Dell, Anne

Magnusson, Staffan

Morris, Howard R.

Petersen, Torben Ellebæk

Sottrup-jensen, Lars

Elsevier - Publisher Connector 

Primary structure of the vitamin K-dependent part of prothrombin 

Volume 44. number 2 FEBS LETTERS August 1974 
PRIMARY STRUCTURE OF THE VITAMIN K-DEPENDENT PART 
OF PROTHROMBIN 
Staffan MAGNUSSON, Lars SOTTRUPJENSEN and Torben Ellebzek PETERSEN 
Department of Molecular Biology, University of Aarhus, 8000 irhus C, Denmark 
and 
Howard R. MORRIS and Anne DELL 
Medical Research Council, University Chemical Laboratory, Cambridge CB2 IEW, England 
Received 2 July 1974 
1. Introduction 
The biosynthesis of normal prothrombin is vitamin 
K-dependent [ 11. During dicoumarol treatment a 
prothrombin is produced [2,3] which can neither be 
adsorbed to barium citrate nor bind calcium ions [4] 
and therefore is not activated in the normal Ca’+, 
phospholipid systems. One reason for determining 
the primary structure of prothrombin has been to find 
the structural modification caused by vitamin K. 
Analyses of carbohydrate and amino acid compositions 
as well as N-terminal sequences of normal [5-71 and 
dicoumarol prothrombin [8-lo] indicated no clear- 
cut difference between the two proteins and no un- 
usual constituents. 
The first clue to the identity of the structural 
modification was the isolation [ 1 l] of the tryptic 
peptides Gly-Phe-Leu-Glx-Glx-Val-Arg and 
Gly-Phe-Leu-Glx-Glx-Val-Arg-Lys from 
positions 4- 10 and 4- 11 in normal prothrombin. 
Both peptides were free of carbohydrate and carried 
two negative charges more at neutral pH than could 
be attributed to normal glutamic acid residues [ 12, 
131. The adsorption of a different tryptic peptide to 
barium citrate [ 141 indicated that this peptide had 
also been modified by vitamin K. Stenflo [ 151 has 
recently confirmed that peptides 4- 10 and 4- 11 
from normal prothrombin carry the extra negative 
charges, and found that the corresponding sequences 
in dicoumarol prothrombin contain normal Glu- 
North-Holland Publishing Company - Amsterdam 
residues. Only the peptide from normal prothrombin 
was found to bind Ca’+. The present paper describes 
the primary structure of residues l-42 of normal 
prothrombin and gives evidence for the structure of 
ten modified glutamic acid residues. 
2. Materials and methods 
Bovine prothrombin [5] was activated by dis- 
solving at 200 mg/ml in buffer containing 20-50 mM 
EDTA at pH 7.5. The detailed conditions of activation 
and of the isolation of the activation products on a 
DEAE-Sephadex A-50 column are described elsewhere 
[12,16]. The A-fragment containing the N-terminal 
156 residues, and prothrombin have both been used 
as source material. The complete primary structure 
of prothrombin will be described elsewhere [ 161. 
Peptides were obtained from digests (and sub- 
digests) of prothrombin and of A-fragment using 
trypsin (T), chymotrypsin (C), elastase (E), thermol- 
ysin (TL), proteinase K (K), and pronase (Pr) under 
standard conditions [ 161, and by partial acid hydrol- 
yses (PA) (formic/acetic acid pH 2.1, 1 lO”C, 15 hr). 
Peptic (P) digests (E:S I:20 molar ratio, 37°C) were 
performed either at pH 2.1,20 hr on heat-treated 
(15O”C, 1 hr) peptides or at pH 1.2,40 hr without 
prior heating. 
Peptides were isolated by a combination of 
standard methods of gel filtration (G-25), chromato- 
189 
Volume 44, number 2 FEBSLETTERS August 1974 
graphy (DEAE-cellulose), high-voltage electrophoresis 
(pH 6.5, 3.5 and 2.1) and paper chromatography 
[ 161. Details of cystine modification, amino acid 
analysis and sequencing methods (DNS-Edman) will 
be given elsewhere [ 161. Paper electrophoretic 
mobilities were determined [ 171. Mass spectrometric 
analyses have been performed on peptides 4- 10, 
S-11, 14-31 (split at 25/26), 14-21 and 31-35, 
according to [ 181 using the permethylation conditions 
of [19]. 
3. Results and discussion 
The sequence of amino acid residues l-42 is given 
in fig. 1. The modified glutamic acid residues having 
an extra acidic group have been tentatively assigned 
the three-letter code Gla (analogous with Cya). In 
hydrolysates (6M HCl, 1 lO”C, 18 hr) Gla-residues 
show up as Glu both on paper electrophoresis and on 
the ammo acid analyzer. Therefore amino acid com- 
position data will be given elsewhere [ 161. No hexos- 
8 9 11 12 13 14 15 17 18 19 
Ala -A&-L$ _GTy c$ecL&uG:&GLA;Val <iFg -Lys -Gly -Asn -Leu -GLA-$ -GLA-Cys -Leu-C?kC?A- 
T1 
, I 
T2 T4 
CONT 
T3’ ’ T4Ei ’ ’ 
’ CT 
, r . , I I 
TL41 TL5 
CCN 
TLl TL2 
TL3 TL5 Pl 
El E2 E3 
ColiT 
E3 Pl E3+2 ’ 
’ ma 
> 
C,TL,Pl 
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 
pro _Cys_Ser -Arg _GLA-GLA-Ala -Phe-GLA-Ala - Leu-GLA-Ser - LeU-Ser -Ala -Thr -Asp-Ala - Phe-Trp 
Td 
T4Ei 
T4E2 ’ - T4ES . . . 
TL5 
TL5 kl ’ ’ ’ TL5k2 ‘TL7 ‘TL81 
TL6’ ’ 
1 
E3 
“” ’ ’ ’ ’ ‘E3P3 ‘E3t4 ’ ‘E3ti5 ’ ’ 
C,TL:P2 
, / 
C;TL,PS ‘Cl’ ’ ’ ’ 
P, 
Prl PA1 
Fig. 1. Amino acid sequence of residues l-42 in bovine prothrombin. GLA = 7-carboxyl glutamic acid. Solid lines indicate amino 
acid composition of peptides. Half-arrows below lines indicate DNS-Edman sequence results. Half-arrows above lines indicate mass 
spectrometric sequence results; complete arrows both. Tryptic peptides Tl-3 were obtained from reduced, carboxymethylated 
A-fragment. T4 was obtained from native prothrombin digested while adsorbed to barium citrate, eluted with EDTA, reduced, 
carboxymethylated, redigested. T4 was subdigested with elastase. Thermolytic peptides TLl-8 were obtained from native 
A-fragment. TLS was subdigested with pepsin and proteinase K. Elastase peptides El -3 were obtained from reduced, carboxy- 
methylated A-fragment. E3 was subdigested with pepsin after heating. Peptides C, TL, P l-3 were obtained from reduced, 
carboxymethylated prothrombin, digested with chymotrypsin, adsorbed to barium citrate, eluted with NazS04, digested with 
thermolysin. The C, TL peptide 14-35 was then subdigested with pepsin at pH 1.2. Peptide Cl was isolated from CNBr-fragment 
l-72 of reduced, carboxymethylated A-fragment. Pronase peptide Pr 1 was obtained from native prothrombin, then adsorbed 
to barium citrate and eluted with Naa SO4, then oxidized with performic acid and subjected to partial acid hydrolysis. 
190 
Volume 44, number 2 FEBS LETTERS August 1974 
Table 1 
Electrophoretic mobilities at pH 6.5 (before and after heating) and pH 2.1 of GLA-containing peptides 
T2 
T3 
TL2 
E2 
T4 
T4 El 
TL5 
C, TL, Pl 
TW K2 
TL6 
C, TL, P2 
C, TL, P3 
Position in pH 6.5 Expected for pH 6.5 (heated pH 2.1 
sequence Mob./Asp Charge Glx=GLA Glx=Glu Mob./Asp Charge Mob./Ser charge 
4-10 0.72 -3 -3 -1 0.32 -1 0.75 +1.5 
4-11 0.46 -2 -2 0 0.22 -1 1.14 +2.7 
5--11 0.44 -2 -2 0 0.21 -1 
5-12 0.52 -2 -2 0 0.0 0 1.11 +2.5 
12-42 0.88 -9 -17 -9 0.23 +0.7 
12-35 0.83 -8 -16 -8 
14-34 1.00 -9 -14 -6 0.53 +2 
14-21 0.99 -5 -8 -4 0.84 -4 
33-34 1.06 -2 -2 -1 0.68 -1 
32-34 0.85 -2 -2 -1 0.57 -1 
22-30 0.92 -6 -6 -3 0.63 -3 0.54 +l 
31-35 0.70 -2 -2 -1 0.45 -1 0.54 +0.5 
The ‘expected’ charges in columns 5 and 6 are based on all Glx residues being either y-carboxyl glutamic acid or glutamic acid. 
amine peaks and no charring on hydrolysis was 
observed, indicating absence of carbohydrate. In the 
DNS-Edman procedure used the hydrolysis conditions 
(6M HCl, 110°C 10 hr) caused the formation of 
DNS-Glu from DNS-Gla. Mobility data for those 
peptides which contain Gla are given in table 1. 
The mobility data at pH 6.5 can be explained by 
two negative charges on each of Glx-residues 7,8, 26, 
27, 30, 33 and on at least one of Glx-residues 15, 17, 
20, 2 1. At pH 2.1 the negative charges attributable 
to Gla were almost completely neutralized. After 
heating all remaining negative charges at pH 6.5 could 
be accounted for by normal Glu-residues (and CM- 
CYS). 
The first sample used for study by mass spectro- 
metry was peptide 14-3 1. A peptide of this size is 
not amenable to examination following acetylation 
and esterification alone, and we therefore prepared 
the acetyl permethyl derivative [ 191. One of the less 
complex spectra is reproduced in fig. 2. The presence 
of two related sequences could be assigned as follows: 
X - Ala - Phe - Y - Ala - 
via signals at m/e 112,225,386,557 and 642, and 
X - Y - Ala - Phe - Y - Ala - 
via signals at m/e 112, 311, 396, 557, 728 and 813. 
250’~ 
V +-Ala- 
I-X+t----Ale----t< Phn ,._ 
21, 
I/ 1 
100 200 300 
x2.5 
396 
3% p 557 ’ 642 728 ’ 813 
%i% L ‘YtJ-h L /1 ii ‘I m/e 700 800 
Fig. 2. Partial mass spectrum (above m/e 100) from the 
acetylated permethylated peptide 14-31. 
The new amino acid derivative Y corresponds to a 
normal glutamic acid derivative with an extra methyl 
substituent. Similarly, X corresponds to cyclised Y, 
i.e. analogue of pyrrolidone carboxylic acid in normal 
spectra [ 181. The partial sequence obtained clearly 
correlates with residues 26-3 1. 
Other spectra of peptide 14-3 1 showed signals 58 
mass units above the signals in fig. 2 indicating that X 
and Y are formed by decarboxylation during derivative 
formation. This has been confirmed by deuterium 
labelling and high resolution mass measurement (full 
191 
Volume 44, number 2 FEBS LETTERS August 1974 
details to be reported elsewhere [20]). 
At this stage in our work, Stenflo proposed that 
residues 7 and 8 from peptide 6-9 (fig. 1) are both 
y-carboxyl glutamic acids [21]. This was based upon 
NMR data, and on the mass spectrum of the acetylated 
permethylated peptide. The spectrum obtained by 
these workers, using our permethylation procedure 
[ 191, showed no evidence of N-methylation. In con- 
trast, using the same procedure, we find the expected 
fully N-methylated and C-methylated derivative of the 
new amino acid with 229 mass units, or the decarboxyl- 
ated product of 171 mass units. Although our mass 
spectrometric data differ, our results substantiate the 
assignment of y-carboxyl glutamic acid made above 
]211- 
In addition to our new assignment of y-carboxyl 
glutamic acid to residues 26,27 and 30 (giving rise 
to X and Y in fig. 2 upon derivative formation), we 
have also examined peptides 4- 10,5- 11, 14-2 1 and 
31-35 by mass spectrometry. We find that y-carboxyl 
glutamic acid is present not only as residues 7 and 8, 
in agreement with Stenflo’s work [21], but also as 
residues 15, 17, 20, 21 and 33. We therefore have 
sound mass spectrometric evidence to show that all 
ten glutamic acids in the N-terminal region of pro- 
thrombin are substituted with a y-carboxyl group. 
Full details of the mass spectra, mechanistic and 
fragmentation assignments, isotope and high resolution 
data on all the peptides studies will be reported [20]. 
In order to detect peptides containing Gla-residues 
we developed a ‘thermal diagonal’ technique in which 
the peptide mixture is subjected to electrophoresis at 
pH 6.5 in the first dimension, then heated, and 
finally subjected to electrophoresis at pH 6.5 in the 
second dimension. Decarboxylated peptides moved 
off the diagonal. 
The mass spectrometric finding that residues 7, 8, 
15, 17,20,21,26,27, 30 and 33 are y-carboxyl 
glutamic acids explains the observed electrophoretic 
mobility data for the peptides*, with heating resulting 
in partial or complete decarboxylation. Apparently, 
then, the function of vitamin K in blood coagulation 
is to cause the post-synthetic modification of all 
glutamic acid residues in the N-terminal parts of the 
* Except with regard to GLA-20 and -21 where full negative 
charge has not yet been observed. 
192 
vitamin K-dependent coagulation factors (X, IX, VII 
and prothrombin) to make them strongly calcium- 
binding. Whether vitamin K plays a more genral role 
in the biosynthesis of other calcium-binding proteins 
remains to be seen. 
Acknowledgements 
S. M., L. S. and T. E. P. wish to thank Laila Br$ns, 
Lene Kristensen, Lone Christensen, Annette Larsen, 
Jette Ulrich and Margit Skriver for technical assistance 
and the Danish Science Research Council and the 
U.S. National Heart and Lung Institute (grant number 
1 RO 1 HL16238-01 HEM) for financial support. 
L. S. received a fellowship from the Svend Coles 
Frederiksen foundation. H. R. M. and A. D. wish to 
thank Mrs M. R. Thompson and Mrs W. Every for 
technical assistance, and the British Medical Research 
Council and 185 1 Trust for financial support. 
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193 


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Primary structure of the vitamin K-dependent part of prothrombin

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