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Genetic Aspects of Hemoglobin Synthesis.

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Genetic Aspects of Hemoglobin Synthesis
By Berthold Schmidt[*’
Dedicated to Professor Fritz Strassmann on the occasion of his 70th birthday
The blood pigment hemoglobin (Hb) occurs in red blood cells in a higher concentration than
the other proteins which are present. It is therefore particularly suitable for the investigation
of genetically induced changes in protein molecules. Unlike the monomeric myoglobin (Mb)
of muscle, the blood pigment is tetrameric. Several types of subunits are known that can tetramerize to hemoglobins having different properties. The information for all hemoproteins
probably originated from the same primary gene. Doublets of this primary gene have developed
differently, and now code different protein species. It is possible to establish from the Hb
molecule the possible consequences of a mutation to the conformation and function of a
protein molecule.
1. Introduction
Hemoglobin (molecular weight 64500) is a chromoprotein
(containing heme as the prosthetic group), which, with a
total content of barely 1 kg in the human organism, is
responsible for the transport of 0, from the lungs to 02consuming tissue, and also to a large extent for the transport of CO, in the reverse direction. The oxygen molecule
reacts reversibly with the heme iron (Fe’+-protoporphyrin)
without oxidizing the iron, which is protected by the coordinate bond of Fe-N(histidine) (Fig. 1).
oc-- -
Fig. 1. Bonding of heme iron in hemoglobin. The coordination number
6 is reached by four pyrrole N and two imidazole N of histidine residues
of the globin chain as ligands.
Hemoglobin (Hb) constitutes about 95% of the dry protein
mass in red blood cells (erythrocytes), each of which contains 2.8 x 10’ molecules of hemoglobin. Hemoglobin can
therefore be investigated for genetically induced microheterogeneities or abnormalities, which cause distinct
[*I Prof. Dr. Dr. B. Schmidt
Physiologisch-Chemisches Institut der Universitat
65 Mainz, Postfach 3980 (Germany)
clinical symptoms, without previous purification steps,
which are always accompanied by partial denaturation.
Hemoglobins can be differentiated electrophoretically by
differences in their migration rates. Sequence differences
in individual peptide chains can be established by means of
fingerprint mapping, which is based on two-dimensional
chromatographic and electrophoretic development of the
hydrolysis products obtained by standardized degradation
with trypsin. The most accurate differentiation can be
achieved by comparative overall analysis or sequence
analysis of the amino acid composition of the peptide
Evolutionary relationships can also be demonstrated by
comparative biochemical paleogenetic studies. Thus it is
practically never possible to isolate traces of proteins from
fossil material, but proteins can be investigated in “living
fossils”, and by comparison of the amino acid sequences,
they can be correlated with recent homologous proteins,
i . e. proteins having the same function.
The genetic “experiments” set up by nature, which lead by
inheritance to pathological symptoms (hemoglobinopathies) of various degrees of severity in man, produce
changes“] in the rate of synthesis and[” in the amino acid
sequence of the hemoglobin molecule. Therefore, it is
possible to deduce the importance of individual amino acids
to the conformation, quaternary structure, and functional
modification of erythrocyte-bound Hb.
In addition to the major component hemoglobin A,, the
erythrocytes of adults also contain 1.5 to 4% of hemoglobin
A, (A =adult). Each hemoglobin type has a pronounced
quaternary structure, and is a tetramer. The three-dimensional structure was established by Perutzf3 for horse
hemoglobin. The spherical molecule (64 x 55 x 50 A3)
consists of two pairs of identical peptide chains. These
globin subunits each contain a heme, and form the tetramer
by noncovalent bonds (weak interactions)[3,5 , 71. Hb A,
consists of two a and two p chains: a2P2.The minor
component HbA, can be writtenas a,&. Duringembryonal
development, an Hb F :a,y, (F= fetal) dominates, and this
is preceded by an a,&, hemoglobin type (during the first
third). Starting with the last third of the embryogenesis up
to the end of the first year of life, Hb F is gradually replaced
by Hb A, (Fig. 2).
Angew. Chem. internat. Edit. 1 Vol. 11 (1972) 1 No. 7
Instability of Hb to oxidation. Hemoglobin (Fe2+)is converted into hemiglobin or methemoglobin (Fe3+), which
is no longer able to transport oxygen.
Fig. 2. Synthesis of the Hb subunits during the embryonal development
(A-B) and after birth (B) [4].
Faults in the structure of the hemoglobin usually manifest
themselves in functional losses and clinical symptoms,
which may appear singly or together and in various degrees
of severity (Table 1):
Increased or decreased 0, binding capacity. An essential
property of Hb is that 0, transport is possible, i.e. 0 ,
uptake at the oxygen partial pressure in the lung and release
of 0, to other heme proteins (0, storage) in the tissue.
A change in the Hb molecule may increase the strength
with which 0, is bound (Table 1, Nos. 11 and 12), so that
the 0, cannot be released where it is needed. Conversely,
if 0, binding is too weak (Table 1, No. 13), an insufficient
0, number of oxygen molecules is bound and transported.
Instability of the Hb can considerably shorten the average
life(120 days) of the erythrocyte as an Hb carrier: hemolytic
anemia. Increased release of immature erythrocytes (reticulocytes) occurs from the red bone marrow, the site of
formation of the Hb-carrying blood cells. The skin also
assumes a yellow color (icterus),which is due to the increased quantities of bile pigments that result from the increased
breakdown of Hb.
Table 1. Abnormalities in the Hb structure [ 2 ] .
Name [a]
Amino acid change [b]
Disturbance occurring
PSI (F 7) Leu
fracture of F-helix
fracture of F-helix
fracture of B-helix
fracture of A-helix
fracture of B-helix
superficial loss of charge
Hb Santa Ana
Hb Freiburg
Hb BorPs
Hb Koln
p66 (E10) Lys
Hb Philly
p35(C 1)Tyr
pS9 (G 1)Asp
Hb Kansas
p l o Z (G
p6 (A 3) Glu
B6 (A 3) Glu
Hb Bristol
p6’ (E 11) Val
+ Asp
HbM Milwaukee
p6’ (E 11)Val
Hb Zurich
Hb Hiroshima
Hyde Park
p8* (F 4) Leu + Pro
p28(B 10) Leu + Pro
(A 3) Glu-deficiency
pZ3(B 5) Val-deficiency
Baa (F 4 ) Leu -+ Arg
06’ (E 11) Val
p9* (FG 5) Val
(HC 2 ) Tyr
donor/acceptor reversal of a
hydrogen bridge, stronger 0,
change of charge, stronger
0, binding
loss of H donor
(hydrogen bridge)
4) Asn
charge reversal, disturbance
of the alplcontact
loss of charge, weaker 0,
discharge on surface
discharge on surface
disturbance of the
sickle cell anemia
p63 His . . . _ _ HO,C
( F 8) His (Prox.)
disturbance of
conformation of
globin subunit
negative charge in heme
pocket stabilizes Fe’+
occurrence of
p63(E 7) His (dist.) + Tyr
p63 (E 7) His
(H 21) His
imidazole replaced by guanidyl-N : danger of
methemoglobinemia occurring
absence of basic point of attack for acid effectors:
stronger 0, binding
[a] Abnormal hemoglobins are usually named after a place or person connected with their first detection.
[b] A symbol ofthe type p9’ refers to the amino acid position 91 of the p chain, while F7 denotes the seventh posi.
tion of helix segment F.
Angew. Chem. internal. Edit. / Vol. I1 (1972) No. 7
2. Genetic Information
have been isolated from Cyclostomi“] (hagfishes, lampreys)[’6z ”I.
It is assumedr9~
l o ] that the information for the present-day
variants of the heme proteins, i. e. of substances that can
bind (myoglobin) or transport (ci$.y,6 subunits of hemoglobin) 0,, is derived from a phylogenetically old gene.
It is concluded from comparisons of amino acid sequences
between heme proteins that are available at present that
this primary gene could have coded a protein containing
154 (or more) amino acids.
From the pool of primary gene, the information for myoglobin (Mb) developed first, characterized by the loss of a
base triplet that was retained in later gene doublets. The
genetic information for the synthesis of early heme proteins
of the Hb type shows the same deletion as for Mb, with the
addition of a further loss, which may have resulted from a
gene displacement. Thus the early monomeric Hb types
show a deficit of 7 amino acids at the C-terminal end of the
chain in relation to Mb. The primitive vertebrate Hb in the
oxygenated state exhibits monomeric character, like Mb.
Most genes are present in the chromosomes of a cell in
several “redundant copies”. In the case of protein synthesis
the cell can thus rapidly synthesize a given type of protein
with the aid of its ribosomal apparatus. This multiplicity
is due to gene duplications that have occurred in the course
of time.
From these identical gene doublets, mutations that affect
individual gene loci separately can lead to modified genes,
which contain modified information for the protein synthesis. Growing independence (migration of gene sections
that have been split off, loss of base triplets) may lead to the
formation of genes that contain the information for homologous proteins instead of for identical species.
The oldest heme protein from the homologous Mb/Hb
group is myoglobin (Mb). This “muscle pigment” is a
monomeric heme protein. Its peptide chain consists of 153
amino acids (molecular weight 17000). The sequence of
sperm whale Mb has been elucidated“ ‘I. The threedimensional structure was determined by Kendrew[”. 1 3 ]
for horse Mb.
Comparison of the amino acid sequence of the globin
subunits of H b with that of Mb reveals differences in
120&5 positions[141.The amino acids agree only in about
30 positions. The variation & 5 arises when sperm whale
Mb is compared with the subunits of all the vertebrate Hb
types investigated so far. It means that differences between
species may be regarded in the same way as an error range
in connection with the statement that the M b chain differs
from the H b subunit in 120 amino acid positions.
From these amino acid differences, Pauling
has tried to
calculate the times required for such changes in the course
of evolution. The basis of his arguments is as follows.
Comparison of the amino acid sequence of the globin
subunits of mammalian Hb (horse, pig, ox, rabbit) shows
differences in 22 positions between the 01 and p chains. If
each chain developed independently, this corresponds to
11 changes per chain. If one assumes that the development
of the higher mammals began about 80 million years ago,
one finds a period of 7 million years for each mutation (i. e.
for each change). For events that happened very far back
in time, it is advisable to reckon on the next higher order
of magnitude, i. e. 10million years per amino acid difference.
On application of the lo7 year value to the myoglobin/
globin difference, it is found that the Mb/Hb dichotomy
occurred (120/2) x lo7= 600 50 million years ago. “Snapshots” of this development are provided by species of
animals that have developed only slightly since their first
appearance. The earliest H b forms can be studied in such
“living fossils”. Monomeric Hb types with 146 amino acids
The gene pool for the primary form of monomeric Hb,
which aggregated only weakly, was probably the basis of
the development of the information for the ci chain, which
also exhibits no tendency to aggregate with its own kind.
On comparison of the amino acid sequences, it is found
that in the a-subunit of Hb, as in Mb and primary Hb, a
deletion has occurred within the chain, as well as shortening by 6 amino acids at the C-terminus. The ci subunit is
characterized by two further “gaps” totaling 6 amino acids,
and giving a typical number of 141 amino acids for the ci
The information for the synthesis of non-ci chains developed
from the gene pool much later than the information for the
ci subunit. The assumption of the later development is based
on the fact that the non-ci chains differ from the 01 chain
only in 85 & 10 positions[’41. Pauling believes that the ci
gene and the non-ci gene separated 380 million years ago.
All the non-a chains exhibit the C-terminal shortening by
6 amino acids in relation to Mb that is characteristic of the
globin subunits. They lack the “old” triplet deletion that
occurs in Mb and primary Hb. It is replaced by a deletion
of two triplets that is characteristic of the non-ci chains.
The characteristic length of the non-ci chains is thus found
to be 154 (primary gene) - 6 - 2 = 146 amino acids.
The oldest non-ci gene is the [3 gene, which contains the
information for the synthesis of the [3 chain. Other non-ci
globins have probably developed from doublets of the p
gene, since they exhibit a greater similarity to the p chain.
The information for the synthesis of the y subunits evolved
first from doublets of the P gene. According to Pauling’s
ideas, 42+6 amino acid differences between the p and y
chains indicates that the development of the y gene from
the [3 gene pool occurred about 150 million years ago. The
y gene remained immediately adjacent to the p gene. 35
million years ago, the 6 gene separated from the [3-y gene
assembly[g].Here again, the time is deduced by the application of Pauling’s ideas to the difference of 10 amino acids
between the 6 and p chains.
The close neighborhood of the @gene family in the
genome[1g1is deduced from experimental findings. A relation must exist between the transcription of and y genes,
such that y-globin suppresses the synthesis of P-globin. If
the synthesis of the y chains slowly dies out, the inhibition
of the P-globin synthesis also vanishes, and this synthesis
increases in proportion to the decrease in the y-globin
Cyclostomi are the oldest vertebrates (still without mandible). They
developed 450 million years ago
Angew. Chem. intemat. Edit.
Vol. I 1 (1972) 1 No. 7
synthesis (cf. Fig. 2)[’01. The p and 6 genes are even closer
together. Investigation of the effect of hormone (erythropoietin‘”]) on the Hb synthesis shows that the rates of
synthesis of the p and 6 chains increase in a constant ratio
of 40:1[221.There are various possible reasons for the
greater rate of synthesis of the p chain: 1. The transport of
the same amino acid may be effected by different
A deficit of one type of tRNA can lead to a
deficit in the synthesis of a protein. 2. It is conceivable that
complications arise in the folding for the younger 6 chain.
3. The mRNA’s or their ribosomal adducts may differ in
their stability for the various globin subunits1’]. It is not
yet known which of these is the true cause.
Closeness of the p and 6 genes is also indicated by the
occurrence of hybrid globin subunits, which have an Nterminal 6 structure and a C-terminal p structure in the
non-a chain. Three such hybrids are known at present[”. 26].
Little is known about an E subunit, which occurs in the
first third of the embryonal Hb synthesis (Fig. 2). It disappears when the synthesis of the y chains begins, just as the
synthesis of they chains disappears later as soon as p chains
are synthesized.
Though up to 120 positions in the sequence of the homologous hemoproteins may be differently occupied, depending
on phylogenetic age, 11 positions are always identically
occupied in all the peptide chains investigated so far[”];
these include the two histidine residues, which guarantee
heme binding, and close to these a proline, which prevents
any interfering helix formation in the vicinity of the heme
binding site.
The globin subunits of the non-a type exhibit the property
of aggregation even in the oxygenated state. Homotetramers and heterotetramers may be formed. Homotetramers of the type p4 appear under pathological conditions, and are much less suitable for the transport of 0,
than the heterotetramer a,p,, which first appears in the
phylogeny of the vertebrates with the formation of the
mandible. An early Hb of this type has been investigated
in the carp, a teleost[18].
3. Changes in the Rate of Synthesis
After the embryonal development, the synthesis of Hb
occurs under physiological conditions only in one type of
cell of the red bone marrow, i. e. the (pro)erythroblasts. In
these nucleated precursors of the (in mammals) later nonnucleated erythrocyte, the information required for the
Hb synthesis can be totally recalled from the genome, i. e.
only here can the requirements be satisfied for all the partial
steps of the Hb synthesis. It is not clear how the cellular Hb
synthesis is terminated. Hb probably represses certain
genome sections after its synthesis, so that further transcription is suppressed. The globin synthesis continues until
intact messenger-RNA is present for all peptide types[”]
and a functioning ribosomal apparatus is available (cf. Fig. 3).
During the transcription, the copies of some genome sections required for the synthesis of the globin subunits are
Angew. Chem. internat. Edit.
Vol. I1 (1972) 1 No. 7
produced in the form of messenger-RNA. From each gene
section there will be different mRNA’s produced, i.e. the
cistron codes an individual peptide chain. The unicistronal
mRNA’s for the synthesis of globin subunits have a remarkably long life of the order of days. They can be isolated
from reticulocytes that have long been without nuclei, and
Hb can be synthesized (starting at the N-terminal end) with
the aid of the mRNA’s from these reticulocytes in proteinsynthesizing in-vitro systems[’*].
In the translation of the mRNA information into a subunits, the polysomes (functionalaggregates of the ribosomes
for the translation of an mRNA) are found to be mostly
pentamers, whereas hexamers can be detected in the synthesis of p subunits. It is possible that the p-mRNA, which
is about 15 nucleotides longer, provides room for an additional ribosome.
The stoichiometric aggregation of a and p chains to form
the complete tetramer takes place at the ribosomal
l e ~ e I [ ’ ~.-As
. the myoglobin synthesis, the complete
a chains separate from the ribosomes and are released into
the cytosol. They do not form tetramers, but they may
clump together in cases of pathological overfilling (pthalassemia). Under physiological conditions, the free a
chains enter into interaction with the longer, still growing
globin subunits of the p family, whereupon ap dimers
detach themselves from the p o l y s ~ m e s [ ~ It
~ - is
~ ~no
longer believed that the p subunits detach themselves
independently and influence the termination of the a subu n i t ~ [ ~ ~The
- ~ap
~ ]dimers
then undergo interaction with
the heme pool [37-391 of the cytos01[~~!Though monomeric a-globin does not bind heme[321,the affinity of heme
for a-globin is greater than that for the p s ~ b u n i t [ ~ ~so* ~ ’ ~ ,
that the a subunits of the dimers first become charged with
the heme, and then the p subunits.
A deficit in preparation of special globin subunits is usually
found if the rate of synthesis of one subunit is decreased.
In a-thalassemia, the a chains are synthesized in insufficient
quantity“]. Since the a-gene occurs in isolation in the
genome[431,the preparation of the non-a chains is not
inhibited. The rate of synthesis of the p chains is then
higher by a factor of 1.5 to 3. Under these conditions, the
non-a chains form homotetramer~[~~],
and an Hb H (p4)
can be detected in the erythro~ytes[~’.~~].
This functionally
inferior Hb form is unstable, and tends to form clumps,
which can be detected as Heinz inclusion bodies (in the
erythrocytes). Since the disturbance of the synthesis occurs
during the embryonal development, self-aggregation of
the y chains occurs here, and Hb Bart (y4) appears[47!
In P-thalassemia, the rate of synthesis of the p chains is
reduced. a chains are synthesized in sufficient quantity
and released into the cytosol. Instead of the formation of
a4 tetramers, however, clumping occurs[481.Because of
the close neighborhood of the p family in the genome, it is
possible that partial filling of the p pool can occur if e. g . the
y gene is not inactivated. The result is a persistence of Hb F,
since the y chains are available to the a chains for tetramer
formation even after birth. The synthesis of 6 chains is also
increased under these conditions, so that where there is a
deficit of Hb A, increased quantities of Hb F and Hb A,
are found.
4. Replacement of Individual Amino Acids
Whereas changes in the rate of synthesis of the globin
subunits probably occur at the ribosomal level, point
mutations, which may lead to the replacement of individual
amino acids, take place in the genome. Each amino acid
is coded in the genome (Fig. 3) by a base triplet. By transcription the triplet information from DNA appears as the
codon in the mRNA. Most amino acids are fixed only by
the first two bases of their codons in the mRNA. For some,
the third base position of the codon may be occupied by
any base, but in most cases one of two bases may appear
without making the coding uncertain.
C i
More than 100 amino acid changes have so far been found
in globin subunits, more ofthese being in the p chaind''~ ' ' 1
than in the u chains, probably because the oxygenation of
Hb is promoted by a change in the conformation of the p
subunit, and changes in the p chains are therefore more
likely to be clinically noticeable. This means that the
mutations that are found most often are not necessarily
identical with those that occur most often, since one looks
for molecular changes only if pathological symptoms
appear. In tolerated mutations, other types of base exchange may occur more
The most common base exchange is the replacement of A
by G in the codon. An example is A.42 (Lys)-GA$ (Glu).
As far as the interactions between the side chains of the
amino acids in the protein are concerned, this exchange
means a charge reversal (Lys=-NH:,
reverse exchange (A in place of G) is also common:
GGg (Gly)-rGAE (Asp). That means a neutral amino acid
with a very low bulk (glycine) is thus replaced by a polar
amino acid (aspartic acid), which can enterahto ionic
interactions or form hydrogen bonds, and which can cause
disturbances by these properties.
If this exchange of two purine bases (A and G) of the codon
is projected into the genome, it corresponds there to "confusion" between the pyrimidine bases cytosine and thymine,
which may also result from a disturbance of the transfer of
amino and methyl groups in the cell nucleus (Fig. 4).
Fig. 3. Scheme of protein synthesis. At the top right, the mobile mRNA
is synthesized enzymatically on an exposed gene segment of the DNA
(transcription). The mRNA migrates to the ribosome, i. e. to the proteinsynthesizing apparatus (translocation). In a checking process (according to the rule of base pairing), the anticodon of the transfer RNA
(tRNA) charged with amino acid moves to the codon of the mRNA
(translation). The newly supplied amino acid (in this case phenylalanine
by tRNA,,,) is attached to the already synthesized peptide residue (in
this case valine-lysine).
For example, the amino acid glycine (Gly) is represented
by the mRNA codon GGX, where X may be A, G, C, or U.
Only one amino acid, i.e. methionine (Met), has an invariable code (the codon AUA).
Consequently, every point mutation, i. e. every base exchange, in the gene need not lead to a change in an amino
acid in the protein chain. If a tolerated base exchange occurs
in the third position of the codon (wobbling), there is no
change in the amino acid. On the other hand, base faults
in the first two positions of the codon nearly always lead
to an amino acid change : GAa +GUG causes the replacement of glutamic acid by valine (GIu+Val), which leads
to the formation of HbS (sickle cell anemia) if it occurs in
position 6 of the chain.
Fig. 4.The two most important pyrimidine bases of the nucleic acids.
They are linked by enzymatic amination and methylation.
The replacement of a polar side chain, which enters into
ionic interactions, will always lead to a change in conformation, particularly ifa charge reversal occurs (Table l,
No. 9).
Hydrogen bonds between peptide structures or between
the side chains of individual amino acids, e.g. between
phenolic hydroxyl (Tyr) and a hetero atom (carboxyl 0,
heterocyclic N), will also collapse if one partner is absent.
5. The Globin Subunit
The globin subunits of Hb have a characteristic conformation, which is very similar to that of Mb["]. 75% of the
secondary structure consists of helix structures, which are
grouped into eight sections A to H (see Fig. 5)i661.
Hydrophobic contacts predominate in the interior of the structure,
particularly between the amino acids leucine, isoleucine,
valine, and phenylalanine['. "I. The replacement of such
amino acids by others with polar side chains can lead to
the breakdown of the conformation. If the same exchange
takes place on the surface of the molecule, the conformation
Angew. Chem. internot. Edit. 1 Vol. 11 (1972)
1 No. 7
is only slightly affected (Table 1, No. 6). On replacement of
one amino acid by another, even the difference in bulk may
be sufficient to labilize the conformation (Table 1, Nos. 7
Fig. 5. Schematic representation of helical (double lines) and nonhelical
(single lines) sections in myoglobin [67]. The numbers indicate how
many amino acids are present in the various sections.
and 8), or the a-helix typical of a section of the conformation may be interrupted (also by the omission of an amino
acid), and this again leads to unstable hemoglobins (Table
1, Nos. I-3,4 and 5).
5.1. Globin-GlobinContacts
If amino acids that are essentiaA to the formation of the
quaternary structure are replaced, the alp' contacts
(Table 1,No. 9) or the alp' contacts (Table 1, Nos. 10-13)
may be labilized.
Instead of leading to breakdown of the quaternary structure, the replacement of an amino acid may result in an
increased tendency to aggregate. Since hydrophobic interactions predominate by farr7]among the secondary valence
forces forming the tetramer, an excess of nonpolar side
chains can lead to higher aggregates. The formation of Hb
polymers results macroscopically in clumping of the Hb
with a corresponding functional loss in the transport of 0,.
The increased tendency to aggregate is particularly noticeable in deoxygenated Hb, since a looser conformation here
facilitates interactions between the subunits. The bestknown example of a pathological Hb type with an amino
acid exchange on the surface of the molecule is sickle cell
Hb (Table 1, Nos. 14 and 15). The replacement of the
negatively charged side chain of Glu by the nonpolar Val
leads to the formation of a hydrophobic contact in excess,
which can induce polyaggregation. The clumping of Hb
results in a decrease in the cell volume, and the characteristic sickle cell shape of the erythrocytes is produced.
5.2. Heme-Globin Contact
In addition to the polar and nonpolar interactions within
a globin subunit, side chains of amino acids in its external
zone must ailow the establishment of contacts between the
subunits so that the quaternary structure can be formed
from four subunits. The interactions between the four
monomers differ in their nature. There is a stronger type
of bond u'p' and uzpz and a weaker globin-globin contact
a'p' and u2p1.In the case alpl, 110 atoms from side chains
of 34 amino acids approach to a distance of < 4 A. In the
case alp', contacts (with a distance of < 4 A) are formed
between 80 atoms of the side chains of 19 amino acidsc7'.
The dissociation of the tetramer into dimeric subunits leads
to the "stronger" a'p' dimer'53]:a2p2+2 a'p'. The difference in the strength of the bonds between the subunits is a
necessary condition for conformational changes in the Hb
molecule, as it allows the two clip' dimers to slip in relation
to each other. This conformational change allows an
allosteric activation of the oxygenation (charging with 0,)
and indirectly of the deoxygenation (release of 0,) of the
Hb in the erythrocyte. In the deoxygenated state, the
conformation of the p chain has a loose structure, while in
the oxygenated state it has a more compact structure. The
conformation compressionfs1 occurs by widening of the
heme pocket and reversible replacement of a coordinate
Fe-N bond by an Fe-0, bond.
The optimum reversible charging with 0, is possible only
on the heterotetramer azp,. The homotetramers p4 and y4
bind 0, more strongly and exhibit no allosteric promotion
of the binding of 0,[541.
Hb F (a,y,) binds 0, more strongly
than Hb A,, since a lower 0, partial pressure is available
for supplying the fetal circulation. If the binding of 0, is
too strong, as in the homotetramers, the release of 0, in
Angew. Chem. internat. Edit. I Vol. 11 (1972)
the tissue, e.g. to Mb, is impossible, and 0, transport is
thus prevented.
I No. 7
The function of Hb is extremely sensitive to disturbances in
the amino acid sequence in the immediate neighborhood
of the heme bonding site. The following are essential :1. One
proline, which suppressesthe formation ofa helical structure
in this region. 2. Two histidines in this region, whose
imidazole N serves for coordination of the Fez+. 3. The side
chains of other amino acids, e. g. p"'
in the
vicinity of the histidine residues. These side chains help to
maintain a hydrophobic medium, in which the planar heme
is embedded (Fig. 6). If this hydrophobic medium is disturb-
.. .... -.-.-....._______.
Fig. 6. Heme-globin contacts [7]. The diagram shows which amino acid
side chains of the p chain interact with the porphin system of the heme
in the p subunit. Continuous lines lead to amino acids lying above the
plane of the heme, and dotted lines to those lying below this plane.
The numbers indicate the positions of the amino acids in the 3! chain.
ed (even disturbances without charge effects are sufficient),
the degradation of the Hb may be induced (Table 1, Nos.
7 and 8). A more striking effect is the labilization on incorporation of amino acids with polar groups. If a negative
charge (Table 1, No. 17) appears in the heme pocket, the
conditions for stabilization of a trivalent Fe are satisfied.
Under physiological conditions, autocatalytic oxidation
of Fez+ (hemoglobin) to Fe3+ (hemiglobin, HbM) of the
order of a few % occurs on oxygenation. The Fe3’ is
reduced again by the hemiglobin reductase, which is
NADP-dependent. The reduction equivalents are provided
by the carbohydrate metabolism of the erythrocytes. If a
negative charge is available to stabilize the Fe3+,no enzymatic reduction can take place, and the hemiglobin is lost
for the transport of 0,.
If the proton of a carboxyl group or the H donor of a
hydrogen bond enters into competition with the electron
gap of the heme Fe for the imidazole N (His), the Fe-N
(His) bond is not formed, and Fe oxidation is possible
(Table 1, No. 16). Loss of the Fe-N (His) bond by replacement of the histidine always leads to oxidation of the Fe
and to the appearance of Hb M. All four conceivable possibilities of His replacement are found in nature; either
the proximal or the distal His in CL and p chains may be
replaced (Table 1, Nos. 18-21). If the imidazole N is
replaced by another ligand containing N, e. g. guanidyl N
(Arg)(Table 1, No. 22), the Fe is more susceptible to oxidation than when bonded to His-N. Under the action of
nitro derivatives (drugs), methemoglobinemia may occur.
6. Effectors
Allosteric changes in the conformation of enzyme proteins
can greatly alter the activity of the enzyme. Substances
that bring about such changes in the conformation of a
protein are known as effectors.
The function of the Hb molecule can also be influenced by
effectors. Oxygenation promotes further 0, uptake after
the manner of allosteric activation (Fig. 7). Deoxygenation
can similarly be promoted. A cavity that remains free in
the molecule of the cr2pz heterotetramer contains basic side
o f
4 Po,+
Fig. 7. 0, binding curves. At the 0, partial pressure prevailing in the
tissue (t), the tetrameric H b is only slightly charged with 02.while
large quantities are bound to Mb, corresponding to the functions of Hb
(0, transport) and Mb (0,storage). The dissociation of 0, from Hb is
particularly favored when it is bound in erythrocytes (- . -), i.e. in the
presence of 2,3-diphosphoglycerate (DPG) [62].
groups (p143His),which allow the attack of CO, (Bohr
- ~such
effect) by formation of carbimino g r o ~ p i n g s [ ~ ~ If
a point of attack is lacking (Table 1,No. 23), deoxygenation
is hindered[58-60!
Investigations on erythrocyte-bound and free Hb showed
differences in the strength with which 0, is bound (see
Fig. 7). The mammalian erythrocyte contains a high concentration of a glycolysis metabolite that occurs only in
catalytic quantities in other glycolyzing tissues ; this substance is 2,3-diphosphoglycerate (DPG). When bonded
molarly 1:1 to basic functions (His) of the heterotetramer
~ ~ ~
f i
~ [a ~
~ ~
in conformation
the erythrocyte such that the bonding of 0, is weakened
and deoxygenationis promoted[63- 6 5 ! In bird erythrocytes,
the function of DPG is taken over by an inositol hexaphosphateL6’J. This allosteric control by acidic effectors is
possible only in the phylogenetically youngest u2pz (a,&,)
heterotetramers[611(Hb F contains an
Ser or y’43 Ser
instead of p143His).The homotetramers p4 and y4 exhibit
no Bohr effect and no loosening of 0, by DPG[541.
Received: March 10,1971 [A 887 IE]
German version: Angew. Chem. 84,649 (1972)
Translated by Express Translation Service, London
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Fluorinated P-Sultones
By I. L. Knunyants and G. A. Sokolski[*'
Fluorinated p-sultones are formed on addition of sulfur trioxi- to fluorinated olefins. Tetrafluoroethanesultone has been studied particularly thoroughly. The title compounds are characterized by the ease with which they undergo ring cleavage to give, e. g., derivatives of a-sulfo
carboxylic acids, of sulfonic acids, of carboxylic acids, and of sulfuric acid. Fluorinated compounds of this type containing an a-hydrogen atom are especially valuable in preparative work.
1. Introduction
The name sultones refers to the inner esters of hydroxyalkanesulfonic acids, which may also be regarded as sulfur
analogs of lactones. As with lactones, there exist a-, p-, y-,
6-sultones and numerous other cyclic sulfonates of this
kind. Among the unsubstituted compounds, the y- and
6-sultoneshaving five- and six-membered rings, respectively,
are the most stable. Unsubstituted p-sultones decompose
Their formation by this reaction has been well studied for
numerous examples. Moreover, a synthesis via sulfene
intermediates has also been described['"!
2. Synthesis of p-Sultones
That sulfur trioxide reacts with olefins was demonstrated
as long ago as the beginning of the 19th Century for the
case of ethylene"b,2! The reaction was subsequently extended to a large variety of substituted ethylened3 61, to a
number of chloroolefin~[~
- 'I1, and fairly recently to fluorooIefins["- '*I.
extremely readily and have not yet been isolated. In contrast, fluorinated p-sultones of type (I), formed by addition
of sulfur trioxide to polyfluoroolefins, are relatively stable.
The reactions of sulfur trioxide with polar reagents of type
A-B can be described by a series of equilibria.
60 60
Prof. Dr. I. L. Knunyants and Prof. Dr. G. A. Sokolski
Institute for Organoelement Compounds of the
Academy of Sciences of the USSR
Moscow V 312, U1. Vavilova 28 (USSR)
Angew. Chem. internat. Edit. 1 Vol. 11 (1972) 1 No. 7
A-B + so,
= A-B-SO,-OO
The initial step in the reaction of sulfur trioxide with olefins
is an electrophilic attack by sulfur trioxide on the double
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synthesis, aspects, hemoglobin, genetics
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