is a doubly altered haemoglobin, resulting from the comblnatmn of the products of two independent mutated genes. The genetical implications of these findings have already been discussed elsewhere2L MATERIALS AND METHODS
Haemoglobin A generous supply of D.K.P.'s blood was made available through the courtesy of Miss J. ATWATER, Philadelphia The haemoglobin solution was prepared as previously described by INGRAM9. After dialysis agamst 0.05 3I veronal buffer at pH 8 6, the haemoglobin solution was centrifuged for 3 ° rain at IOO,OOO g m a Spinco preparatlve ultracentrifuge and the supernatant used Immediately for starch block electrophoresis.
Starch block electrophoresis The electrophoresis on starch block was carried out following the method of KUNKEL10,11. 5 ml of haemoglobin solution (47 mg/ml) were applied to each block. o.05 M veronal buffer pH 8.6 was used to wash the starch and to fill the electrode vessels. A voltage of 350 V across the starch block (30 cm) and the paper wicks (2 ', 5 cm) was applied for 48 h in the cold (2°). After that time, four bands were clearly separated, three migrating toward the anode and one toward the cathode. The bands were eluted from the starch with distilled water, discarding the zones where overlapping was observed. Three starch blocks were run and the homologous fraction pooled together.
Concentration of the haemoglobins The method described by INGRAMAND STRETTON12 was applied. The starch block eluates were dialysed against o.oi M phosphate buffer pH 6.8 and concentrated by adsorption on columns of carboxymethylcellulose whose dimensions were I × 5 cm. The columns were washed with the same phosphate buffer and then eluted with small volumes (4-5 ml) of 0.05 M veronal buffer pH 8.6. 129 mg of Hb-A, 98 mg of Hb-Gl, hna, 85 mg of Hb-C and 52 mg of Hb-X were obtained. The total yield was about 50 °o. The purity and the electrophoretic mobility of each fraction were determined by starch gel electrophoresls, according to SMITHIESla B~och~m.B~ophys Acta, 48 (t96I) 253-265
CHEMISTRY OF HAEMOGLOBINS A, G, C, X
255
Digestion Tryptic digestion of the heat denatured haemoglobins was performed in a thermostated automatic titrator as previously described 9.
F~ngerprinting The peptlde mixtures obtained by tryptic digestion were analyzed by the original fingerprinting technique 9. In order to get a better resolution of the peptides, the digests were also examined by a modified fingerprinting technique 14, in which a pyridineisoamyl alcohol-water (35:35:30) so Ivent15 was used for chromatography.
Pept~de preparation For quantitative preparation of peptides, the tryptlc digests from 2- 5 mg of haemoglobin were applied per inch of a line on a sheet of W h a t m a n 3MM paper. Votatile buffers p H 6.4 (see ref. 9) or 4.7 were used. The buffer of pH 4.7 was prepared with pyridine-acetic acid-water-butanol (25 : 25 : 19oo : 50, v/v). Electrophoresis was carried out as described by INGRAM9, but applying 3o V/cm to the paper on a watercooled brass plate. The papers were dried, a guide strip was cut and the peptides located with o.2 °'o nmhydrin in acetone. The bands corresponding to peptides were cut out from the paper and eluted with I N acetic acid.
End group analysis of peptides Dinitrophenylation of peptides was performed as described by SANDER AND THOMPSON1~, dissolving the peptides in I ° o aqueous trimethylamine and adding a 5°0 solution in alcohol of I-fluoro-2,4-dinitrobenzene. The identity of the DNPamino acids was determined in a two dimensional paper chromatographic system, using for the first dimension the tert-amyl alcohol solvent of BLACKBURN AND LOWTHER17 and for the second the 1. 5 M phosphate solvent of LEVY18. EOMAN stepw~sedegradation of peptides The method of SJOQUIST 19 w a s followed with minor modifications. At each step the peptides were reacted twice with phenylisothiocyanate in triethylamine buffer. Identification of the PTH-amino acids was achieved using the chromatographic solvent system described by Sj/3QUIST2°.
Quantitative arn,no acid analys~s The peptides to be analyzed were hydrolyzed in 5.7 N HC1 at i i o ° for 16 h. Amino acid composition was determined by means of a Spinco model MOORE ANn STEIN automatic amino acid analyzer. RESULTS AND DISCUSSION
Fingerprints The haemoglobins separated from D.K.P.'S blood were subjected to the fingerprinting analysis. The photographs of the four fingerprints, obtained with the modified technique 1~ are shown in Figs. I and 2. The fingerprint of Hb-A shows a pattern of peptldes similar to that described by INGRAM9. The fingerprint of D.K.P.'s haemoglobin component characterized by an electrophoretic m o b l h t y equal to that of Hb-C shows a pattern of peptides equal to B~och~m B,ophys .4cta, 48 (I961) 253-265
d
~o
k_
e0 Fxg l Photograph of f i n g e r p r i n t s q of h a e m o globins \ a n d Gphlla Electrophoresls was m the usual buffer, but on a (ooled bras~ plate at about 3° V/cm for 2 h -Xscendlng chlomatogr a p h v la is in p y n d m e lsoainyl alcohol water (35 35 3 " , v / v )
>
t~
>
CHEMISTRY OF HAEMOC~LOBI~IS A, G, C, X
257
e" O ~ 0
~r5
B~och~m. Bwphys. Acta, 48 (I96r) 253-265
258
C. BAGLIONI, V. M. INGRAM
c~ ®
C32e +
(-)
0 ~, ~
Hb-A
(+)
e
~,
3b (: ~ )
¢b
t.,0,
=
:
÷
(-)
Hb-G
(+)
Fig. 3. Tracing of f i n g e r p n n t s of haemoglobins A and Gphtla Peptldes A- 3, A-9, G-3b, G-3b ox and C--9a are marked m black. Peptlde A- 4 is shaded.
~0
.- . 3b(:gb) ......
--~'
3 b o×
4a :-x..
90
+
(--)
1.4b -X
(-)
Hb-¢
(+)
(+)
Ftg. 4- Tracing of fingerplants of haemoglobins X and C Peptldes G-3 b, G-3b ox a n d G-9a are marked m black, peptldes C-4a and C-4b are shaded
Bzo~htt~z Btoph>s 4c/a, 48 (1961) 253-265
CHEMISTRY OF HAEMOGLOBINS A, G, C, X
259
t h a t described b y HUNT AND INGRAM21 for Hb-C. One can see (Figs. I and 2) the absence of peptide A- 4 and the appearance of two new peptides, C-4a and C-4b, resulting from the cleavage b y trypsin of the peptide analogous to A- 4 where In haemoglobin C a lysine substitutes for a glutamic acid residue. In the Figs. 3 and 4 are shown the tracings of the fingerprints, the peptide A- 4 in Hb-A and the peptides C-4 a and C-4b in Hb-C are indicated by shading. In the fingerprint of the Hb-Gpuna, peptide A- 3 is missing from the band of uncharged peptldes; instead three positively charged peptldes, called G-3b, G-3b ox, and G-9a, which are not present in fingerprints of Hb-A, are evident. The reasons for indicating these peptides in this way will be made clear below. Later reexamination by ionophoresls at p H 4.7 of the area of the fingerprint which has peptides G-3b and G-3b ox showed the absence of peptide A- 9. Therefore in haemoglobin G peptldes A- 3 and A- 9 have disappeared to be replaced by G-3b ,G-3b ox and G-9a (see Figs. I and 3 )and G-3a (see later). Peptide 3 is known to contain histidlne and methlonine, since it gives the reactions on paper which are characteristic for these amino acids 22. B y staining Hb-G fingerprints with sulphanilic acid 22 for histidine and with platlnic iodide 22 for methionine or cystelne, it was shown that peptide G-3b gives a positive reaction while peptide G-9a gives no reaction for those amino acids. Amino acid analysis (see below) showed the a m m o acid reacting positively with platinic iodide to be methionine. The meth~onine contained In peptide G-3b undergoes partial oxidation during the tryptic digestion and the fingerprinting, a proportion of it being transformed into methionine sulphoxide. The resulting peptlde has an electrophoretic mobility equal to the peptide containing methionlne, but behaves differently in chromatography, having a lower RF. This phenomenon is known to occur in other peptides, for example, peptides A-25 (containing methionlne) and A-2 4 (containing the sulphoxide), which have the same electrophoretic mobility but a lower RF in chromatography. In addition peptide A- 9 shows sometimes a partial separation into two components, which contain respectively the oxidized and the reduced form of methionine. It is easyto differentiate the two peptides, because the one containing methionlne sulphoxlde, while having all the properties in common with the peptide containing methionine, gives no reaction with the platlnic iodide reagent 22. The peptide G-3b containing methionlne sulphoxlde is mdlcated as G-3b ox (ox for oxidized). The fingerprint of H b - X shows the abnormalities characteristic of both Hb-C and Hb-G Both peptides A- 3 and A- 4 are absent. In addition to a normal pattern of peptides four new positively charged peptides are present, namely the two described b y HUNT .a~ND INGRAM21 in Hb-C and the two found in the Hb-Gl, htia fingerprint (Fig i). In the Figs 3 and 4 are shown the tracings of the fingerprints of haemoglobin Gl~hlla and X; peptides A-3 in Hb-A fingerprints, G-3b and G-9a in Hb-G and H b - X fingerprints are marked in black. The peptldes C-4a and C-4b In the H b - X fingerprint are shaded H b - X shows two abnormalities, being a combination of the pattern characteristic of Hb-C and that of Hb-G. However, these findings do not show whether the chemical change, which takes place in Hb-G, is situated on the a- or on the B-chain of haemoglobin. The fingerprint of the isolated a-chain has indeed shown that a peptlde with the properties of peptlde 3 belongs to the a-chain, while no peptide with similar properties was found in the B-chain fingerprint 23. Btoch~m B~ophys Acta, 48 (I961) 253-265
260
C BAGLION1, V. M INGRAM
Two sets of analyses of the amino acid composition of all the tryptic peptldes from the a-chain of haemoglobin A have so far been reported by \:ON HILSE AND BRAUNITZER 24 and by HILL AND I{ONIGSBERG ~a. The latter authors also determined the N-terminal amino acid of each peptide. Therefore, we decided to analyze peptide A-3 and peptides G-3b and G-9a, to ascertain whether peptide A- 3 IS indeed an ~-cham peptide and to discover the ammo acid substitution which had occurred in our HbGPwla and in Hb-X.
Peptides C-4a and C-4b Preparation and analysis ofpepSdes. The peptldes C-4a and C-4b were prepared by paper electrophoresis accordmg to HUNT AND INGRAM21. Amino acid analysis of peptide C-4a showed the presence of glutamic acid and lysine only. Amino acid analysis of peptide C-4b gave lysine, histidine, threonme, proline, valine and leucine in equal amount These compositions agree with the compositions reported by HUNT AND INGRAM21 and imply a lysine for glutamic acid substitutmn in the expected peptide of the fl-cham It was concluded that the haemoglobin present in D.K.P.'s blood is authentic Hb-C, since it has the same electrophoretlc mobility of Hb-C and shows the same amino acid substitution.
Peptides A-3 and A-9 Peptide A-3 was prepared by electrophoresls at pH 6.4 of a Hb-A dlgestg; the band of uncharged peptides (see Fig. 5) was cut from the paper and eluted. The eluate was then separated by electrophoresis at pH 4.7, applying 30 V/cm for 4 h. The separation at pH 4-7 IS shown in Fig. 6. After the second electrophoretlc separation, peptide A- 3 was obtamed almost
,
O0 @000¢ 09 00 000
22
21
20
t9
18
17-15
14-)0
9
~ band
23
;~4-25
26
Fig 5- Comparison by one-dimensional paper electrophoresls9 at pH 6 4 of the tryptlc digests of haemoglobins A, C, Gphtta and X Peptides are marked as in Fig 3 a n d Flg. 4
O0 <+>
<_>
0 0 N@ t
2
5-6
3
@ 4
Fig. 6. Paper electrophoresm a t p H 4.7 of the trypelc peptides isolated from the neutral bands (see Fig. 5) of haemoglobms A and Gpwt~. Peptides containing hlstldine (posture reaction 2~ with P a u l y ' s reagent) are shaded
B*och~n Blophys. Acta, 48 II96I) 253 265
CHEMISTRY
O F H A E M O G L O B I N S A, G, C, X
261
pure. As a check of the purity the N-terminal amino acid was determined by SANGER'S methodiC; only DNP-valine was obtained. The amino acid composition of peptide A- 3 was determined (see Table I) and was found to be equal to the amino acid composition given by HILL AND KONIGSBERG25 for the a-chain peptide called by them Ta6, except for our finding of 2 histldme reslI
TABLE AMINO
ACID COMPOSITION
OF
THE
PEPTIDES
Peptutes A rrllno a c i d
Lys His Asp Thr Ser Pro Ala Val Met Leu Total residues
A-3
A "9
lb ( 2G-G-9b)
G_.9a
i.ii 2.13 5 7° 0.90 i 84 i.o9 6.78 3.22 (I) 4 oI
1.98 (2.59) 5 72 i 28 i 94 1.3 6.56 3.17 0.84 4.28
o 91 2 28 4oi -i 83 i.o 4 4.92 2.03 (I) 3.oo
1.9I -i 09 0.87 --2.I3 0.96 -i oo
21
8
28
29
dues compared with 3 reported by these authors. The N-terminal amino acid was reported to be valine 25, in agreement with our findings for peptide A-3. The amino acid composition of peptide A-3 is slightly different from the composition of a peptide found by HILSE AND BRAUNITZER24 in the tryptic digest of the a-chain and indicated by them as peptide I I ; our peptide A- 3 has 2 histidines instead of 3 and one more aspartic acid residue. Peptide A-3 is the largest peptide which is present in tryptic digests of haemoglobin apart from the "core." Its 28 amino acids constitute a large unit, which has no equivalent m the B-chain of haemoglobin, nor can it be confused with any other peptide. The absence of peptide A- 9 from the tryptic digests of Hb-G and Hb-X was rather surprising, since it could indicate that two peptldes are in some way changed by the same mutation. Peptide A- 9 is known to be present in variable amount in different tryptic dlgests of the same haemoglobin preparation; it is usually observed as a rather weak spot, which gives positive reactions for hlstidine and methionine. By fingerprinting the tryptic digest of the isolated a-chain, HUNT23has shown that peptide A- 9 is an a-chain peptide On the other hand the amino acid composition of the a-chain of Hb-A gives only two methionine residues per chain 24,25HUNT2Zhas instead found three methionme containing peptides in the fingerprint of the a-chain. These facts led us to suppose that peptide A- 9 has a definite relationship with peptide A- 3, since both give positive reactions for methionine and histidine and both disappear in Hb-Gl,hila. The peptide A- 9 was prepared by electrophoresis at pH 6.4 of the tryptic digest of Hb-A (see Fig. 5) and subjected to quantitative amino acid analysis (see Table I) Bzochtm. B*ophys
,4cta, 4 8 ( I 9 6 I ) 2 5 3 - 2 6 5
262
c. BAGLIONI, V. M. INGRAM
The amino acid composition of A- 9 was found to be almost equal to the composition of peptide A-3, except for the lvsine content, which is double in peptlde A- 9. When subjected to end group analysis by SANGER'S method '~, peptide A- 9 gives di-DNPlysine. With the exception of the higher value for hlstidlne, the analysis agrees well with the idea that peptlde A- 9 ---- Lys-(A-3). TABLE II N-TERMINAL AMINO ACID BY SANGER'S METHOD18
Peptzde
A- 3 A-9 G--3a G-9a G-3b ( = G-9b)
N-termmal
Valme Lyslne Vahne Lvsme Aianlne
Peptides G-3b and G-9a The peptldes G-3b and G-9a were prepared by electrophoresis at pH 6. 4 of the tryptic digest of Hb-G and Hb-X (see Fig. 5). Further check of purity by electrophoresis at pH 4.7 was considered to be necessary for peptide G-3b, since it overlaps in electrophoresis at pH 6.4 with the peptide A- 9. The eluate of the band containing the peptide G-3b was run side by side with a sample of peptide A- 9 isolated from the tryptic digest of Hb-A by electrophoresls at pH 6.4. No trace of peptide A- 9 was found to contaminate our peptide G-3b. The peptides G-3b and G-9a were consequently used without further purification. Analysed by SANGER'S end group method le, peptide G-3b gives DNP-alanine and peptide G-9a gives di-DNP-lysine.
Peptide G-3a The neutral band isolated by electrophoresls at pH 6. 4 of the tryptic digest of Hb-G (see Fig. 5) was eluted and subjected to the electrophoretic separation at pH 4.7. The neutral band from Hb-A was run along side (see Fig. 6). As expected, no trace of peptide A-3 was observed in Hb-G digest; a new faint peptide, not present in Hb-A, was instead observed. This peptide gives no reaction for histidine and methionine. It is also present in Hb-X. The yield of this peptide G-3a is rather poor and it was found to be not sufficiently pure in several preparations to give an accurate amino acid composition. The analysis of this peptide G-3a is rather similar to that of peptide G-9a, but showed only one lysine residue. Subjected tg end group analysis by SANGER'S method :e, peptide G-3a gives DNP-vahne. CONCLUSIONS
In order to prove unequivocally that peptides A-3 and A- 9 and that peptides G-3a and G-9a are the same peptides, except for the additional N-terminal lysine in A-9 and G-9a, the EDMAN analysis of the N-terminal sequence was undertaken according to SJOQUIST1~,so. The results are reported in Table III. The peptides A-3 and G-3a show the same N-terminal amino acid, valine ; peptides A- 9 and G-9a also show Bzoch~m Bzophys Acta, 48 (196:) 253-265
CHEMISTRY OF HAEMOGLOBINS A, G, C, X
263
the same amino acid, lysine. Except for the N-terminal lysine of peptides A- 9 and G-9a, all these peptides have the common N-terminal sequence Val. Ala.Asp. The presence of lysine in N-termmal position is rather unexpected, since trypsin is supposed to split the peptide bond between the carboxyl group of lysine and the amino group of the next amino acid in the peptide. We would expect, however, that TABLE I I I STEPWISE
DEGRA.DATION OF
Pept*de
~st step
2nd step
A-3 A-9 G-9a G-3 a
Valme Lysme Lysme Vahne
Alanme Vahne Vahne Alamne
P E P T I D E S 19
~rd step
Aspartlc Alanme Alanme Aspartic
4th step
-Aspartic Asparttc --
an N-terminal lysine residue is split at a very slow rate, if at all, by trypsin 26. Since the lysine of peptides A- 9 and G-9a is not in N-terminal position in the whole a-chain (the N-terminal amino acid of the a-chain is valine), we have to suppose that somewhere along the polypeptide chain there is a sequence such as L y s . L y s or Arg. Lys. The trypsin will alternatively split the peptide bond between the two basic amino acids or between the lysine and the next amino acid. When the first split occurs between the two basic amino acids, the lysine will occur in the N-terminal position of the following peptlde where it will be removed only slowly by the trypsin. This explanation allows us to describe the relationship between the peptides A-3 and A- 9, G-3 a, G-9a and G-3b (see Table IV). The amino acids suffering the mutational change in Hb-Gphna and Hb-X are underlined. In this figure amino acids whose sequence is unknown are placed inside brackets. The provisional assignment of amide groups to two of the four aspartlc acid residues in peptide G-3b is based on the electrophoretlc behaviour of this peptide and of peptides A-3 and G-3a at pH 6. 4. Peptide G-3b is basic at this pH. Since it contains 2 histidine and I lysine residue TABLE IX"
Pept,de A -3 Val. Ala. Asp(Leu,Ala,Thr)Asp[NH,] • Ala(HlsvAspi, Asp[NH2]~,Ser2,Alaa,Pro,Val2,Met,Leu3) Lys
Pept~de G-3a Val. Ala. Asp(Leu, Ala,Thr) Lys
Pept,de G-3b Ala(Hls2,Asp~,Asp[NH~]2,Ser2,Ala2,Pro,Val2,Met,Leu3) Lys Pept~de A-9 Lys. Val. Ala- Asp(Leu,Ala,Thr) Asp [NH~] • Ala(Hls z,Aspz,Asp [NH~]z,Ser vAla4,Pro,Val2,Met,Leua) Lys
Pept,de G-9a Lys- Val. Ala. Asp(Leu, Ala,Thr) Lys (Peptlde G-9b ~ G-3 b)
B,och,m. Biophys Acta, 48 (I96I) 253-265
264
C. BAGLIONI, V. M. INGRAM
not more than 2 aspartlc acid residues can be free. Again, peptlde G-3a IS uncharged; hence the first aspartic acid residue in A- 3 must be free The second aspartlc acid residue in A- 3 on the other hand is hkely to be covered by an amide group, since it~ substitution by lysine in the whole haemoglobin G molecule gives rise to a change in electrophoretic mobility corresponding to one charge unit per a-chain Therefore, and also because peptlde A- 3 is uncharged, we would expect two free and two covered aspartlc acid residues in peptlde G-3b These arguments wait to be supported by the determination of the complete sequence of peptide A- 3. The amino acid compositions of peptides G-3a and G-3b together give the composition of peptide A-3, except for one aspartic acid residue missing and one addlt,onal lvsme residue present in G-3a G-3b Similarly, the sum of G-9a and G-3b has the same relation to A- 9" A-3 = G-3a + G-3 b + L y s - - A s p [NH2] A-9 = G-9a + G-3b + L y s - - A s p [NH2~
The extra lysine present in Hb-G provides the additional cleavage site for the action of trypsin and therefore peptides A- 3 and A- 9 are split into two peptides, G-3a and G-3b, G-9a and G-9b ( = G-3b) respectively. The new lysine in Hb-G probably substitutes an asparagine residue. The evidence for an asparagine and not an aspartlc acid being substituted by lysine rests on the consideration of the charge difference between Hb-Gputl~ and Hb-A. Double the observed charge difference would be expected from an aspartic acid to lysine substitution, by analogy with the behaviour of Hb-C NOTE ADDED IN PROOF
Since this paper went to press additional information has become available which together with the results in the present paper, shows that the asparagme residue of peptide A- 3 and A-9, which undergoes the mutational change, occupies position number 68 from the N-terminus of the peptide chain (BRAUNITZER e~ al.2S; CRAIG et al.*9). RAPER e~ al. 8° have reported an analogous situation in which four haemog l o b i n s - A , G, C and G / C - - a r e found in one person, a double heterozygote for the G and C abnormalities. Their characterisation of the four haemoglobins rests on experiments involving dissociation and reassociation of the various component haemoglobins. Received January 28th, 1961 ACKNOWLEDGEMENTS
This work has been supported by grants from the National Science Foundation and from the National Institute for Arthritis and Metabolic Diseases, U.S. Public Health Service. REFERENCES
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