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Evolutionary Aspects of the Structure of Proteins.

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probable from the chemical point of view. However,
this conflict would be resolved if the blue color in the
commelin, as in the larkspur, were due to the combination of a polysaccharide with the anhydro-base cf
the delphinidin glycoside, and if the magnesium were
simply an impurity that is difficult to remove. It is
probable, therefore, that ffayashi et al. 12x1 are the first
to have isolated a member of this class of macromolecular pigments.
The bonding of anthocyanins to pectins is probably
also involved in the coloring of fruits. Thus it has been
known for some time that the pigment of red grapes
can be dissolved more easily in order to obtain an
intensely red wine if an enzyme that attacks pectins, e.g.
vinibon [301, is added.
We thank the Deutsche Forschungsgemeinschaft and the
Fonds der Chemischen Industrie for their support of this
work.
Received: May 31st, 1966
[A 532 IE]
German version: Angew. Chem. 78, 834 (1966)
Translated by Express Translation Service, London
[30] E. Vogt: Der Wein. 3rd Ed. Eugen Ulmer, Stuttgart 1595,
p. 129.
Evolutionary Aspects of the Structure of Proteins
BY PROF. R. ACHER
LABORATOIRE DE CHIMIE BIOLOGIQUE, FACULTE DES SCIENCES, PARIS (FRANCE)
The evolution of protein structures is discussed using cytochrome c, hemoglobin, and neurohypophyseal hormones as examples. Although these substances have different biological
functions, their evolution is controlled by the same general rules: their primary structures
vary at the level of the species, order, or class, but this variation is restricted by the fact
that the biological activity o f the protein must not be impaired. Alterations (i.e. substitutions,
deletions, or additions of amino acid residues) can therefore occur only in certain positions
Gf the peptide chains, although with dq7erent frequencies. The total number of alterations
thus represents only the final state of a protein and does not take into account successive
substitutions which may have taken place at the affected sites. It can therefore give only
a rough indication of the phylogenetic distance between two species. The nature of the
substituting residues, on the other hand, is a useful guide to zoological cognateness, since
it allows the identiJ5cation of transition molecules which simultaneously contain amino
acid residues from the protein of the evolutionary ancestor and from the protein of the
evolutionary descendant.
Although normally observed on the morphology of
organisms, evolution must ultimately be due to changes
at the molecular level, since every hereditary modification in the phenotypes results from a genetic mutation,
i.e. a change in deoxyribonucleic acid (DNA). This
does not mean, however, that the “laws” of the evolution of organisms apply to molecular evolution. Thus,
whilst genetic mutations are random and anarchic, the
evolution of organisms seems to be oriented in a certain
direction by natural selection, which filters the multitude
of genetic mutations and makes new beneficial characteristics dominant. The question, however, is to what
extent is morphological “orthogenesis” connected
with molecular “orthogenesis”, i.e. to what extent is
morphological evolution parallelled by chemical
changes. This question can be answered only by selecting
the operative molecules from various species and by
arranging them in the order attributed to phylogeny. If
we can then recognize a stepwise change in the molecules that can be correlated with the steps of anatomical
evolution, we can begin the more direct, biochemical
investigation of the mechanism of evolution.
The new task in this connection is to examine the
chemistry of the genes, which are responsible for the
798
transmission of existing characteristics and for the
appearance of new characteristics through mutations.
It is now accepted that genes are deoxyribonucleic acids
and that their specific action depends on the sequence
of the constituent nucleotides. If these deoxyribonucleic
acids could be isolated and analysed, we could directly
compare the genetic materials of various species. While
this is not yet possible, we can isolate and characterize
the “products” of the genes, i.e. enzymes and, more
generally, proteins. It has emerged from the experiments
of Yanofiky et al.[ll that the sequence of the amino
acid residues in proteins corresponds to the sequence
of nucleotides in DNA (concept of colinearity). We
may therefore study mutations as reflected in the
corresponding proteins.
Proteins are characterized not so much by the number
and the nature of the constituent amino acids, as by
their sequence. Mutational changes in this structure
can occur by insertion, deletion, or substitution of the
amino acid residues. A unit change can therefore be
defined as one which affects a single residue in the
chain. We can also determine the variation coefficient,
C.Yanofsky, B. C. Carlton, J . R. Guest, 0 . R . Helinski, and
U.Henning, Proc. nat. Acad. Sci. USA 51, 266 (1954).
[l]
A n g e w . Chem. internat. Edit.
/ Vol. 5 (1966) / No. 9
i.e. the ratio of the number of changed residues to the
number of unchanged residues. Comparison of homologous proteins of fishes, amphibians, reptiles, and
mammals shows that a structural pattern remains
unchanged in all these classes. This usually involves
the length of the peptide chain, the prosthetic group,
and the nature of the amino acids in certain positions.
This unchanged structural pattern is probably linked
with the biological role of the particular protein.
number of the mutable sites. Thus it is not the
total number of substitutions in the chain, but the
nature of the amino acids on sites with frequent
variations that enables us to trace the evolution of a
protein, and it should be possible to characterize in
certain zoological groups transition molecules with
structures intermediate between those of proteins of
more primitive species on the one hand, and of more
highly evolved ones on the other (Fig. 1, right).
Interestingly, the length of the peptide chain of homologous proteins is the most stable parameter. All the
cytxhromes c of the vertebrates so far investigated
consist of a chain with 104 amino acid residues, and all
the neurohypophyseal hormones of fishes, amphibians,
birds, and mammals contain nine amino acid residues.
Evolutionary changes seem, therefore, to be restricted
to substitutions of amino acid residues in the chain,
insertion and deletion occurring only rarely.
Since the evolution of the vertebrates is the best known,
and since proteins from vertebrates are relatively easy
to obtain, they have been the subject of most studies.
For practical reasons, the proteins selected must be
produced by the organisms in sufficient amounts, they
must be amenable to rapid purification, and they must
have a relatively small molecular weight permitting
complete structural analysis in a reasonably short time.
Hemoglobins and cytochromes c (abundant and easy to
purify), and the neurohypophyseal hormones (structurally simple) fulfil these conditions. Although they
have different biological functions, they agree in that
their molecules vary from species to species or from
class to class.
Furthermore, investigations of the mutations of tryptophan synthetase [I]. hemoglobins [21, and tobacco
mosaic virus (TMV)[31 have shown that a genetic
change generally affects only one amino acid residue
at a time. We may therefore consider an amino acid
substitution as a unit evolution. .If this is correct, the
evolution of the vertebrates should be accompanied by
an increase in the number of substitutions. Substitutional
similarity will then be a criterion of interspecific
kinship, and a scheme of evolution can be based on the
number of substitutions (Fig. 1, left), provided that
1
2
3
4
1
2
3
4
Ala-Val-Thr-Leu
Ala-Val- Thr-Leu
Ancestral molecule
Ancestral molecule
1
Gly-Val- Thr-Leu
-
i
I
-
Ma-Gly-Thr-Leu
-1
_-
Ma-&- T h r - Leu
9 - I l e -Ser-Leu
Ala-Ile-Ser-Leu
G 2 - e - Thr-Leu
1
--I
9 - I l --e-Ser-Met
--
1
_-
1
M a - Phe- Ser-Leu
--
Fib. I. Evolution of proteins having e q u a l (left) or u n e q u a l (right)
frequencies of substitution a t various sites in t h e chain (schematic).
mutation affects all positions of a peptide chain with
equal probability. However, this is not the case, since
in the proteins of vertebrates some positions are never
affected while others are involved with various frequencies. The biological function of the molecule does
not allow just any substitution, and it is probably the
position in the chain which determines whether an
amino acid will be a permanent feature or substitutable,
and if so, with what probability. The number of
immutable positions often is equal to or exceeds the
[2] C. Baglioni in J. H.Taylor: Molecular Genetics. Academic
Press, New York, London 1963, Part I. p. 405.
[3] A.Tsugita and H. Fraenkel-Conrat in J . H.Taylor: Molecular Genetics. Academic Press, New York, London 1963.
Part I, p. 477.
Angew. Chem. internat. Edit. 1 VoI. 5 (1966)
No. 9
I. Cytochrome c
Among the proteins involved in the respiratory chain
of aerobic organisms, cytochrome c is one of the easiest
to purify, since it is easily soluble and, like most basic
proteins, easy to process on catisn exchangers. Cytochrome c has been isolated from vertebrates, invertebrates, microorganisms, and some plants. In a number
of cases it has been crystallized. Cytochrome c of the
heart of vertebrates has been investigated in particular,
since this organ contains a relatively large amount of
the enzyme. The cytochromes c from various classes of
vertebrates all contain 104 amino acid residues, a single
prosthetic group (heme), have an isoelectric point close
to p H 10 and a redox potential close to +250 mV. Furthermore, they all react with mammalian cytochrome
oxidase. Obviously their function has suffered no change
in the course of evolution, although their structures
have been modified by amino acid substitutions. It has
not yet been possible to relate the steps of chemical
change with the steps of morphological evolution,
although the number of amino acid substitutions
increases as we approach the top of the evolutionary
ladder.
1. The Structural Pattern
We denote by the term “structural pattern” the totality
of structural features that do not show any interspecific
variation. The structural pattern of the cardiac cytochromes c of 12 species belonging to three classes of
vertebrates 14.51 is characterized by a chain of 104 amino
[4] E. Margolinsch and E . L . Smith i n V . Bryson and H . 1.Vogel:
Evolving genes and proteins. Academic Press, New York, London 1965, p. 221.
[S] E. L. Smith and E. Margolinsch, Fed. Proc. 23, 1243 (1964).
799
acid residues, about half of which are identical in all the
proteins in question, and by a heme prosthetic group
fixed in the 14,17-positions, so that the two thioether
bridges of the chain are always separated by two amino
acids (see Fig. 2). Though the chain length varies
slightly in invertebrates and microorganisms, it is
constant in vertebrates, so that evolution operates
here only through amino acid substitutions.
establishing a kinship among various species of vertebrates. Thus, the cytochrome c of the tunny, a bony
fish, differs from that of mammals only by 17 to 21
residues, the difference between those of man and horse
being as high as 12 residues. Such a comparison takes
into account only the “final” states and gives the
minimum number of substitutions that must have taken
place. The a c t u a l number is surely considerably higher,
10
14
Gly - - - -Gly -- -Phe
--- CyS
1
17
20
- -CyS * H i s - T h r . Val
L H e r n d
21
30
-Gly - Gly -H i s . L y s -Gly * P r o
Glu
40
-
Asn Leu
-Gly - -Gly.Arg-
-
50
41
Gly.Gln.Ala-
Gly-
60
- Lys - - -T r y -
-Tyr--Ala.Asn-
61
80
70
-----G l u - T y r . L e u - A s n . P r o - L y s
81
.Lys-Tyr.Ile.Pro.Gly-Thr.Lys.Met
100
90
---Gly101
--_-
---Lys
---Arg
- Asp .Leu - - T y r -Leu - Lys
104
Fig. 2. Structural pattern of the cytochromes c of vertebrates: Thc dashes represent mutable sites (after 141).
2. Substitutions
a) T o t a l N u m b e r of S u b s t i t u t i o n s
Figure 2 shows that positions 70 to 80 on the peptide
chain are exempt from substitution. This may be because mutations have never affected this region, or
because this region is indispensable to the enzymatic
activity and the molecules inactivated by substitutions
were eliminated during evolution. On the other hand,
the substitutable positions outside this region are fairly
regularly distributed along the peptide chain, indicating
that substitutions affect the sites singly.
Table 1 shows that the difference in the n u m b e r of
substitutions in the cytochromes c is small between
mammals of the same order, but significantly greater
between mammals of different orders. However, the
total number of substitutions is not sufficient for
Tab1el.Number of substitutions in the cytochromes c of sowe mammals.
Species compared
Man
~
ape
I
I
Orders compared
I
Primates - primates
l 1
Cow - pig - sheep
Cow - horse
Arfiodactyla-Artiodactyla
ArfiodacfylaPerissodactyla
Man - cow
Man - horse
Primates - Arfiodactyla
Primates - Perissodacfyla
Man - dog
Man - rabbit
800
I Primates - Carnivora
I
Primates
- Rodentia
Number of
substitutions
l3
0
10
12
I 10
for some sites may have suffered a series of substitutions
in the course of evolution, only the last one of which is
detected. The observed m i n i m u m number of substitutions is thus only a rough indication of interspecific
kinship, having a greater validity only within small
zoological groups.
b) N a t u r e of t h e S u b s t i t u t i o n s
Depending on whether the substitution involves two
chemically equivalent or non-equivalent amino acids,
we distinguish between substitutions which p r e s e r v e
and others which a l t e r the chemistry of the chainc41.
Chemically equivalent amino acids are, for example,
isoleucine and valine, serine and threonine, or lysine
and arginine. Substitution between any of these pairs
generally does not change the chemical properties of
the protein, its biological properties being changed only
to a very small extent. By contrast, the replacement of a
charged residue by a hydrophobic residue such as that
of lysine by isoleucine, represents a considerable modification and may lead to a molecule with novel properties. Both preserving and modifying substitutions are
detectable in the cytochromes c.
Since the prosthetic heme group plays an important
part in the electron transfer, substitutions in its neighborhood are of particular interestI61. Figure 3 shows
the preserving and the modifying substitutions affecting
these positions in the cytochromes c of 12 vertebrates,
in yeast, and in the invertebrate Samia Cynthia [41.
[6] S. Paleus and H . Tuppy, Acta chem. scand. 13, 641 (1959).
Angew. Chem. internat. Edit.
1 VoI. 5 (1966) 1 No. 9
Thr Thr
Glu
Ala
Ile
S e r Leu
Asn
Met A r g
-
.
.
.
.
-
.
V a l . Gln Lys * C y S M a . Gln C y S H i s Thr V a l . Glu Lys
11
12
13
J 18
16
19
20
21
22
.Yeme
Fig. 3. Substitutions near t h e h e m e g r o u p in a series of 14 cytochromes c.
T h e base line s h o w s t h e sequence a s f o u n d in seven species ( a f t e r [61
a n 3 141).
11. Hemoglobins
Hemoglobin is an abundant protein which can be easily
purified and which constitutes almost the entirety of the
soluble proteins present in the red cells of the hlood.
For its isolation the erythrocytes are separated from
the plasma by centrifuging, washed with an isotonic
solution, and finally lysed with distilled water. Unlike
the red cells of mammals, those of lower vertebrates are
nucleated, and the purification must then involve the
elimination of the nucleic acids. The molecular weights
of the hemoglobins of numerous species belonging to
different classes of vertebrates were determined by
ultracentrifugation. The values found are between 64000
and 68000, except in the case of Cyclostomata, the
most primitive vertebrates, where it is 17000[71. It
appears, therefore, that the hemoglobin molecule increased in size at the very beginning of the evolution
of the vertebrates.
Study of the primary structure of hemoglobin, i.e. of the
amino acid sequence, received a new impetus when it
was found that the protein is a “tetramer” formed from
four peptide chains each having a molecular weight of
17000, and that the molecule can be dissociated simply
by lowering the pH. Horse hemoglobin has been
separated by electrophoresis and chromatography at
pH 2 into two types of peptide chain, cc and p, native
hemoglobin containing two of each. This has led biochemists to consider two independent gene-protein
systems (corresponding to the a and the p proteins)
and to postulate the duplication of a n ancestral molecule.
1. Multiplicity of Chains
The chains of the hemoglobins of mammals have been
generally separated by ion-exchange chromatography
on Amberlite IRC-50, a carboxylic acid resin, at p H 2.1
with a urea buffer gradient. In the case of the hemoglobins of lower vertebrates, however, it is better to use
countercurrent distribution with sec-butanol and dilute
dichloroacetic acid as the mobile and the stationary
171 V . M . Ingram: The hemoglobins in genetics and evolution.
Columbia University Press New York, London 1963.
A n g e w . Chem. internat. E d i t .
/ VoI. 5 (1966) 1 No. 9
phases, respectively[gl. Two types of chain have
been found in the hemoglobins of various species,
such as the horse, the rabbit, the pig, the llama, and the
cow [81, a great number of primates 1101, and the carp 191.
The hemoglobin of adult man contains 90 % of hemaglobin Al,consistingoftworand twopchains,and2-3 %of
hemoglobin A2, consisting of two cc and two 8 chains.
The human fetus has hemoglobin F, consisting of two
a and two y chains. The human body thus produces
four hemoproteins (cc, p, 8, y). The amino acid sequence
in the cc, p, and y chains is now fully known [7-111. The a
chain contains 141 residues, the other three contain 146.
Comparison between the p and they chains shows that
only 39 out of the 146 sites differ, i.e. the degree of
homology is 73 %. The 39 substitutions seem to be
randomly distributed along the chains without forming
blocks. Chains p and 8 are likewise similar. As mentioned above, the a chain, which is always present in
human hemoglobin, differs from the other three by
containing only 141 amino acid units. Although the
homology between the cc and the p chains is only 44 %
(64 identical sites), it is still sufficiently great to indicate
a common ancestry. In this case the variations are not
restricted to substitutions but also involve deletions
and insertions.
Detailed investigations on ten apes [lo] have revealed
the presence of two a and two p chains. The simian cc
chain differs from the human a chain by 0 to 6 substitutions, but the simian p chain differs from the
human p and fetal y chains by 3 to 23 and by 9 to 36
substitutions, respectively.
The two hemoglobins found in the erythrocytes of the
horse may be considered to be homologous with the
human A1 and A 2 types. As in humans, the a chains of
both hemoglobins contain 141 residues and are identical.
The p chains, on the other hand, contain 146 residues
and are different. More than one type of hemoglobin
has been found in the cow, the sheep, and the mouse 191,
and this plurality appears to be the rule.
As in humans, the hemoglobins of all the adult mammals
investigated (cow, horse, sheep, rabbit, pig, and llama)
consist of two a chains and two p chains, and can be
represented by the general formula a2p2. As in man,
the fetal hemoglobin found additionally in e.g. cow,
sheep, and goat, consists of cx chains, identical to those
in the adult hemoglobin, and y chains which differ
from the adult p chains. The general formula of fetal
hemoglobin is therefore ~ ( 2 ~ 2 .
In each group the chain length seems to be the most
stable property (see Table 2). On the other hand, the M
chain exhibits appreciably fewer substitutions than the
[S] G. Braunitzer, K. Hilse, V. Rhdloff, and H . Hilschmann,
Advances Protein Chem. 19, 1 (1964).
[9] G . Braunitzer, V . Braun, K . Hilse, G . Hobom, V . Rudloff, and
G. v. Wettstein in V. Bryson and H . J . Vogel: Evolving genes and
proteins. Academic Press, New York, London 1965, p. 183.
[lo] J . Buettner-Jauusch and R. L. Hill in V . Bryson and H . J .
Vogel: Evolving genes and proteins. Academic Press. New York,
London 1965, p. 167.
[ l l ] W . A . Srhroeder, Fortschr. Chem. org. Naturstoffe 17, 322
(1959).
801
p
and y chains. This is illustrated in Fig. 4 for the
amino end of the chains.
common hemoglobin. The hemoglobin of the lamprey
has been isolated in pure form. It contains only one
peptide chain consisting of 160 amino acid residues.
Its structure is more like that Of
n’yoglobin
than that of the v. or the p chain of vertebrate hemoglobin [91.
Table 2. Number of residues in the C L , ~and
,
y chains of the hemoglobins
of some mammals
Species
I x
Man
Horse
cow
Lemur
Camel
I
B
1
I Y
146
141
141
141
146
I45
146
I46
2
3
I
4
5
146
2. The Theory of Duplication
145
Inspection of the number and the sequence of amino
acid residues in the hemoglobin chains suggests that
6
7
8
9
10
12
11
13
14
15
16
. . .. . . . .
Val -Leu-Se - ~ - A l a - A s p - L y s - T h r - A s n - V a l - L y s - A l a - A l a - T r y - G l y - L y s . .
cow
(ff)
- A l a - A s p - L y s - m -Am-Val-Lys-Ala-Ala-Try-Gly-Lys..
.... . ..
Horse
(a)
-Ma-Asp-Lys-Thr-Asn-Val-Lys-Ala-Ala-Try-a-Lys..
..... ..
1
2
3
5
4
6
7
8
9
10
11
12
13
14
16
15
17
Man
-Pro-Glu-Glu-Lys-
-Ma-Val-Thr-Ala-
-Try-Gly-Lys..
. ..
cow
-Pro-Glu-Glu-Lys-
-Ma-Val-Thr-Ala-
-Try-Gly-Lys..
...
1
2
3
4
5
6
7
&
9
10 1 1
12
13
14
15
16
17
I
Man
-Glu-Glu-Asp-Lys-Ala-
Thr-Ile -Thr-Ser-Leu- Try-Gly - L y s . .
. . .. .
cow
-Glu-Glu-[I-Lys-Ma-
Ala-Val -Thr-Ser-Leu- Phe-Ala - L y s . .
. . .. .
Fig. 4 Comparison of the amino ends of the a , 8, and y chains in
human, bovine, and equine hemoglobins. Boxes denote substitutions,
empty boxes deletions.
Similar remarks seem to apply to other classes of vertebrates in that the adults frequently contain two or
more hemoglobins formed by two types of chain, so
that the general formula cc2p2 still applies. Thus, in the
classes A v e s and Pisces, the hen[121 and the carp[131
have two and three hemoglobins separable by electrophoresis or chromatography. Unlike the p chain, the v.
chain in the predominant hemoglobin of the carp has
an acetylated amino group at one end [I31 and, unlike
the mammalian u chain (141 residues) contains 142
residuesL91. As with the cytochromes c of the vertebrates, the length of the peptide chains is probably the
most constant feature.
Since the Cyclostomata constitute the most primitive
class of vertebrates, their proteins should be closest to
the ancestral type. The lamprey is one of the few
remaining representatives of this class. The molecular
weight of its hemoglobin is about a quarter of that
found in vertebrates[141. In this respect, it is closer to
myoglobin found in the muscles of vertebrates than to
[12] CI. Paul, A . G . Schnek, and J . LPonis: Chromatography
Symposium 11, 1962, p. 133.
[13] K . Hilse, V . Sorger, and G . Braunitrer, Hoppe-Seylers Z .
physiol. Chem. 344, 166 (1966).
[14] T. Svedberg and I . B. Eriksson-Quensel, J . Amer. chem. SOC.
56, 1700 (1934).
they are derived from a single ancestral molecule. Gene
duplication is thesimplest way to explain the formation cif
new genes. Ingram [*I has advanced this explanation to account for the four types of chain in normal human hemoglobins. As regards their molecular weight, amino acid sequence, etc., these chains resemble the myoglobin of muscles. Accordingly, the gene controlling the biosynthesis of
myoglobin should be the oldest and the ancestor of the
genes now controlling the biosynthesis of the cc, (3, y,
and 8 chains (Fig. 5).
+ MYOGLOBIN
M
f f -
*ff
Fig. 5. Evolution of the hemoglobin chains. The dots denote gene
duplications (after IS]).
Differentiation of the myoglobin gene M would have
given rise to two genes, one of which continues to
control the synthesis of myoglobin, the other leading
via mutations to the formation of the u chain. Duplication of theu genewould give two daughter genes, one
Angew. Chem. internat.
Edit. J Vol. 5 (1966) 1 No.9
the amino acid sequence of the corresponding protein.
As time proceeds, the two protein structures become
more and more dissimilar.
Investigations on the pathological variations of human
hemoglobins have revealed a great number of abnormalities.
Genetic studies on several generations
have shown that these abnormalities result from
mutations. The number of the abnormal hemoglobins
(B, C, D) has turned out to be so large (about 40) that
now a combination of letters (B, C, D) and places of
origin is used for denotation. An important feature is
that mutation affects almost always only one amino
form of which continues to control the production of the
M chain, the other after modification causing the
synthesis of the y chain. The same mechanism would
operate in the successive formation of the p and 6 chains.
This scheme accounts for certain genetic and chemical
observations: as implied by the similarity in the amino
acid sequences of the corresponding proteins, genes y,p,
andsareseen to bederivedfromoneanother,in thatorder,
while thecc gene, which is the most stable, is considered as
the oldest of the four. The only reason for placing the y
gene before the and 6 genes is that it occurs in the
fetus, the other two being found only in the adult.
p
1
2
Val. L e u . .
16
30
0
0
68
116
141
. Asp.
0
0
58
57
0
. L y s . . . G l u . . . Gly.
His..
NH,.
. . Glu.. . Arg
0
Hb I
.Asp.
GIu. NH,
Hb GHonolulu
.
0
Hb Norfolk
.Asp.
. Tyr.
Hb MBoston
0
.L y s .
GPhiladelphia
0
.Lys.
Hb OIndonesia
1
2
0
3
Val. His. Leu..
6
7
2fi
63
0
0
0
0
. Glu.
Hb S
.V a l .
Hb C
. L 0y s .
Iib GSan ~ o s e
Glu.
Glu.. . His..
67
121
. Val..
0
146
. Glu..
0
. His
.Gly.
Q
Hb E
Lys.
Hb MSaskatoon
.Tyr.
0
.Glu.
Hb MMilwaukee
Hb Dpunjab
. GIu.
( = D Y)
Hb Z u r i c h
Hb 'Arabia
NH2.
0
.Arg.
0
.Lys.
Fig. 6. Substitutions in a b n o r m a l h u m a n henroelobins d u e to t h e iiiutalions in the series .* ( a b o v e )
a n d I3 (below) (after 181).
3. Mutations
The hypothesis of duplication explains the formation
of two twin geces, each controlling the synthesis of one
protein. Initially, the two proteins were also twins.
However, each gene suffers some mutations independently of the other. These mutations result in changes in
Angew. Chem. internat. Edit.
1 Vol. 5 (1966) 1 No. 9
acid residue at a time. Gene p is characterized by an
appreciably greater number of mutations than gene dc.
The substitutions induced in the protein molecules by
mutations are randomly distributed along the chains
(see Fig. 6). Furthermore, insertions and deletions must
be very rare, since the chain length has been found to
be constant.
803
111. Neurohypophyseal Hormones
The neurohypophysis produces hormones which have
relatively simple structures. For this reason, they were
among the first peptide hormones to be characterized.
Oxytocin and vasopressin were isolated from cattle [15-171.
1. Duality of the Hormones
la1
lsnlncin
Tests for oxytocic and pressor activities in the hypophyseal extracts give positive results with all vertebrates, though the levels of activity may differ considerably (Table 3). The neurohypophyseal (NHP)
hormones have been isolated from six mammals (man,
Table 3. Biological activities of posterior pituitary powders from
various species of vertebrates
Mammals:
Vasopressin
activity
[unitsI mg
dry powder]
Oxytocic
activity
[unitslmg
dry powder]
Species
1 .o
1.4
1.1
cow
Pig
Horse
Sheep
Whale
0.9
0.8
0.5
1.4
0.8
0.8
2.5
Birds:
Hen
0.4
0.4
Amphibians:
Frog
Toad
I .2
I .o
Bony fishes [a]:
Bib
Pollack
Hake
Cod
Carp
0.6
Cartilaginous fishes [a]: Ray
White skate
0.5
0.5
0.7
0.05
0.005
0.007
I
1 .o
0.8
0.5
0.5
0.3
0.2
must be extracted. Analysis has shown that mammals
contain oxytocin and vasopressin, amphibians mesotocin and vasotocin, bony fishes isotocin and vasotocin,
and cartilaginous fishes glumitocin and probably vasotocin. On the various chromatograms, oxytocin, mesotocin, isotocin, and glumitocin occupy the same position. They are the first to be eluted, and are followed by
vasotocin or arginine-vasopressin (Fig. 7).
0.04
0.001
0.001
cow, pig, horse, sheep, and whale), one bird (hen), one
amphibian (frog), five bony fishes (bib, pollack, silver
hake, cod, and carp), and four cartilaginous fishes
(rays). Purification was always carried out by selective
adsorption of the hormones onto a protein, neurophysin. The resulting complex was then isolated by
fractional precipitation and dialysis. Treatment with
trichloroacetic acid precipitated the protein, the hormones remaining in solution. The last step was chromatography on Amberlite CG-50. Some of the chromatographic results are shown in Figures 7a to 7d.
Two neurohypophyseal hormones were found in each
of the five classes of vertebrates investigated. The purity
of the substances was checked by amino acid analysis.
In the case of the lower vertebrates, the small size of the
hypophysis necessitated the processing of a great
number of glands to obtain sufficient material for the
determination of the chemical structure. To isolate
1 to 5 mg of hormones, about 5000 to loo00 glands
[15] V. DuVigneaud: A trail of research. Cornell University
Press, Ithaca 1952.
[I61 H.Tuppy and H . Michl, Mh. Chem. 84, 1011 (1953)
[17] R. Acher and C. Fromageot, Ergebn. Physiol., biol. Chem.,
exp. Pharmakol. 48, 286 (1955).
804
Fig. I. Chromatograms of the neurohypophyseal hormones of
vertebrates (Amberlite CG-50, ammonium acetate gradient) (after [18]).
(a) mammals (whale); (b) amphibians (frog); (c) bony fishes (hake);
(d) cartilaginous fishes (ray).
2. Characterization of the Hormones
All these hormones are peptides having nine amino
acid residues (Table 4).
Mammals: The N H P hormones of the cow and the pig have
been known since 1953, those of the horse, sheep, whale[lsl,
and man "91 having been characterized more recently. All
species contain the same oxytocin, five of the six containing the same arginine-vasopressin. Lysine-vasopressin, in
which a lysine residue takes the place of an arginine residue,
has been found only in the pig.
Birds and amphibians: The neurohypophysis of the hen, the
only bird investigated, contains oxytocin and vasotocin,
that of the edible frog (Rana esculenta) containing mesotocin
and vasotocin [181.
Bony fishes: Isotocin and vasotocin are the two N H P hormones found in four marine species of Teleostei: the bib
(Gadus luscus), the pollack (Pollachius virens), the hake
(Merluccius merluccius), and the cod (Gadus morhua), and in
one freshwater species of the Teleostei, the carp (Cyprinus
carpio).
Cartilaginous fishes: Glumitocin and probably vasotocin are
the two N H P hormones found in four species of Elasmobranchs (Raia clavata, Raia batis, Raia fullonica, and Raia
naevus). The first hormone has been characterized chemically, the second only by pharmacological methods.
[18] R . Acher, J . Chauvet, M.T. Chauver, and D . Crepy, Bull
SOC. Chim. biol. 47, 2279 (1965).
Angew. Chem. internat. Edit. J Vol. 5 (1966) 1 No. 9
Table 4. Neurohypophyseal hormones of some vertebrates.
I
Cartilaginous fishes
2
3
4
5
6
7
8
9
Cys.Tyr.Ile.Ser.Asn.CyS.
Pro.Gln.Gly(NH2)
I
I
Vasotocin (?)
Glumitocin
Bony fishes
1
2
3 4 5
6
7 8
9
CyS.Tyr.Ile.Ser.Asn.CyS.Pro.Ile.Gly(NH2)
-
I
1
-
I
2
3
I
Tsotocin
Amphibians
1
-
2
3
4
1
-
I
2
3
4
9
8
-
I
2
3
5
5
4
5
6
7
8
9
-
Vasotocin
6
7
8
9
1
2
3
4
5
6
7
8
9
CyS.Tyr.Phe.Gln.Asn.CyS.Pro.Arg.Gly(NH2)
I
I
CyS.Tyr.ile.Gln.Asn.CyS.Pro.Leu.Gly(NH2)
I
I
I
7
-
Oxytocin
Pig
6
CyS.Tyr.Ile.Gln.Asn.CyS.Pro.Arg.Gly(NH~)
Mesotocin
Mammals (except pig)
5
Vasotocin
I 2 3 4
5
6
7
8 9
CyS.Tyr.lle.Gln.Asn.CyS.Pro.Ile.GIy(NH2)
I
4
-
CyS.Tyr.1le.Gln.Asn.CyS.Pro.
Arg.Gly(N H2)
Arginine-vasopressin
6
7
8
9
1
= -
CyS.Tyr.Ile.Gln.Asn.CyS.Pro.Leu.Gly(NH2)
2
3
4
5
6
7
8
9
CyS.Tyr.Phe.Gln.Asn.CyS.Pro.Lys.Gly(NH~)
-
I
I
Oxytocin
Lysine-vasopressin
a) T h e A n c e s t r a l H o r m o n e
3. Phylogeny of the Hormones
Although the number of species investigated (about 20)
is very small in comparison with the 50000 or so
known vertebrates, the following general conclusions
may be drawn since almost all the classes of vertebrates
were represented :
(i) The neurohypophysis of each species contains two
dominant hormones.
(ii) All these hormones have a common structural
pattern characterized by nine amino acid residues with a
disulfide bridge connecting the amino acids in positions
1 and 6.
(iii) The structure is the same within a given zoological
class, and varies by only one or two residues between
the classes.
The twofold nature of the hormones of the neurohypophysis recalls the chain multiplicity of the hemoglobins
and suggests a gene duplication. It must be assumed
that this duplication occurred at a very early stage
during evolution, since even the Pisces contain two
hormones. In this connection, the Cyclostomata, and
especially the lamprey, are of interest. Column-chromatographic and paper-electrophoretic experiments on
the pituitary glands of the lamprey revealed only one
neurohypophyseal hormone [201, which gave a positive
result in pharmacological tests for vasotocin. In addition
the lamprey may contain a second hormone, but one
which is not detectable by the pharmacological test
used. However, this hormone would have to differ
greatly from those so far identified, since it is not
adsorbed onto neurophysin. Apart from this, it seems
1
2
3
4
5
6
7
8
9
CyS-Tyr-O-O-Asn-CyS-Pro-O-Gly(NHz)
I
I
Ancestral molecule
Bony fishes
Isotocin
Amphibians
Mesotocin
Ser, Ile,
Vasotocin
Gln, Ile8
Vasotocin
Gln, Leu,
Arg-Vasopressin
Glnl Leu,
Lys-Vasopressin
4
4
4
Mammals
(except pig)
Pig
(iv) The changes or mutations seem to affect preferentially “privileged” positions on the chain, in particular
positions 4 and 8.
Angew. Chem. internnt. Edit.
1 Vol. 5 (1966) 1 No. 9
Oxytocin
Ilea Arg,
4
Phe, Arg,
4
4
Oxytocin
Ile, Arg,
Phe, Lys,
Fig. 8. Hypothetical scheme of the evolution of neurohypophyseal
hormones. One gene duplication and a series of subsequent single
substitutions i n oositions 3, 4, or 8 produce two molecular
“lines”.
805
feasible to envisage the gene duplication taking place
somewhere between the Cyclostomata and the fishes.
This would mean that two “lines” of molecules are
formed, which then undergo diversification independently of each other. One line (isotocin, mesotccin,
oxytocin) seems to be specialized in reproduction, the
other (vasotocin, vasopressin) being specialized in the
regulation of the salt and water balance (see Fig. 8).
b) M o l e c u l a r L i n e s
Assuming that the hormones are derived from one
another in the same chronological order as the species
to which they belong, we can draw up the genealogy of
these molecules in juxtaposition to the phylogenealogy
proposed by Simpson [211. The cartilaginous fishes and
the bony fishes appeared at about the same time some
300 million years ago. However, while the cartilaginous fishes gave rise to no other class of vertebrates,
the bony fishes gave rise to the amphibians, these to the
reptiles, and these to the birds on the one hand and
mammals on the other. It must therefore be examined
whether molecular evolution as manifested by the
chemical structure of the neurohypophyseal hormones
follows the order: bony fish - amphibians - mammals.
Considering the NHP hormones involved in the regulation of the water and mineral balance, we note that
vasotocin is present in all the vertebrates, with the
exception of mammals which contain vasopressin
instead. The transformation of vasotocin into vasopressin requires only the substitution of phenylalanine
for isoleucine in position 3 (cf. Fig. 8). Although
chemically this is merely an interchange between two
hydrophobic groups, it precipitates great biological
changes: Vasotocin increases the water permeability of
the frog bladder, exerts a contracting action on the
rat uterus, and stimulates the re-absorption of water
at the renal level. When vasotocin is transformed into
vasopressin by the substitution Ile3 + Phe3, the first
two actions disappear almost entirely, while the third
[19] A. Light and V . DuVignearrd, PIOC.So<. exp. Biol. Med. 93,
692 (1958).
[20] R . Acher, J . Chauvet, M . T . Chauvet, and D . Crepy, unpublished.
[21] G. G . Simpson: The Meaning of Evolution. Oxford University Press, London 1950.
806
one increases considerably, so that in mammals vasopressin is a powerful antidiuretic.
In addition to the above substitution, porcine vasopressin contains lysine instead of arginine position 8.
This second substitution causes practically no chemical
change, and only a small biological change. In fact,
lysine-vasopressin is the antidiuretic hormone in the
pig, just as arginine-vasopressin is in other mammals.
More numerous - and thus more informative - transformations are found in NHP hormones in reproduction. Thus the bony fishes contain isotocin, the amphi bians mesotocin, and the mammals oxytocin. The
formation of mesotocin from isotocin involves a single
substitution in position 4 (glutamine for serine);
the formation of oxytocin from mesotocin involves the
substitution of leucine in position 8 (see Fig. 8). Mesotocin in amphibians thus represents biochemically an
intermediate stage between isotocin and oxytocin: the
isoleucine in position 8 is still a piscine feature, but
glutamine in position 4 is a mammalian one. From a
general point of view, this mixed chemical structure is
the molecular equivalent of mixed anatomical structures
used by evolutionists to establish the connection between
two zoological groups, and mesotocin may therefore be
called a “transition molecule”, i.e. a molecule forming
a chemical and, ultimately, a phylogenic link between
two genera, orders, and classes.
Although it involves an inference from the above
particular case to the general, it seems reasonable to
assume that proteins have evolved by a series of
single substitutions of amino acids. In fact, this is in
agreement with findings on the mutations of various
proteins (hemoglobins, tryptophan synthetase, TMV
protein), where the changes generally affect only one
residue at a time. The transition molecules should
provide a clue to the steps of transformation of proteins.
It seems logical that the probability of molecular
changes increases with time and with increasing chain
length. This means that the larger the peptide or
protein in question, the more one must restrict the size
of the zoological group to be investigated, as otherwise
one is confronted with undecipherable complexity.
[ A 539 LEI
Received; June 6th, 1966
German version: Angew. Chem. 78, 856 (1966)
Translated by Express Translation Service, London
Angew. Chem. internat. Edit. 1 Vol. 5(1966) 1 No. 9
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