On the Heterogeneity of Chicken Hemoglobin.

Transcription

1 Arch. histol. jap. Vol. 25, n. 4 (May 1965). P Ist Dept. of Anat. (Prof. M. MORI), Fac. of Med., Kyushu Univ., Fukuoka. On the Heterogeneity of Chicken Hemoglobin. (Received March 15, 1965.) It is in general accepted that hemoglobin is detectable within the karyoplasm as well as in the cytoplasm of erythroid cells in various vertebrates. There are different interpretations on the occurrence of hemoglobin within the nucleus, that is: the intranuclear hemoglobin may be, 1. synthesized within nucleus as well as in cytoplasm (D'AMELIO and SALVO 1959, 1961, O'BRIEN 1960, WILT 1962), 2. emigrated from the cytoplasm through pores of nuclear membrane (DAVIES 1961, GRASSO 1962), or 3. trapped into the nucleus during mitosis (THORELL 1947, WILKINS and DE CARVALHO 1953). As previously reported (MIYOSHI 1961) on the appearance of hemoglobin in erythroid cells of developing chicken, it is suggested that most amount of the nuclear hemoglobin may originate from the cytoplasmic hemoglobin. Furthermore, it is well known that there are a few components in chicken hemoglobin separable by electrophoresis or column chromatography. The investigations, however, have not thoroughly been done on the chemical properties of nuclear hemoglobin (D'AMELIO and SALVO 1959) Present work deals with electrophoresic analysis of hemoglobin of chicken remained in nuclear fraction after hemolysis, and some results obtained on the nature of heterogeneity of chicken hemoglobin will be discussed. Blood was obtained by heart-puncture from groups of chickens just after hatched and adult chickens of white Leghorn breed. After pooling of citrated blood (50-60 ml), the red blood cells were washed several times with saline (0.9% NaCl solution containing M CaCl2) and resuspended in the saline to make a volume equal to that of the original blood. Preparation of nuclear and cytoplasmic fractions of hemoglobin. According to LAN and DOUNCE (1943), the hemolytic reagent was 6% saponin in 0.11M phosphate buffer (ph 6.9). Red blood cells were lysed by adding 1ml of the reagent to every 20ml of cell suspension. After laked completely in five minutes, the hemolysate was centrifuged, and the supernatant was removed by suction and used as cytoplasmic fraction of hemoglobin. The sediment was thoroughly washed with the saline to separate the water extractable hemoglobin. The sediment was after then homogenized 405

2 406 M. MIYOSHI: containing dilute hemoglobin was obtained by centrifugation and used as nuclear fraction of hemoglobin. To obtain high concentration of hemoglobin solution, the supernatant was poured into a cellophan bag and dialysed against 30-50% gummi arbicum in veronal buffer (ph 6.8) (ERNERBECK 1951) Purification of hemoglobin samples. Crude hemoglobin solution of each fraction mentiened above was mixed with a equal volume of cold ether and centrifuged to separate superior layers of ghost and ether from inferior layer of hemoglobin solution. Hemoglobin was, further, crystallized by dialysis through cellophan against 2.8M were disolved in distiled water and analysed in following experiments. In addition, a part of crude hemoglobin solution was dialysed against saturated (NH4)2 SO4 solution hemoglobin. A little amount of gruel-like pellet was obtained, although crystallization of hemoglobin did not occur in this procedure. Therefore, hemoglobin in the supernatant and the pellet was also crystallized in the same manner as mentioned above. Paper electrophoresis and densitometry. All the hemoglobin samples were electrophoretically examined by means of a horizontal type of paper electrophoresis appara- No. 51 A. The constant voltage was conducted at volts for 10 hours. A solution used as a fluid medium was 0.12M tris EDTA boric acid buffer (ph 9.1). Following to electrophoresis, paper strips were dried in air and cleaned with mixture hemoglobin in the patterns. Spectrophotometry. The same spots of hemoglobin which were separated in electrophoresis were cut off from five strips and immmersed in 5ml of distiled water for one hour to extract hemoglobin from paper. These extract solutions were centrifuged and spectrum analysis of the supernatant were carried out with automatic ab- length. The scanning speed was 3m per minute, In addition, the same fluid were spectrophotometer DU type. As shown in electrophoretograms (Fig. 1, A and B), chicken hemoglobin migrated towards anode and was distinctly resolved into two components at room temperature than the faster moving small component on strips of both stained (A) and unstained (B). Of hemoglobin in nuclear and cytoplasmic fractions prepared from red blood cells in newly hatched chicken, the two components characteristic of chicken hemoglobin were also demonstrated in both nuclear and cytoplasmic fractions (Fig. 2, A and B). There was no obvious distinction on electrophoretograms of nuclear and cytoplasmic fractions. The migrated patterns of these components of hemoglobin were quite sim-

3 Heterogenity of Chicken Hemoglobin. 407

4 408 M. MIYOSHI: Fig. 2. Paper electrophoretograms of the nuclear and cytoplasmic fractions of hemoglobin from newly hatched chickens (Tablel, No. 10) ilar to those in adult chicken. In densitometric analysis on strips of these nuclear and cytoplasmic fractions, there was, however, a difference in relative concentration of main and small componts between them. Fig. 3 reveals the relative quantity of the two components in nuclear and cytoplasmic fractions, which were analysed as a pair in electrophoresis (listed as Nuclear fraction. SC:MC=24.2:75.8% Cytoplasmic fraction. SC:MC=28.48:71.6% Fig. 3. Densitometry of unstained strips of nuclear and cytoplasmic fractions of hemoglobin component, MC main component.

5 Heterogenity of Chicken Hemoglobin. 409 No. 10 in Table 1). As shown in the integrated recordings in Fig. 3, the main component accounted for 75.8 per cent of total hemoglobin in nuclear fraction, whereas it was 71.6per cent in cytoplasmic fraction. These results showed that hemoglobin in nuclear fraction contained higher 4.2 per cent of main component than that of cytoplasmic fraction. The higher concentration (about 5per cent in average) of main component in nuclear fraction than in cytoplasmic fraction was also confirmed by all other data which were listed in Table 1. On the other hand, crude hemoglobin solutions were dialysed for long time (24-36 hours) against saturated ammonium sulphate solution Table 1. Densitometric recordings of relative concentration of hemoglobin components on unstained strips. 1, 2, 3; 4, 5, 6; and so forth are of the same blood sample each. procedure, two fracticns of hemoglobin were obtained, those were hemoglobin in either the supernatant or the gruel-like pellet. The hemoglobin obtained from this supernatant fluid showed only one component on electrophoretic pattern, which accorded with migrating spot of main component mentioned above (Fig. 4). However. there were two components of hemoglobin in the pellet (Fig. 4). Fig. 4. Paper electrophoretograms of hemoglobin in the pellet (P) and the supernatant (S). These fractions of hemoglobin were obtained when crude hemoglobin solution was dialysed against ammonium sul- The two components of hemoglobin separated in electrophoresis were subjected to similarity among the absorption curves, which were recorded to each of those components as well as original solution of hemoglobin (Fig. 5, A and B). The absorption

6 410 M. MIYOSHI: A Fig. 5. Spectrophotometry of each components of nuclear fraction of hemoglobin solution, but not in those of two components extracted from strips, for it was covered up by spectrum extinction of medium itself. By means of electrophoresis using supporting media, such as filter paper (CABAN- NES and SERAIN, DATTA et al., JOHNSON and DUNLOP, RODNAN and EBAUGH, SARA et al., SYDENSTECKER et al., VAN DER HELM and HUIS- MAN), agar gel (MATSUDA et al., NAKAMURA, WILT, YAMAUCHI), or starch gel (AMBS and THORELL, D'AMELIO and SALVO 1959, 1961, WILT), it has been reported that chicken hemoglobin is separated into two components; the main (C1) and the small (C2) components. The column chromatography, however, shows the presence of three components (C1, C2, C3) in this hemoglobin (MATSUDA and

7 Heterogenity of Chicken Hemoglobin. 411 and that this property is more characteristic of small component. But the real implihemoglobin. With automatic (A) and BECKMAN spectrophotometer (B). small component. Arrow shows tryptophan notches. B TAKEI). In the present study, two components (C1, C2) were identified on filter paper, but no any other component was migrated. A dilute tris EDTA boric acid buffer (0.12M, ph. 9.1) (MIYOSHI) was more suitable as a fluid medium for distinct separation of those components in chicken hemoglobin than that of high concentrated (0.5M) and low ph ( ) (ARONSON, LONG). However, the third minor component (C3) migrating towards more anodic than the small component (C2) is reported by FRASER (1963). This resolution of third component may be due to his application of cellurose acetate as a supporting medium. It is reported by DRABKIN (1949) and WILT (1962) that chicken hemoglobin does not salt out in ammonium sulphate solution. In the present study, the hemoglobin did not crystallized when dialysed against saturated solution of ammonium sulphate (ph 4.4), but a certain amount of pellet was obtained. In electrophoretic assay, two component (C1, C2) were separated from hemoglobin in this pellet, while there was only one component (C1) in the supernatant. These results may suggest that the solubility of chicken hemoglobin in water is affected to some extent by existence of sulphate ion

8 412 M. MIYOSHI: cations of this phenonmenon are unknown at present. Furthermore, it has been suggested that the proportion of two component is essentially the same in ratio to hemoglobin from mature chickens of various breed (VAN PER HELM and HUISMAN) and that this proportional concentration of two types of hemoglobin is retained from the time of hatching to the adult (FRASER). There are, however, considerable differences among data reported about it elsewhere. The content of main component to whole hemoglobin varies from 85 per cent (VAN- RER HELM) to 60 per cent (AMPS and THORELL). In the present study, two components were found in either nuclear or cytoplasmic fraction of hemoglobin from newly hatched chickens. There was no any difference in the migrating patterns of hemoglobin between them. But, in all samples subjected to densitometry, there was the increase of about 5 per cent of main component in nuclear fraction as compared with cytoplasmic fraction. These results verify the suggestion of RUHENSTROTH-BAUER (1957) that after hemolysis considerable amount of hemoglobin remains attached to the stroma. Therefore it might be supposed from the results obtained here that the ghost hemoglobin is also composed of two components, the concentration of which are different in proportion from that of the dissolved cytoplasmic fraction. And also, the difference of the proportion in the data reported by many authors may depend on gradient extraction of the ghost hemoglobin into the water soluble fraction. These suggestions are supported by the evidence that when nucleus is isolated inn non-aqueous medium, the non water extractable hemoglobin in nucleus of chicken red blood cell is chiefly composed of main component (D'AMEELIO and SALVO). However, there is no evidence that this ghost hemoglobin is synthesized in nucleus (DAMELIO and SALVO 1959, 1961) and is identical with the structural hemoglobin of nucleus (TOOZE and DAVIES). Spectrophotometric analysis of main and small components showed the typical absorption spectra characteristic of oxy-hemoglobin. Every absorption curve revealed and extended to the two fractions of hemoglobin obtained by dialysis against ammonium sulphate solution. The nature of absorption curve of chicken hemoglobin are consistent with the report of MATSUDA and TAKEI (1963), but not with the statement of YAMAUCHI (1960), in which all absorption peaks are shifted to long wave length. The nuclear fraction of hemoglobin was obtained from chicken red blood cells by hemolytic procedure and the heterogeneity of this hemoglobin was studied by means of paper electrophoresis. Chicken hemoglobin was separated into two components; main and small components. There was no any significant difference between the migrating patterns of the nuclear and the cytoplasmic fractions of hemoglobin. But in densitometric analysis, there was the increase of main component in nuclear fraction as compared with cytoplasmic fraction. These results suggested that the ghost hemoglobin remained in

9 Heterogenity of Chicken Hemoglobin. 413 nuclear fraction after hemolysis is composed of two components, the concentrations of which are different in proportion from those of dissolved cytoplasmic fraction. The supernatant of hemoglobin solution which was dialysed against the saturated ammonium sulphate solution contained main component only, whereas the pellet exhibited two components. It might be supposed that the solubility of chicken hemoglobin, especially the small component, is lowered by sulphate ion. The main and small components showed the absorption spectra characteristic of oxy-hemoglobin and marked trytophan notches. Author is much indebted to Professor M. MORI for his advice and encouragement. References. Ambs, E. and B. Thorell: On the type of hemoglobin in the neoplastic cells of virus induced fowl erythroleukemia. J. nat. Cancer Inst. 25 (1960) P Aronson, T. and A. Gronwall (1958): Improved separation of serum protein in paper electrophoresis. A new electrophoretic buffer. Scand. J. clin. lab. Invest. 9 (1958). P Cabannes, R. and Ch. Serain: Etude electrophoretique des hemoglobines des mammiferes domestiques d'algerie. C. r. Soc. Biol. 149 (1955). P D'Amelio, V. and A. M. Salvo: On the nuclear hemoglobin of chicken red blood cells. Exp. Cell Res. 18 (1959). P Further studies on the embryonic chick hemoglobin. An electrophoretic and immunoelectrophoretic analysis. Acta Embryol. Morphol. Exp. 4 (1961). P Davies, H. G.: Structure in nucleated erythrocytes. J. bioph. biochem. Cytol. 9 (1961). P Datta, R. et al.: Electrophoretic behaviour of avian hemoglobin. Nature 181 (1958). P Drabkin, D. L.: Arch. Biochem. 21 (1949). P (Cited from) Drabkin: Spectrophotometric studies. XV. Hydration of macrosized crystals of human hemoglobin, and osmotic concentrations in red cells. J. biol. Chem. 185 (1950). - Drabkin, D. L. and J. H. Austin: Spectrophotometric studies. II. Preparations from washed cells; nitric oxide hemoglobin and sulfhemoglobin. J. biol. Chem. 112 (1935). P Ernerbeck, H.: Die elektrophoretische Darstellung normaler menschlichen Liquor. Scandinav. J. Clin. Lab. Invest. 3 (1951). S

10 414 M. MIYOSHI. Fraser, R. C.: Hemoglobin formation in the chick embryo. Exp. Cell Res. 25 (1961). P Electrophoretic characteristics and cell content of the hemoglobin of developing chick embryos. J. exp. Zool. 156 (1963). P Grasso, J. A.: Observation on the development of erythrocytes in mammalian fetal liver. J. Cell Biol. 14 (1962). P Johnson, V. L. and J. S. Dunlop: Electrophoretic separation of hemoglobin from chickens. Science 122 (1955). P Lan, T. H. and A. L, Dounce: Isolation and properties of chicken erythrocytes nuclei. Science 97 (1943). P Long, C. (Editer): Biochemist's handbook. New york, Van Nostrand press, Matsuda, G. et al.: Comparative biochemistry of hemoglobins, I. Comparison of a few properties of various hemoglobins. Acta med. Nagasaki. 7 (1963). P Matsuda and H. Takei: The studies on the structure of chicken hemoglobin. I. Chromatographic purification of chicken hemoglobin. J. Biochem. 54 (1963). P Miyoshi, M.: Appearance of hemoglobin in the earliest erythroblast. Arch. histol. jap. 21 (1961). P An application of tris EDTA boric acid buffer on the paper electrophoresis of hemoglobin in the chicken. (Abst.) Arch. histol. jap. 23 (1963). P Nakamura, E.; Das mehrfache Hamoglobin. II. Avian. Lymphatol. 4 (1960). S O'Brien, B. R. A.: The presence of hemoglobin within the nucleus of the embryonic chick erythroblast. Exp. Cell Res. 21 (1960), P Rodnan, G. P. and F. G. Ebaugh, jr.: Paper electrophoresis of avian blood. Proc. Soc. exp. Biol. Med. 95 (1957). P Ruhenstroth-Bauer, G.: Die Struktur der Saugererythrozyten. Handbuch der gesamten Hamatologie. 2. Aufl. Bd. 2. Munchen, S Saha, A. et al.: Paper electrophoresis of avian and mammalian hemoglobin. Science 125 (1957). P Sydenstecker, V. P. et al.: Electrophoretic behaiviour of some animal hemoglobins, Proc. Soc. exp. Biol. Med. 95 (1956). P Thorell, B.: Studies on the formation of cellular substances during blood cell production. Acta. med. stand Suppl. P Tooze, J. and H. G. Davies: The occurrence and possible significance of hemoglobin in the chromosomal regions of mature erythrocyte unclei of the newt, Triturus christatus christatus, J. Cell Biol. 16 (1963). P van der Helm, H. J. and T. H. J. Huisman: The two hemoglobin components of the chicken. Science 127 (1957). P Wilt, F. G.: The ontogeny of chicken embryo hemoglobin. Proc. nat Acad. Sci. 48 (1962). P Wilkins, M. H. F. and S. De Carvalho: The violet light microscope. A method for visual estimation of heme in living cells. Blood 8 (1953). P Yamauchi, M.: Untersuchungen uber die Agargel-Elektrophorese. Lymphatol. 4 (1960). S. 121.