K. LAKI 
479 
rate of clotting of human, dog, rabbit and pig 
fibrinogen falls between those of sheep and 
goat. These thrombins apparently confuse va- 
line with glycine, aspartic and glutamic acids at 
position 13. The question arises why do these 
thrombins confuse glycine, aspartic and glu- 
tamic acids with valine? The answers may be 
that residues located at positions further away 
may have an effect at position 13. 
Inspection of Table I shows that what distin- 
guishes the human, dog, rabbit and pig peptides 
from the others, is that at position 16 these do 
not have serine. Also, that human, dog, rabbit 
and pig do not have aspartic acid residues at po- 
sition 18. We may assume that thrombin can 
discriminate strictly the amino acid residues at 
position 13 only if serine is located at position 
16, or if aspartic acid is located at position 18. 
For the purpose of further speculation, let us 
assume that it is position 18 which influences 
position 13 because the conclusions coming 
from this assumption may be checked. In order 
that aspartic acid at position 18 may influence 
the action of thrombin, the peptides at this posi- 
tion also would have to interact with thrombin. 
Since aspartic acid carries a negative charge, 
we may attribute the influence of aspartic acid 
at position 18 to a positively charged group on 
the thrombin molecule. 
In the case of chymotrypsin, it is well estab- 
lished tlhat there is indeed a positively charged 
group on the molecule that influences the activ- 
ity of the enzyme. This group has been identified 
as the N-terminal isoleucine.^'^-^^ Accord- 
ing to current views, the amino group of isoleu- 
cine forms a salt bridge with the aspartic acid 
residue located next to the serine residue of the 
active site. This "activation salt bridge" is sup- 
posed to regulate the activity of the enzyme. 
Thrombin also has N-terminal isoleucine.^** The 
aspartic acid at position 18 may then be as- 
sumed to interfere with this "activation salt 
bridge" by competing for the amino group of 
the terminal isoleucine. 
From these considerations, we may conclude 
that if peptide A contains aspartic acid residue 
at position 18, this residue, by forming an elec- 
trostatic (or hydrogen bond) with the N-ter- 
minal isoleucine of thrombin, enables it to 
discriminate among the different residues that 
occur at position 13, probably because in such a 
case, the peptide chain is tied down at position 
18. These considerations suggest that peptide A 
may be attached to thrombin at positions 13, 18, 
and perhaps at 16, as well. 
The dissociation of the ct-amino group of the 
N-terminal isoleucine in chymotrypsin is known 
to influence the acylation step of this enzyme. 
Because of the great similarity of thrombin to 
trypsin and chymotrypsin,'"*^-^- we may tenta- 
tively conclude that the changes brought about 
in peptide A at position 18 during evolution 
afi'ect the acylation step in the thrombin-fibrino- 
gen interaction. At the beginning, our assump- 
tion was that the structure of peptide A has an 
effect on the rate with which thrombin splits off 
this peptide from fibrinogen. This assumption 
led to interesting consequences indicating that 
it was justified. 
During evolution, thrombin also underwent 
modifications. This is discernible from the qual- 
itative and the quantitative response with 
which thrombins adjust to the changes in fibrin- 
ogen: (1) different thrombins respond differ- 
ently to changes taking place in fibrinogen ; (2) 
also, different thrombins magnify these changes 
to a different extent. 
SUMMARY 
The experiments discussed show that during 
evolution both fibrinogen and thrombin have 
changed. When the different rates of clotting 
were compared to the amino acid sequence of 
the peptides released in the clotting reaction, a 
correlation could be detected between the speed 
of reaction and the amJno acid residues at a cer- 
tain locus in the peptides. The observations sug- 
gest that the peptidylation (acylation) of 
thrombin in the enzyme substrate interaction is 
influenced by the amino acid residues placed at 
a given position by the evolutionary change. 
REFERENCES 
1. Gilliam, M., and Shimanuji, H. Progress re- 
port: studies on honey bee blood. Amer. Bee J. 
107:256, 1967. 
2. Levin, J., and Bang, F. B. A description of cellular 
coagulation in the Limulus. Bull. Johns Hopkins 
Hosp. 115:337, 1964. 
