Re-evaluation of the presence of multiple haemoglobins during the ontogenesis of the chicken: Electrophoretic and chromatographic characterization, polypeptide composition and immunochemical properties

By M. SCHALEKAMP 0 1 2 M. SCHALEKAMP 0 2 D. VAN 0 2 R. SLINGERLAND 0 2 0 Medical Faculty Rotterdam , Rotterdam 1 Author's address: Department of Anatomy HB12, Medical Faculty Rotterdam , P.O. Box 1738, Rotterdam , The Netherlands 2 From the Department of Anatomy and Embryology of multiple haemoglobins during the ontogenesis Electrophoretic and chromatographic characterization, polypeptide SUMMARY Haemolysates of red blood cells from embryos of several developmental stages ranging from 2 to 21 incubation days and from post-hatching chickens of various age groups were analysed by ion-exchange chromatography, agar- and starch-gel electrophoresis, immunoelectrophoresis with specific antisera and polypeptide chain electrophoresis. With these methods two adult (Ax and A2) and six embryonic (Ej-Eg) haemoglobin types were identified. Antisera specific for the major adult haemoglobins (Ax and A2) as well as antisera specific for the major embryonic haemoglobins (E3, E4) could be prepared. Throughout embryogenesis the haemoglobin types contribute in varying amounts to the total haemoglobin pattern. Three periods of haemoglobin synthesis could be recognized, the transition between these periods occurred at the 6th and 12th incubation day. The first period is characterized by the presence of two major embryonic haemoglobins (E3 and E4) and two minor embryonic haemoglobins (E2 and E5). During the second period E3 and E4 are largely replaced by a major adult haemoglobin (A2) and a new embryonic haemoglobin (Ex). The third period is characterized by the appearance of a second adult haemoglobin (Ax) and a new minor embryonic haemoglobin (E6) with a concomitant decrease of E2 and E5. At the time of hatching two embryonic haemoglobins (Ex and E6) are still present. Besides Ax and A2, several minor haemoglobin fractions were inconsistently found in adult chickens. Evidence has been obtained that these additional fractions are reflecting a so called minor heterogeneity or separation artifacts. The haemoglobins Al5 A2 and Ej-E6 show different polypeptide chain compositions. Three embryo-specific chains could be demonstrated (/? E2E5,yE4 and #E3). The production of the polypeptide chains appears to be correlated with the aforementioned periods of haemoglobin synthesis. The genetic and morphological implications of the findings are discussed. - During ontogenesis activation and repression of genes take place continuously, causing the process of differentiation. The factors involved are still difficult to study due to complexity of the developmental events. Emergence of new proteins is thought to be a rather direct expression of gene activity. A system in which changes in protein composition are readily demonstrable would therefore be useful as a model. Haemoglobin maturation heterogeneity may be such a system. Embryonic and foetal haemoglobins, different from adult haemoglobins, occur in representatives of several vertebrate classes (Manwell, 1960; Ingram, 1963; Manwell, Baker, Rolansky & Foght, 1963). In a number of avian species, embryonic haemoglobins have been demonstrated: turkey and partridge (Manwell, Baker & Betz, 1966), white Peking duck (Borgese & Bertles, 1965), and house sparrow (Bush & Townsend, 1971). The presence of embryonic haemoglobins in chickens is still controversial. Some authors (Fraser, 1961, 1964, 1966; Wilt, 1962; Simons, 1966) state that the changes in haemoglobin composition as observed during ontogenesis are essentially quantitative, others (D'Amelio & Salvo, 1959, 1961; D'Amelio, 1966; Manwell et al. 1963; Manwell et al. 1966; Hashimoto & Wilt, 1966; Schiirch, Godet, Nigon & Blanchet, 1968; Denmark & Washburn, 1969) report the presence of distinct embryonic haemoglobins, but do not agree about the number of haemoglobin types and the time of their appearance. There is no agreement even about the number of haemoglobins present in adult chickens (Godet, Schiirch & Nigon, 1970). These discrepancies are possibly due to the fact that most authors have used only one or two different techniques. Therefore, a thorough re-analysis of the haemoglobin types present in adult and embryonic chickens seems to be necessary. In the present study the different haemoglobin types have been characterized by agar- and starch-gel electrophoresis, by chromatography on cation and anion exchangers, by immunochemical techniques and by the analysis of the polypeptide chain composition. Furthermore, the developmental stages at which the haemoglobin types appear and disappear in the circulation have been determined. MATERIALS AND METHODS Preparation of the haemolysate. Blood samples were obtained from the wing vein of White Leghorn chickens of various age-groups, ranging from 1 day to 3 years post-hatching and from the vitelhne vein or the heart of White Leghorn embryos at various developmental stages, ranging from 2 to 21 incubation days. Care was taken to avoid contamination with yolk, as washing of the blood cell suspension with saline does not sufficiently remove the yolk particles. For the 3- to 5-day embryonic stages the haemolysates from 300 animals were used. For the later embryonic stages haemolysates from 3-30 animals were pooled. Posthatching samples were obtained from individual chickens. The blood cells were suspended and washed three times in a tenfold volume of ice-cold saline. They were allowed to lyse for 12 h in 0-025 M-NaCl at 4 C. After centrifugation the haemoglobins in the supernatant were converted into the CO-form. Samples were stored at 4 C until analysed. Analyses were started within 48 h after preparation of the haemolysate, as haemoglobins are known to alter by ageing as well as by freezing and thawing (Manwell et ah 1966). Electrophoresis. Agar-gel electrophoresis was carried out using 0-05 M barbiturate buffer, pH 8-6, following the method of Wieme (1959). Vertical starch-gel electrophoresis was performed according the Smithies (1959) using a discontinuous buffer system containing 0-017 M Tris, 0-07 M EDTA and 0-025 M boric acid, pH 8-9 (gel) and 0-3 M boric acid and 006 M-NaOH, pH 8-2 (electrode vessels), or as described by Scopes (1963),'who used 0-25 M sucrose, 0-0015 M citric acid and 0-020 M Tris, pH 9-1 (central part of the gel), 0-010 M boric acid and 0-06 M Tris, pH 8-6 (ends of the gel) and 0-3 M boric acid and 0-06 M-NaOH, pH 8-2 (electrode vessels). All analyses were carried out in the cold room at 4 C. In order to trace the haemoglobins, the agar- and starch-gel strips were stained by a standard peroxidative procedure, in which benzidine in acetic acid and hydrogen peroxide were used (Dessauer, 1966). Amido black or Ponceau S were used to visualize other protein components. Relative electrophoretic mobilities in agar were calculated according to Wieme (1959), using as standards 1 % human serum albumin (Behring), mobility 100, and 1 % dextran (M.W. 135000), mobility 0. Column chromatography. Carboxmethyl (CM)-cellulose was used as cation exc (...truncated)