Review Article J Med Genet: first published as 10.1136/jmg.10.1.50 on 1 March 1973. Downloaded from Journal of Medical Genetics (1973). 10, 50. Fetal and Embryonic Haemoglobins P. A. LORKIN MRC Abnormal Haemoglobin Unit, University Department of Biochemistry, Cambridge Haemoglobin has been the subject of intensive form a nearly spherical molecule with extensive research for many years and is one of the most areas of contact between unlike chains; the two thoroughly understood of all protein molecules. main types of contact are denoted alp, and alg2 The amino-acid sequences of haemoglobins from The tetramer exhibits cooperative behaviour or many species of animals have been determined haem-haem interaction. As each haem combines (tabulated by Dayhoff, 1969) and the molecular with oxygen the affinity of successive haems in- structures of horse and human haemoglobins have creases. The oxygen affinity curve of the tetramer been determined in great detail by x-ray crystallo- is sigmoidal and may be represented approximately graphy (Perutz et al, 1968a and b; Perutz 1969). A by the Hill equation:* mechanism of action of haemoglobin has been pro- = kpo2n posed (Perutz, 1970a and b and 1972). The y haemoglobins of higher organisms share a common +kpo2n tetrameric structure built up of two pairs of unlike Oxygen affinity data are usually presented in copyright. chains; the a chains containing 141 amino-acid terms of P102, the partial pressure of oxygen re- residues and the non-a chains containing generally quired to attain half saturation with oxygen, and of 145 or 146 amino acids. In man, five types of n, the exponent of the Hill equation. These para- non-a chains have been recognized, /3, y, 8, E. and 4, meters respectively give measures of the oxygen corresponding to different stages of ontogenesis. affinity and of the degree of haem-haem interaction. The globin chains ofhaemoglobin and of myoglobin For normal haemoglobin n is approximately 3. have a similar overall tertiary structure composed of Myoglobin, isolated chains, and tetramers composed http://jmg.bmj.com/ segments of a helix interspersed with non-helical of one type of chain such as Hb H (4) show no regions forming a compact structure of great com- haem-haem interaction, their oxygen dissociation plexity. Amino-acid residues whose side chains curves are hyperbolic and the n value is 1. are of a polar nature, ie, those which bear an electric The haemoglobin tetramer has a lower oxygen charge or a strong dipole movement are located at affinity, more appropriate of its physiological func- the outer surface of the molecule in contact with the tion, than single chains. The cooperative behaviour whereas the of the tetramer renders it more efficient in releasing aqueous surroundings, non-polar on September 23, 2021 by guest. Protected hydrocarbon side chains are, in general, located oxygen in response to relatively small changes in within the interior of the molecule. The haem oxygen tension. The tetramer exhibits other groups are accommodated within deep cavities or physiologically advantageous properties; the oxygen pockets lined with non-polar side chains and are affinity varies with pH (Bohr effect) in such a manner anchored in position by links between the haem iron as to facilitate the release of oxygen in the tissues. and the imidazole side chains of histidines. The Haemoglobin binds CO2 at the N terminus of the non-aqueous environment of the haem promotes / chains (Kilmartin and Rossi-Bernardi, 1969) and reversible binding of oxygen and protects the iron assists its transport from the tissues to the lungs. from oxidation to the ferric form characteristic of Furthermore its oxygen affinity is reduced in methaemoglobin which is incapable of reversible the presence of organic phosphates particularly oxygen binding. 2,3 diphosphoglycerate (2,3 DPG) and inositol The four chains of the tetramer fit together to * Hill equation: y =fraction of haemoglobin in oxy form, P02 Received 27 November 1972. partial pressure of oxygen, k and n are constants. 50 Fetal and Embryonic Haemoglobins 51 J Med Genet: first published as 10.1136/jmg.10.1.50 on 1 March 1973. Downloaded from hexaphosphate (IHP) (Benesch and Benesch, 1969). postulated that a gradual divergence of the structure This provides an important method of regulating of the polypeptide chains led to their ability to the oxygen affinity in vivo. The properties can be associate with each other to form dimers and tetra- accounted for in terms of the molecular structure mers and the appearance of haem-haem interaction (Perutz, 1970a and b). and the Bohr effect. It seems likely that the Many species of animals contain multiple haemo- 'ancestral molecule' was probably longer than globins which can be separated by electrophoresis. modern globin chains because of the need to postu- The components are often present in unequal pro- late deletions and the fact that the globin chains of portions eg, in man Hb A and Hb A2 are present in more primitive animals eg, lamprey are longer than the ratio of about 40: 1. Some of the components those of higher animals. It is a matter of contro- may be chemically modified forms of other com- versy whether the differences in amino-acid se- ponents; for example the minor component Hb A,, quences of haemoglobin chains of different animals of human blood consists of Hb A modified by may be attributed to the action of Darwinian natural attachment of a carbohydrate residue at the N selection or to the action of genetic drift and the terminus of the chain (Bookchin and Gallop, fixation in different species of amino-acid substitu- 1968). Other multiple components represent the tions which cause no significant change in the pro- products of separate gene loci and contain globin perties of the haemoglobin and are selectively chains which differ by one or more amino-acid neutral (review by Harris, 1971). Some parts of substitutions, thus the : and 8 chains ofhuman Hb A the amino-acid sequence of the globin chain show and Hb A2 differ by 10 amino-acid substitutions. much less interspecies variability than others and Fingerprinting has revealed differences between some residues are invariant in all species. In the haemoglobins of different species (Muller, 1961) general, the amino acids whose side chains form the and in many cases the exact amino-acid sequences lining of the haem pocket and the surfaces of the have been determined (Dayhoff, 1969). In general ail92 interface show very little variability and any each species has one or more haemoglobin compon- substitutions are of a conservative nature ie, one ents unique to itself. The more closely related two amino acid is replaced by another of a similar charac- species are, the more similar are their haemoglobins. ter. In contrast, amino acids located at the outer In man and chimpanzee the major adult haemo- surface of the molecule show wide variability. copyright. globin Hb A is identical and the minor component In studying the haemoglobins of animals it is Hb A2 differs by only one amino-acid substitution practicable to examine the specimens from only a (de Jong, 1971). In various species of the genus small number of animals. The situation is different Equus haemoglobin components differ by one with the human population where blood samples amino-acid substitution (Kitchen and Easley, 1969). from many people have been examined by electro- When the amino-acid sequences of the globin chains phoresis during the course of routine haematological http://jmg.bmj.com/ of haemoglobins and myoglobins from many species examinations and surveys. are compared they show a great degree of homology These studies have given a measure of the degree if gaps are introduced at the appropriate positions ofintraspecic variation ofhaemoglobin in the human (Braunitzer et al, 1964). population and have led to the discovery of over 160 Such comparisons of globins and other proteins, different mutant forms ofhaemoglobin (reviewed by especially cytochrome c (Dayhoff, 1969), have pro- Huehns and Shooter, 1965; Lehmann and Carrell, vided a novel basis for the construction of phylo- 1969; Lehmann, 1972). The study of human genetic trees based on data quite independent of haemoglobin mutants has afforded much valuable on September 23, 2021 by guest. Protected conventional zoological characteristics. Further- information. Studies of the inheritance of abnor- more these comparisons have lent support to the mal haemoglobins showed that the a and f chains speculation that the haemoglobin and myoglobin were specified by separate gene loci, and this is also chains may themselves have diverged and evolved true for y and 8 chains and presumably also for the from a more primitive 'ancestral molecule'. The E and 4 chains. Some recent evidence (discussed general manner in which this may have come about below) suggests that, at least in some individuals, has been outlined by Ingram (1961), who postulated the a-chain locus is duplicated. The y-chain locus that an initial single gene locus specifying a globin is also duplicated and the polypeptide chains speci- chain may have undergone duplication followed by a fied by different loci differ by at least one amino- gradual divergence of the two genes by independent acid substitution (Schroeder et al, 1968). mutations and deletions. Successive duplications The study of rare pathological haemoglobins in may have given rise to the genes for the families of which the physiological functions are impaired has polypeptide chains observed in each species. It is played an important role in the understanding of 52 P. A. Lorkin J Med Genet: first published as 10.1136/jmg.10.1.50 on 1 March 1973. Downloaded from the normal functioning of haemoglobin (Perutz and at 12 months (summarized by Huehns and Beaven, Lehmann, 1968).