DISTRIBUTION OF EGG WHITE PROTEINS IN CHICKEN BLOOD SERUM AND EGG YOLK*

Transcription

1 DSTRBUTON OF EGG WHTE PROTENS N CHCKEN BLOOD SERUM AND EGG YOLK* BY MARGARET E. MARSHALL AND H. F. DEUTSCH (Prom the Departments of PhzJsiological Chemistry and Physical Chemistry, University of Wisconsin, Madison, Wisconsin) (Received for publication, May 29, 1950) The four proteins, ovalbumin, conalbumin, lysozyme, and ovomucoid, which comprise the major portion of the proteins of chicken egg white have now been isolated. Quantitative immunochemical investigations of these proteins (l-2) have led to the development of reagents and methods suitable for the study of their distribution in the tissues of the chicken. Such a study has been carried out with egg yolk and embryo and adult chicken serum. n addition conalbumin was isolated from adult chicken serum, and its properties were compared with those of the analogous protein of egg white. Materials and Methods Conalbumin from egg white was prepared by the procedure of Bain and Deutsch (3) and repeatedly reprecipitated at ph 7.4, P 0.005, and 25 per cent. ethanol to remove impurities which were detectable immunologically in the first preparation studied (1). Rabbit antisera to this protein, ovalbumin crystallized six times, lysozyme crystallized seven times, and ovomucoid reprecipitated four times (4) were used. The quantit,ative precipitin reactions were carried out in the manner described by Cohn et al. (1). The supernatants to the specific precipitates were divided in half. One portion was tested for antibody excess by the addition of 3 to 6 y of antigen N and the other for antigen excess by the addition of 0.1 ml. of antiserum. The total nitrogen content of the antigens was determined on the precipitates obtained with 5 per cent trichloroacetic acid. Sedimentation analyses were carried out on 0.6 per cent protein solutions in 0.15 M NaCl in the Svedberg oil turbine ultracentrifuge at 185,- 000 x g. The diffusion constant of the serum conalbumin was determined in 0.1 M NaCl at 1 in an electrophoresis apparatus provided with a cylindrical lens, schlieren optical system. The rectangular slit recommended by Ander- *This work was supported in part by a grant from the Wisconsin Alumni Re search Foundation. 1 The sedimentation experiments were carried out by Mr. E. 11. Hanson.

2 2 EGG WTE PROTENS son and Alberty (5) was used. The initial boundary was sharpened by the technique of Kahn and Poison (6). Chicken blood was obtained at a local abattoir. Embryo serum was obtained in a manner previously described (7). A fraction of the yolk proteins suitable for immunological study was isolated as follows: Yolks, carefully separated from the white and washed with saline, were emulsified in twice their volume of water and the insoluble material was removed by centrifugation. The supernatant solution was adjusted to ph 7.0 to 7.2 with 0.5 M NaHC03, cooled too, and cold 50 per cent et,hanol was added to a final concentration of 10 per cent (final temperature -3 ). The yellow precipitate which formed was removed by centrifugation at 0 and discarded. The proteins in the supernatant were recovered by precipitation at ph 5.0 in 25 per cent ethanol at a temperature of - 6. The precipitate was suspended directly in the desired solvent in the cold and the ethanol removed by dialysis. This fraction represents approximately 15 per cent of the total yolk protein. The concentration of iron and the iron-binding capacity of chicken serum were determined by the method of Laurel1 (8). Measurements of the extinctions of the iron-phenanthroline complex were made at 510 rnp in a Coleman spectrophotometer, model 11. The iron-binding capacity was also determined by the procedure of Schade and Caroline (Q), adapted for this purpose by Cartwright and Wintrobe (10). n this case, the light absorption of the iron-protein complex was measured in the Beckman spectrophotometer at 490 mp. These two methods give essentially the same average values for the iron-binding capacity of human serum. Results Standardized rabbit antisera to ovalbumin, conalbumin, ovomucoid, and lysosyme were treated with varying quantities of adult chicken serum, chick embryo serum, and the yolk protein fraction. No lysozyme or OVOmucoid could be detected in these systems. Relatively large amounts of conalbumin were present; ovalbumin appeared only in traces. The results of the reaction of a rabbit antiovalbumin y-globulin preparation with ovalbumin, serum from 13 day-old chick embryos, and the yolk fraction are plotted in Fig. 1. n the case of the embryo blood serum and the yolk fraction, the values of the abscissa have been multiplied by and , respectively. The points for the reaction of these antigens, when plotted in this manner, coincide with the ovalbumin curve through the equivalence zone (10.2 to 12.7 y of ovalbumin N) and allow one to calculate the percentage of ovalbumin in the system. Because the amount of ovalbumin in these sources is so small, it was not possible to study the precipitin reaction in the region of a large excess of antigen. The

3 M. E. MARSHALL AND H. F. DEUTSCH 3 amounts of ovalbumin in embryo blood serum, the yolk fraction, and in the serum of male chickens and laying and non-laying female chickens as determined by this immunological method are shown in Table. The results of the reaction of rabbit anticonalbumin serum with conalbumin, serum from 13 day-old chick embryos, and the yolk fraction are plotted in Fig. 2. The amounts of protein nitrogen from the latter two systems added to 0.5 ml. of antiserum have been multiplied by and 0.040, respectively. At the equivalence zone and in the region of. antigen excess, the total amount of precipitate obtained varies considerably, depending upon the source of antigen. This result may be due to the presence of impurities in the conalbumin used for immunization, FG. 1 FG. 2 FG. 1. Quantitative precipitin reactions of rabbit antiovalbumin serum with ovalbumin (O), 13 day-old embryo serum (A), and yolk fraction (X). FG. 2. Quantitative precipitin reactions of rabbit anticonalbumin serum with conalbumin (O), 13 day-old embryo serum (A), and yolk fraction (X). The concentration of conalbumin, determined immunologically in the antibody excess zone, is shown in Table. n order to obtain additional evidence for the similarity of the components assayed immunologically with the corresponding egg white proteins, samples of protein were pipetted from both limbs of the electrophoresis cell after the system in question had been sufficiently resolved. The electrophoresis patterns of adult serum and of the yolk protein fraction are shown in Fig. 3. From these data, it can be seen that the fractions which gave positive reactions with the ovalbumin and conalbumin antisera have approximately the same mobilities as these egg white proteins. The same is true for the blood serum of 13 day-old embryos for which electrophoretic patterns have been presented previously (7). Hektoen and Cole (11) had concluded that, at least immunologically, conalbumin

4 4 EGG WHTE PROTENS and chicken serum albumin are identical. Their serum albumin preparation must not have been pure, since the serum albumin isolated elect.rophoretically in this experiment did not react with anticonalbumin serum. Although the r-globulin fraction separated electrophoretically from chicken serum reacted with anticonalbumin serum, the y-globulin prepared by TABLE Quantitative mmunochemical Determinations of Chicken Ovalbum.in and Conalbumin in Various Systems System assayed Ovalbumin Conalbumin 13 day embryo serum.... Serum of laying hen.... non-laying hen.... Male chicken serum.... Yolk fraction.... Chicken egg white FG. 3 FG. 4 wr cent total #rot& FG. 3. Electrophoretic patterns of (top section) adult chicken serum in Verona1 buffer, ph 8.6, JG 0.1, after 368 minutes at 3.3 volts cm.- ; (lower section) yolk fmction described in the text in Verona1 buffer, p 0.1, after 410 minutes at 3.3 volts cm.-. The levels from which the samples were successively removed are designated by arrows; the presence of conalbumin or ovalbumin is indicated by +CA or +EA. FG. 4. A, descending electrophoretic pattern of the male chicken r-globulin fraction in Verona1 buffer, ph 8.6, p 0.1, after 180 minutes at 6.5 volts cm.-. B, sedimentation diagram of the same protein fraction after 77 minutes at 185,000 x Nichol and Deutsch (12) from the serum of female chickens did not contain a component with the sedimentation constant of conalbumin (szou, = 5.4 S). When their fractionation scheme was repeated with serum from male chickens, however, a y-globulin preparation was obtained which on electrophoretic study gave evidence of two proteins and which on ultracentrifugal analysis showed an szow = 5.6 S component that constituted

5 M. E. MARSHALL AND H. F. DEUTSCH 5 50 per cent of the fraction. By immunological assay, this material contained 29 per cent conalbumin. The sedimentation and electrophoretic diagrams of this fraction are shown in Fig. 4. The smto = 8 S component of the ultracentrifuge pattern represents the chicken r-globulin. t was not possible to concentrate the serum conalbumin to more than about 30 per cent of the total protein by modification of the procedure used DAQRAM 1 Preparation of Serum Conalbumin 3 liters chicken serum; ph adjusted to 7.4 with 0.5 M HOAc; cooled to 0 ; 50% EtOH added to 30%; temperature --So; centrifuged Ppt. suspended in ice water and lyo- Supernatant discarded philized Powder dissolved in 560 ml. water; sufficient (NH&SO4 added to final concentration 1.6 M and final volume 2 liters; centrifuged Discarded ppt. Adjusted ph of supernatant to 5.0* with 0.15 M H&Oa; dialyzed in sufficient solid (NH&S04 to final concentration 2.4 M; centrifuged Discar ded ppt. Filtered supernant; adjusted ph to 6.8* with 2.5 M NH,OH; dialyzed in sufficient solid (NH,)&04 to final concentration 3.1 M; centrifuged /----- Suspended ppt. in water; dialyzed against running tap water overnight with toluene added, then against distilled water; lyophilized Discarded supernatant * Diluted to 0.3 M (NH)&O~ to measure the ph. by Nichol and Deutsch for the isolation of the chicken y-globulin. A combination of ethanol and ammonium sulfate fractionation gave a preparation containing 87 per cent conalbumin by immunological analysis, with a yield of 30 per cent. The scheme for the fractionation is shown in Diagram 1. The component in this preparation with the lowest mobility at ph 8.6 was separated electrophoretically in amounts sufficient for its characterization. t gave a single boundary in the ultracentrifuge with = 5.3 S and showed only one component on electrophoresis from ph s2aw

6 6 EGG WHTE PROTENS 5.1 to 6.3 and at ph 8.6. The isoelectric point of this protein, determined on two different preparations, in a buffer (0.1 ionic strength) containing 0.02 M sodium cacodylate and 0.08 M NaCl is ph 5.7. Conalbumin under the same conditions has an isoelectric point at ph 6.1 (3). A single determination was made of the diffusion constant of the serum conalbumin. The values when calculated by the method of moments and the height-areamethod are 7.1 and 6.0 X lo- cm.2 sec.-, respectively. A higher value of D, than of DA is an indication of molecular heterogeneity. t is possible, therefore, that the true value for the diffusion constant of this protein may be as low as 5.66 X 10-7, the diffusion constant reported for egg white conalbumin (3). This question cannot be decided until a more homogeneous preparation is available. From the sedimentation FG. 5. Quantitative precipitin reactions of rabbit anticonalbumin serum with egg white conalbumin (0) and serum conalbumin (0). constant, the diffusion constants, and an assumed part.ial specific volume2 of a molecular weight between 72,000 and 85,000 can be calculated. The molecular weight of the conalbumin from egg white, determined in a similar manner3 is 90,000. The significance of the difference between the molecular weights of the two proteins cannot be evaluated until the diffusion constant of the serum preparation is known more accurately. The differences in the physical properties might be due to modifications of the proteins resulting from the methods employed in their preparation. This electrophoretically isolated serum protein was allowed to react with the rabbit antiserum to egg white conalbumin. The curves plotted 2 The partial specific volume was assumed to be 0.743, since this gives a molecular weight for egg white conalbumin of 90,000, the value calculated from the ironbinding capacity. 3 The sedimentation constant of the reprecipitated conalbumin was found to be spas = 5.3 S rather than 5.4 S (3).

7 M. E. MARSHSLL AND H. F. DEUTSCH 7 from the data for the reactions of the conalbumins from both sources are shown in Fig. 5. n both cases, there was an equivalence zone in the neighborhood of 34 y of antigen N added to 0.5 ml. of antiserum. The discrepancy between the curve for egg white conalbumin and that for the serum protein can be interpreted to mean either that the serum protein still has 5 per cent impurity or that there actually is some slight difference between the two proteins. A second preparation of the serum conalbumin, when allowed to react with the antiserum, gave p0int.s falling between the two curves of Fig. 5, while another preparation of egg white conalbumin, which was demonstrated immunologically to be less pure than the first, gave points which coincided exactly with the curve for the serum protein through the point of maximum precipitation. t is probable, t herefore, that the conalbumins from chicken serum and egg white are identical immunologically. Cross-reacting proteins, in general, give much lower amounts of specific precipitate than does the homologous antigen. Schade and Caroline (13) have shown that a factor in egg white, identified as conalbumin by Alderton et al. (14), is capable of binding iron to form a colored complex. The mechanism of this iron binding is apparently the same as that by which the metal-binding protein of human serum combines with iron (15,16). The latter protein has been shown to be probably exclusively responsible for the transport of iron in human serum (17). Since the concentration of the serum conalbumin in chicken serum is of the same order of magnitude as that of metal-binding P-globulin of human serum, it was of interest to investigate whether serum conalbumin functions in the transport of iron in chicken serum. The iron-binding capacities of the egg white and serum conalbumin were determined by the method of Laurel1 (8) with the protein dissolved in a buffer containing 0.05 M H,BOs and M NaHC03, made up to ph 8.1 with NaOH. Both proteins bound 7.6 y of Fe per mg. of N. This corresponds to 2 atoms of iron per molecule of protein on the basis of a molecular weight of 90,000 and a nitrogen conversion factor of 6.14 (3). The metal-binding protein of human serum also binds 2 atoms of iron per molecule of protein (18). The addition of conalbumin from either source to chicken serum increased the iron-binding capacity of the serum by the amount expected from the iron-binding capacity of the added protein (see Table ). The iron-binding capacity of chicken serum, determined by the method of Laurell, was then compared with that to be expected from the concentration of conalbumin determined immunologically. (The validity of the immunochemioal method for determining conalbumin has been established by the analysis of mixtures of a known amount of pure serum conalbumin and of the iron-conalbumin complex with serum. The conalbumin added can be recovered within the experimental error, which is

8 8 EGG WHTE PROTENS approximately 3 per cent.) t can be seen from Table that in every case the iron-binding capacity is greater than that calculated from the concentration of conalbumin. This is true even in the embryo serum in which the concentration of conalbumin relative to the other serum proteins is from 2 to 3 times greater than in adult chicken serum. TABLE Concentration of Serum ron and ron-binding Capacity of Chicken Serum Sample Serum iron Pool 1 (adult, female) 1 + egg white conalbumin (314 mg. pe 100 ml.) Chicken 1 + serum conalbumin (140 mg. pe 100 ml.) Pool 2 (mixed adult) 3 (laying hens) 4 (males) 5 (non-laying hens) 16 day embryo Embryo > 16 days Chicken egg white conalbumin (82.8 ml per 100 ml. serum) Chicken 3 injected with 250 y Fe 450 ron-binding capacity y per cent - y #e* cent $ % $ ron-binding capacity conalbumin present 0i y per cent t 600t t * Determined by the method of Laurel1 (8) (unless otherwise indicated). t Sum of the total iron-binding capacity of the serum plus that of the added conalbumin. $ Determined by the method of Cartwright and Wintrobe (O). The method of Cartwright and Wintrobe for measuring the iron-binding capacity of human serum is difficult to apply to chicken serum because the latter tends to form a precipitate with the addition of increasing amounts of iron. One can, however, estimate approximately the saturation level of the serum for iron. t can be seen from Table that this method gives lower values for the iron-binding capacity of chicken serum than does the method of Laurel& but that the total amount of iron bound

9 M. E. MARSHALL AND H. F. DETJTSCH 9 is still too great to be accounted for exclusively by the conalbumin present. f either egg white or serum conalbumin is added to the serum, the additional iron bound can be accounted for on the basis of 2 atoms bound per molecule of conalbumin added. Two adult male chickens were injected with 250 and 450 y of iron (FeS04 in a solution of ascorbic acid), respectively. n both cases, the serum was saturated by the method of Cartwright and Wintrobe and the total serum iron was greater than the amount which could be bound by the conalbumin present (see Table ). Even in viva, therefore, conalbumin cannot be exclusively responsible for the capacity of chicken serum to bind iron. SUMMARY The distribution of the egg white proteins, ovalbumin, conalbumin, lysosyme, and ovomucoid in egg yolk and in embryo and adult chicken serum was studied. Only ovalbumin and conalbumin were detectable immunologically. Their concentrations were determined quantitatively by the precipitin reaction. Conalbumin was isolated from adult chicken serum. The diffusion constant and isoelectric point of this preparation differed from those of the conalbumin from egg white, but its molecular weight was of similar magnitude. mmunochemically, it appeared to be identical with its egg white counterpart. The iron-binding capacities of both proteins are the same. Conalbumin is not present in sufficient amounts in chicken serum to account for the total iron-binding capacity of this system. BBLOGRAPHY 1. Cohn, M., Wetter, L. R., and Deutsch, H. F., J. mmunol., 61, 283 (1949). 2. Wetter, L. R., and Deutsch, H. F., Arch. Biochem., 28, 399 (1950). 3. Bain, J. A., and Deutsch, H. F., J. Biol. Chem., 172,547 (1948). 4. Fredericq, E., and Deutsch, H. F., J. Biol. Chem., 181, 499 (1949). 5. Anderson, E. A., and Alberty, R. A., J. Phys. und Colloid Chem., 62,1345 (1948). 6. Kahn, D., and Polson, A., J. Phys. and Colloid Chem., 61, 816 (1947). 7. Marshall, M. E., and Deutsch, H. F., J. Biol. Chem., 185, 155 (1950). 8. Laurell, C.-B., Acta physiol. &and., 14, suppl. 46 (1949). 9. Schade, A. L., and Caroline, L., Science, 104, 340 (1946). 10. Cartwright, G. E., and Wintrobe, M. M., J. Clin. nvest., 28, 86 (1949). 11. Hektoen, L., and Cole, A. G., J. nfect. Dis., 42, 1 (1928). 12. Nichol, J. C., and Deutsch, H. F., J. Am. Chem. Sot., 70, 80 (1948). 13. Schade, A.,., and Caroline,,., Science, 100, 14 (1944). 14. Alderton, G., Ward, W. H., and Fevold, H.,., Arch. Biochem., 11, 9 (1946). 15. Schade, A. L., Reinhart, R. W., and Levy, H., Arch. Biochem., 20, 170 (1949). 16. Fiala, S., and Burk, D., Arch. Biochem., 20, 172 (1949). 17. Rath, C. E., and Finch, C. A., J. Clin. nvest., 28, 79 (1949). 18. Surgenor, D. M., Koechlin, B. A., and Strong, L. E., J. Clin. nvest., 28, 73 (1949) *

10 DSTRBUTON OF EGG WHTE PROTENS N CHCKEN BLOOD SERUM AND EGG YOLK Margaret E. Marshall and H. F. Deutsch J. Biol. Chem. 1951, 189:1-9. Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at #ref-list-1