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J. Embryol. exp. Morph. Vol. 19, 1, pp. 95-101, February 1968 With 2 plates Printed in Great Britain 95 Ultracentrifugal and electrophoretic analysis of the water-soluble fraction of chick embryo yolk By P. CARINCI & L. MANZOLI-GUIDOTTI1 From the Institute of Histology and General Embryology, University of Bologna INTRODUCTION Water-soluble proteins, mainly livetins (a-, /?-, y-), are present in hen egg yolk (Martin, Vandegaer & Cook, 1957). They represent 5 % of fresh yolk solids (Saito, Martin & Cook, 1965). During the later stages of incubation the watersoluble proteins (water-soluble fraction, WSF) undergo a marked increase in relative proportion; after 15 days of incubation they form over 15% and at 18 days over 40% of yolk-residual solids (Saito et ah 1965). This proportional increase of the WSF is tentatively explained by the passage of egg-white proteins into yolk (Mclndoe, 1960; Saito et al. 1965). Indeed some egg-white proteins (ovalbumin, conalbumin and lysozyme) have been found in the yolk from 14 to 15 days of incubation (Saito & Martin, 1966; Carinci, Wegelin & Manzoli-Guidotti, 1966). At these stages ovalbumin is present in the yolk in such a great quantity that it is difficult to detect qualitative and quantitative changes of other proteins without resorting to further fractionations. We have studied these changes by ultracentrifugal and electrophoretic analysis of the WSF, of its globulin components and of the egg-white proteins separated by the same procedures. MATERIAL AND METHODS We have used White Leghorn hen fertile eggs provided by the Corticella agricultural station (Bologna). Two independent sets of experiments were performed on non-incubated eggs and on eggs after 10, 15 and 21 days of incubation (38-39 °C). Yolk was obtained by puncturing the vitelline membrane or yolk sac and was then diluted with 0-16 M-NaCl (1:2, w/w). Care was taken to avoid contamination of the yolk with egg-white and, for the incubated eggs, with embryonic fluids from the developing embryo. 1 Authors' address: Institute of Histology and General Embryology, University of Bologna, Bologna, Italy. 96 P. CARINCI & L. MANZOLI-GUIDOTTI The WSF was prepared according to Martin et al. as described previously (Carinci et al. 1966) and then was exhaustively dialysed at 4 °C against 0-16 MNaCl. Albumen was removed in toto, diluted with 0-16M-NaCl (1:2, w/w), and carefully homogenized with a Waring Blender. To separate globulin, aliquots of WSF and albumen were mixed with a saturated solution of ammonium sulphate to 45-50 % of saturation. The procedure was repeated four times. The final precipitate was dissolved in M-NaCl (these fractions will be referred as WSFp and Ap) and dialysed exhaustively against M-NaCl at 4 °C for ultracentrifugal and electrophoretic investigation. In this way we obtained a material from non-incubated egg albumen free from albumin contaminations (electrophoretic analysis) and with sedimentation properties comparable to those described by Kaminski (1954). The yolk and albumen fractions (WSF, WSF P and Ap) obtained at all stages of incubation were examined, in M-NaCl (pH 6-5) and at 1 % protein concentration (micro-Kjeldahl), in a Phywe model U 50 L analytical ultracentrifuge at 20 °C and 167 241 g. Calculated sedimentation coefficients (in Svedbergs, S), referred to 1 % protein concentration, were corrected for temperature, viscosity and partial specific volume. Corrections were not made for Johnston-Ogston effect or differences in the refractive index increments. The relative peak areas were estimated from enlarged tracings (x 15). Electrophoretic analysis was carried out using cellulose polyacetate (Gelmann Sepraphore III, 1 x 6 | in. strips) in tris barbital-sodium barbital buffer, pH 8-8, ionic strength [i = 0-05, 300 V. The electrophoregrams were stained with Amido Schwartz (1 % in methanol:acetic acid: water, 45:45:10, v/v) and then scanned, in some cases, with a Chromoscan densitometer (Joyce-Loebl). RESULTS Water-soluble fraction The ultracentrifugal examination of WSF of non-incubated egg (WSF0) and at 10 days of incubation (WSF10) yields two peaks after 35 min centrifugation (Plate 1, fig. a). The sedimentation coefficients permitted us to identify the faster peak as y-livetin and the slower one as a-+/Mivetin (Saito et al. 1965). The electrophoretic patterns of WSF0 and WSF10 show the three well-known zones produced by a-,/?-,y-livetin; in addition we have noticed a fourth zone at low concentration and with a mobility similar to that of ovalbumin, by comparison with egg-white (Plate 2, fig. c). The ultracentrifugal examination of WSF15 also shows two peaks of the same sedimentation coefficient as those of WSF0 and WSF10, but with much less of the fast component (see Table 1). The electrophoretic pattern of WSF15 is more complex. There are 7-8 zones PLATE 1 /. Embryol. exp. Morph., Vol. 19, Part 1 \ J A v K Sedimentation patterns of water-soluble fraction (a, b, c), water-soluble fraction precipitate (e, /»g), albumen precipitate (d), and water-soluble fraction + albumen precipitate (h). See text for experimental details. P. CARINCI & L. MANZOLI-GUIDOTTJ facing p. 96 J. Embryo/, exp. Morph., Vol. 19, Part 1 PLATE 2 Electrophoretic patterns of water-soluble fraction (c, e), water-soluble fraction precipitate (d, f), egg-white (a), egg-white precipitate (b), and water-soluble fraction + albumen (g). See text for experimental details. P. CARINCI & L. MANZOLI-GUIDOTTI Analysis of embryonic yolk 97 with anodic mobihty and one irregular zone with cathodic mobility. This pattern is qualitatively similar to that of total egg-white (Plate 2, fig. a). Among the anodic zones, a-livetin and ovalbumin, which is divided into three components (Fevold, 1951), can be definitely identified. Comparison of the electrophoretic pattern of the WSF15 with that of egg-white shows that the other anodic zones have very similar mobilities to those of ovomucoid, G3, G2 and conalbumin, while comparison with that of WSF0 shows that the 5th band has a mobility very similar to that of y#-livetin and the 7th band to that of y-livetin. Table 1. Rate coefficients and relative proportions of protein components Incubation (days) WSF (a) S % (b) S % WSF P (fl) S % s % (c) 0 10 15 21 2-6 79 2-8 80 2-7 95 2-6 100 6-9 21 6-9 20 6-9 5 — — 2-4 21 30 26 2-7 34 2-9 30 7-6 79 — — 7-4 74 — — 71 57 16-2 9 7-5 59 15-4 11 31 95 16-3 5 30 87 15-7 13 3-3 87 16-2 13 — — — A S % (6) % S = Svedbergs at 20 °C and 1 % protein concentration; proportion from uncorrected areas of Schlieren peaks, (a), (b), (c) iindicate individual peaks of the various fractions examined Analytical ultracentrifugation of WSF21 (1 % protein concentration) shows a single peak, even after 90min centrifugation (Plate 1, fig. e). A second fast sedimenting component can be detected in low amounts at 2-5% protein concentration. The electrophoretic pattern of WSF21 is, however, quite similar to that at 15 days. Water-soluble fraction precipitate The WSFp0 and WSF pl0 show two peaks after 30 min centrifugation (Plate 1, fig. e). Their sedimentation coefficients correspond to those of fast and slow 7 JEEM 19 98 P. CARINCI & L. MANZOLI-GUIDOTTI sedimenting components of the total WSF, but the slow component is in a lower proportion (see Table 1). Electrophoretically there is evidence of /?- and y-livetin, the latter divided into two bands (Mok & Common, 1964), in the following proportions: /?livetin 15%, y-livetin 31 %, y-livetin 50%. a-livetin was not demonstrable. We have therefore in WSF P a loss of a-livetin and a strong reduction in the concentration of /?-livetin. This is due to the solubility properties of these proteins at the (NH^SC^ concentration employed by us (Gorini & Lanzavecchia, 1955; Williams, 1962). This explains the diminution of the relative proportion of the slow component in the ultracentrifugal pattern. WSFp at 15 and 21 days yields the same ultracentrifugal pattern. On reaching speed, two peaks appear; the slower peak divides into two peaks after 15 min centrifugation (Plate 1, figs. / , g). Rate coefficients and relative proproportion of the three components are given in Table 1. Electrophoretic examination of both WSF pl5 and WSF p21 shows two anodic migration bands and, inconsistently, one cathodic migration band (Plate 2,fig./ ) . The cathodic migration component has a mobility similar to that of lysozyme. One anodic migration component moves as y-livetin and the other moves a bit faster; a- and /?-livetin are not present. Albumen precipitate The albumen precipitate in non-incubated eggs and at 10 and 15 days incubation exhibits two peaks by ultracentrifugal analysis (Plate 1, fig. d). The sedimentation rate and relative proportions are given in Table 1. Electrophoretic analysis demonstrates two zones of moderate anodic migration, which presumably correspond to the G 3 and G4 fractions of albumen. A cathodic migration zone is not consistently found (Plate 2, fig. b). Water-soluble fraction + total albumen WSF0 was combined with non-incubated egg total albumen to give an ovalbumin final concentration of 40 % of total proteins (Carinci et al. 1966). The electrophoretic pattern of this combination (WSF0 + Ao) is quite similar to that of WSF15 and WSF21 (7-8 zones of anodic migration) Plate 2, fig. g). With analytical ultracentrifugation only one peak (S = 2-9) is observed, as for WSF21. The precipitate obtained from this mixture (WSFQ + A,,) using (NH4)2SO4 dissolved in M-NaCl, shows two peaks after 14 min centrifugation. The slower peak later splits into two peaks (Plate 1, fig. h). Sedimentation coefficients of these components are respectively S = 2-2, 4 8 % ; S = 7-3, 4 4 % ; S = 15-1, 8%. These sedimentation coefficients are in close agreement with those of WSFp at 15 and 21 days incubation. Electrophoresis gives three anodic migration bands on staining with Amido Schwartz. Analysis of embryonic yolk 99 DISCUSSION In the first incubation period the qualitative and relative quantitative protein composition of chick embryo yolk WSF remains unchanged. Indeed, the electrophoretic and ultracentrifugal patterns of WSF10 are very similar to those of WSF0 with regard to both total WSF and the globulin fractions. These are in agreement with previous observations (Biagi, 1964; Saito & Martin, 1966; Carinci et ah 1966). However, the electropherogram presence of a component of WSF0 migrating as ovalbumin, heretofore unreported, is to be noted. From 15 to 21 days incubation the yolk protein composition undergoes marked changes. By electrophoresis on cellulose acetate WSF shows 4-5 new zones with anodic mobility .The second, third and fourth zones are due to the presence of ovalbumin readsorbed from albumen. The examination of the precipitate obtained by (NHJaSC^ treatment (globulins) further clarifies the WSF composition during the second period of incubation. These components thus separated have different ultracentrifugal properties from those of WSF0 globulins. A new component is present (S = 15-16), not previously shown, with a sedimentation coefficient value in the range of albumen macroglobulin. It is very likely that the macroglobulin is reabsorbed from eggwhite into the yolk but it cannot be excluded that this may be an aggregate of yolk protein. Another component is y-livetin (5 = 7). Finally, the slow sedimentation component (S = 2-3) in the last period of incubation does not correspond to /?-livetin, which it did in the early phase of incubation as demonstrated by electrophoresis. In summary, the changes in the yolk WSF observed in the final phase of incubation are due partly to the selective utilization of yolk proteins (disappearance of /#-livetin) and partly to the appearance of new proteins absorbed from eggwhite. Almost all egg-white albumins and globulins are found in the yolk. These represent the major proportion of yolk protein in the last phase of incubation as demonstrated by the relative quantitative values. It is therefore probable that a large part of the reserve albumen protein is reabsorbed by the chick embryo after passage into the yolk. Since yolk is rich in soluble enzymes (see Bellairs, 1964), it is possible that the reserve proteins are broken down into simple units, especially amino acids. Indeed, the active absorption of amino acids by yolk-sac entodermic cells is well established (Holdsworth & Hastings Wilson, 1967). Since small quantities of albumen proteins are found in the circulating fluids of the developing chick embryo, it is possible that their use occurs in distinct ways in response to embryonic requirements. 7-2 100 P. CARINCI & L. MANZOLI-GUIDOTTI SUMMARY 1. Ultracentrifugal and electrophoretic analysis of chick embryo yolk watersoluble fraction (WSF) has been carried out using fertile non-incubated eggs and at 10, 15 and 21 days incubation. 2. Ultracentrifugal and electrophoretic analysis of WSF globulins has been carried out at the same incubation stages. 3. The ultracentrifugal and electrophoretic patterns of both the WSF and its globulin components at 10 days incubation are very similar to those observed in non-incubated eggs. 4. Ultracentrifugal and electrophoretic patterns at 15 and 21 days incubation undergo marked changes due to the appearance of egg-white proteins in the yolk. 5. The significance of these findings in relation to embryogenesis is discussed. RIASSUNTO Ricerche sul vitello delVembrione di polio: analisi alVultracentrifuga ed alVelettroforesi della frazione idrosolubile 1. E' stata studiata, mediante analisi all'ultracentrifuga analitica ed all'elettroforesi, la composizione della frazione idrosolubile del vitello dell'uovo di polio non incubato ed al 10°, 15° e 21° giorno di incubazione. 2. E' stata esaminata inoltre, con le medesime techniche, la composizione delle globuline della frazione idrosolubile per i medesimi stadi di incubazione. 3. II quadro sedografico ed elettroforetico della frazione idrosolubile in toto e delle sue componenti globuliniche in 10° giorno di incubazione e sovrapponibile a quello osservato nell'uovo non incubato. 4. In 15° e 21° giorno il quadro sedografico ed elettroforetico e profondamente modificato in parte per la comparsa di proteine provenienti dall'albume. 5. II significato di tali dati ai fini delPembriogenesi viene discusso. REFERENCES R. (1964). Biological aspects of the yolk of the hen's egg. Adv. Morph. 4, 217. (1964). The proteins of the high density fraction of yolk during embryogenesis. Biochim. biophys. 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Changes in the protein content of yolk during chick embryogenesis. J. Embryol. expl. Morph. 8, 47. MOK, C. C. & COMMON, R. H. (1964). Studies on the livetins of hen's egg yolk. I. Identification of paper electrophoretic and immunoelectrophoretic livetin fractions with serum protein antigens by immunoelectrophoretic analysis. Can. J. Biochem. 42, 871. SAITO, Z. & MARTIN, W. G. (1966). Ovalbumin and other water soluble proteins in avian yolk during embryogenesis. Can. J. Biochem. 44, 1966. SAITO, Z., MARTIN, W. G. & COOK, W. H. (1965). Changes in the major macromolecular fractions of egg yolk during embryogenesis. Can. J. Biochem. 43, 1755. WILLIAMS, J. (1962). Serum proteins and the livetins of hen's egg yolk. Biochim. J. 83, 346. MCINDOE, (Manuscript received 3 August 1967)