Ontogeny of haemoglobin in the chicken

Ontogeny of haemoglobin in the chicken 0 Author's address: Department of Biology, Carleton University , Ottawa 1 , Canada. 5 JEEM l6 1 Authors' address: Laboratory of the Marine Biological Association of the U.K. , Citadel Hill, Plymouth, Devon, England 2 From the Department of Biological Science, The Florida State University; the Research Division , Thornber Bros., Halifax , Yorkshire; and the Department of Zoology, University of Illinois Study of an individual protein type such as haemoglobin during development provides a convenient way to study the embryological expression of a small number of genes. Knowledge of the quaternary structure of the protein is necessary because a number of proteins have been found to be made up of more than one type of polypeptide chain, and each polypeptide chain with a different amino acid sequence is coded from a distinct gene (cistron). For example, human foetal haemoglobin is a2y2; human adult haemoglobin is a2/?2. Both chemical and genetical studies indicate that the a chains of both haemoglobins are coded from the same cistron, whereas the embryological change involves a 'switchover' from the y- to the /?-chain cistron (reviewed by Ingram, 1963). Embryonic, larval or foetal haemoglobin, distinct from adult haemoglobin, occurs in representatives of several vertebrate classes (reviewed by Manwell, 1960, 1963). However, as the following summary of the literature shows, the presence of a distinct embryonic haemoglobin in the chicken is controversial. Fraser (1961) and Wilt (1962) claim that there are basically only two haemoglobins in the domestic fowl and that during ontogeny there is only a change in relative amounts of the two haemoglobins, although Fraser (1964) has recently reported finding a third haemoglobin appearing 2 weeks before hatching and continuing to occur in the adult chicken. Among the criteria used by Fraser (1964) is that one of the haemoglobins has a higher content of methionine than the others, whereas other workers report the absence of this amino acid from chicken haemoglobin, whether it is resolved into two (van der Helm & Huisman, 1958) or three (Alekseenko & Orekhovich, 1964) components. Huisman and colleagues (Huisman, Van Veen, Dozy&Nechtman, 1964; Huisman & Van Veen, 1964) state that similar haemoglobins are found in embryonic and adult chickens, although they examined no material earlier than 14 days of incubation and they did report the existence of a trace basic haemoglobin component in late embryos - (foetuses) and newly hatched chicks which does not occur in adult chickens. They find that the differences in the oxygen affinity of late embryo and adult chicken haemoglobins, such as those reported by Hall (1934), are due to differences in the concentration of phosphate ion, which also influences the electrophoretic behaviour of the minor (anodal) chicken haemoglobin component. However, D'Amelio & Salvo (1961) found two distinct embryonic chicken haemoglobins present only in the first few days of incubation; by from 9 to 11 days of incubation they observed that the embryonic haemoglobins were largely replaced by the adult type of haemoglobins characteristic of foetuses and adults. Using starch-gel electrophoresis, Manwell, Baker, Roslansky & Foght (1963) obtained results very similar to those of the Italian workers. Up to 6 days of incubation chicken embryos of three different breeds contain a major and a minor haemoglobin, both different from the major and minor haemoglobin of late foetuses, chicks and adult chickens. Thus, the 'switchover' from embryonic haemoglobins to adult haemoglobins is very rapid in the chicken; by 7 days of incubation approximately half of the haemoglobin is of the adult type. Two other independent biochemical criteria for the distinctness of embryonic and adult chicken haemoglobins have been supplied (Manwell, Baker, Roslansky & Foght, 1963): (a) tryptic peptide patterns ('fingerprints') of the haemoglobins from 5-day embryos and from adult chickens differ by several peptides; (b) oxygen equilibria of 5-day embryo and adult haemoglobins, dialysed against the same phosphate buffers (thus avoiding the influence of any possible phosphate ion difference, as reported by Huisman et al. (1964), are very different. Recently Deuchar & Dryland (1964) found two distinct embryonic chicken haemoglobins, using cellulose acetate electrophoresis, and Borghese & Bertles (1965) have found a similar ontogenetic change for the haemoglobins of the duck. It is clear that some of the controversy arises from the failure to sample embryos sufficiently early (e.g. Huisman & Van Veen, 1964; Huisman et al. 1964). However, that does not explain the results of Fraser (1961, 1964) or Wilt (1962). Fraser (1964) mentions using the methaemoglobin cyanide derivative, often preparing the haemoglobin by freezing and thawing. It is known that there are differences in the electrophoretic mobility of various haemoglobin derivatives (Chernoff & Pettit, 1964a) and that freezing and thawing can blur the electrophoretic resolution of haemoglobin (Manwell, Baker & Childers, 1963; Huntsman et al. 1964); in addition, in the presence of appropriate ions, freezing and thawing can result in hybridization of different types of lactate dehydrogenases (Kaplan, 1964) or haemoglobins (Manwell, unpublished studies). Accordingly, in the present paper we report the results of additional studies on embryonic and adult chicken haemoglobin, including studies on chemical modification, polypeptide chain composition, electrophoresis in additional buffer systems, and measurements on oxygen-binding properties of purified and dialysed haemoglobins. Also, as the erythrocyte represents an easily isolated 'pure' cell type, studies have been made on electrophoretic properties of several erythrocyte enzymes to see if the haemoglobin 'switchover' is paralleled by other ontogenetic changes in red blood cell proteins. MATERIAL AND METHODS Haemoglobin preparation, vertical starch-gel electrophoresis, and oxygen equilibrium determination are as described elsewhere (Manwell, 1963; Manwell, Baker, Roslansky & Foght, 1963). All haemoglobin preparations were kept in the carbon monoxide form during purification and electrophoresis, except in specific electrophoretic comparisons of oxyhaemoglobin, methaemoglobin and methaemoglobin cyanide, or where the sample was destined for oxygen equilibrium studies. It was necessary to pool 5-day embryos to provide enough haemoglobin for determination of the oxygen equilibrium under reasonable conditions of haemoglobin concentration (2-5 %); however, no individual embryonic or adult variation has been observed in screening on an individual basis a total of 98 adults and 78 5-day embryos, plus 70 embryos of other ages and 18 newly hatched chicks; the breeds and strains surveyed are: New Hampshire, Columbian, New Hampshire x Columbian, White Leghorn, Silkie, Light Sussex type (Thornbers' 'strain 6') Thornber 404 (...truncated)