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Free Amino Acid Content of Infertile Chicken Eggs1
Free Amino Acid Content of Infertile Chicken Eggs EUFEMIO D. DUCAY, LEO KLINE AND STANLEY MANDELES
Western Regional Research Laboratory? Albany, Calif. (Received for publication September 28, 1959)
T
MATERIALS AND METHODS Identification of Amino Acids. Proteinfree extracts were made from white and yolk by the absorption dialysis method of Hunter et al. (1955). In this method a *A laboratory of the Western Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture.
slurry of ion-exchange resin such as Dowex-50 is separated from the biological material by a cellophane membrane so that only diffusable cations are adsorbed on the resin. The adsorbed substances can be eluted from the resin by suitable solvents. In our study, extracts prepared were next chromatographed on Dowex-50 columns according to the directions by Hirs, Moore and Stein (1954). The presence and identity of specific amino acids indicated by this initial separation were confirmed by paper chromatography (Hardy et al., 1955; McFarren, 1951), and specific color tests (Block, Durrum and Zweig, 1958) where applicable. Determination of Total Content of Amino Acids. The chloramine-T method of Cohen (1939) was used for estimation of total amino acid content in protein-free filtrates prepared by the method of Folin and Wu (1919). The absorption dialysis method was not used for this purpose because the Folin and Wu method was faster and easier to use. RESULTS Identification of Amino Acids. The results of analysis of a typical extraction of white and yolk from fresh eggs stored at 4°C. for one week are shown in Table 1. It may be seen that in addition to the amino acids cited by Lea and Rhodes (1953), serine, proline, isoleucine, methionine, histidine, lysine, and phenylalanine were found. We were unable to detect cysteine, cystine, or cysteic acid on our paper chromatograms. We have found that extracts prepared by our method do not respond to the nitroprusside test for cysteine
831
Downloaded from http://ps.oxfordjournals.org/ at NERL on April 13, 2015
HE free amino acids in infertile chicken eggs have received comparatively little attention since the observation by Pucher in 1927 that egg white contains 3.3 mg. amino acid N/100 g. and yolk contains 49 mg. amino acid N/100 g. His data also indicated that during storage of infertile eggs at incubator temperature, presumably 37°C, the free amino acid content of white increased two-fold. These observations were reported in a short note with no information on methods used. More recently, Lea and Rhodes (1953), in a study of impurities in yolk phospholipid preparations, identified 11 free amino acids in yolk. They used paper chromatography and made no attempt to identify all of the amino acids in yolk nor to determine total concentration. As part of an investigation of basic composition of chicken eggs, we have examined fresh infertile eggs for content and distribution of free amino acids, and have identified at least 16 which occur free in both white and yolk. In addition, we have observed a large difference in total concentration of free amino acids initially between yolk and white and increased concentration in white during storage of shell eggs at various temperatures.
832
E. D. DUCAY, L. KLINE AND S. MANDELES TABLE 1.—Free amino acids in egg
TABLE 2.—Initial free amino acid content of
yolk and white
infertile eggs
Amino acid
+ + + + + + + + + +
This report
+ + + + + + + + + + + + + + + + *
Additional color tests (7) Sakaguchi
Pauly
Isatin Periodate: Nessler Periodate: Nessler a-nitroso-£-naphthoI
Determination
Breed
1 2 3 4
Inbred Inbred Inbred Inbred
1 2
White Leghorn White Leghorn
p. moles li. moles amino amino acid C0 2 acid CO2 per ml. per ml. egg white egg yolk
Cross Cross Cross Cross
0.23 0.20 0.14 0.42
40 41 38 40
0.54 0.53
38
Ehrlich
+
* Tryptophane indicated by positive test for indole. ** Cysteine concentration <10 _ 6 M.
and cystine as given by Toennies and Kolb (1951) but are positive to the more sensitive NaN 3 test (Feigl, 1954) for sulfhydryl. Tryptophane could not be detected in extracts of white and yolk prepared by the absorption dialysis method, most probably because of the low pH of these extracts. A positive test was obtained for the presence of indole with Ehrlich's reagent (Horn and Jones, 1945) when a Zn(OH) 2 filtrate was made of white and yolk, indicating that free tryptophane may well be a constituent. Estimation of Total Free Amino Acids. In order to estimate the free amino acid content of white and yolk, very fresh infertile eggs were obtained from commercial sources at different times of the year and were separated within one day of being laid. Analyses for total free amino acid concentration in composite samples of whites and yolks, usually separated from 36 eggs, are shown in Table 2. The free amino acid concentration in the yolk ranged from 38 to 41 [/.M/ml. and was much lower in the white, varying from 0.14 ort 0.54 jiM/ml. As shown in Table 3, a gradual increase in concentration of free amino acids in the
whites occurred during storage for 2 months or more. In the experiment represented here, clean fresh infertile eggs were obtained on day of lay from a local ranch and divided into 2 groups. One group was partially oiled by spraying an emulsion directly on the eggs in the filler flats. The other group remained untreated. Half of the eggs in each group were held at 1°C. while the other half was kept at 15°C. It can be seen from the results in Table 3 that there is a tenfold increase in concentration of free amino acids in the white over a period of 80 days. The eggs used for these TABLE 3.—Rise in amino acid content of egg
white in infertile eggs Days of storage
Untreated eggs 0 9 20 36 77 83 Oiled eggs 0 9 20 35 77
Storage tempera- Storage temperature=l°C. ture = 15°C. li moles amino acid CO: per ml. white*
/i moles amino acid CO2 per ml. white*
0.24
0.24 0.41
0.85 1.0
1.3 1.9
2.3 0.24 0.65 0.81
0.24 0.33 0.86 2.3
*L100 ml. of separated white used per each analy-
Downloaded from http://ps.oxfordjournals.org/ at NERL on April 13, 2015
Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine Tryptophane Cysteine Citrulline
Lea & Rhodes
833
AMINO ACID CONTENT OF EGGS TABLE 4.—Change in amino acid concentration in white and yolk during storage Temperature = 25° in days 0 1 2 6 10 20
acid CO2 per ml. white*
acid CO2 per ml. yolk**
0.42
39.5 38.5 39.0 39.1 41.7 39.9
0.53 0.80 0.88 1.34 Temperature = 4° 0.42 0.54 0.65 0.69 0.78 0.94 1.23
39.5 39.2 38.7 38.3 39.5 40.6 39.4
* 100 ml. of separated white used for each analysis. ** 75 ml. of separated yolk used for each analysis.
studies were found to be microbiologically sterile throughout the whole study. The concentration of free amino acids in yolk apparently remains constant during storage, as shown in Table 4. In the experiment represented here, very fresh eggs were divided into 2 groups, one of which was held at 4°C. and the other at 25°C. No special oiling procedures were applied to the eggs. Although the yolk may be the source of free amino acid increase in white, the concentration in yolk is so high compared to concentration in white that the corresponding decrease in concentration in yolk cannot be detected within the experimental error of our procedure. If a diffusion mechanism is operative, the slow diffusion of amino acids into the white was expected on the basis of the work of Straub and Hoogerduyn (1929), and Needham (1931), who showed that the vitellin membrane was less permeable than ordinary dialysis membranes then in use, even though the isolated vitellin mem-
DISCUSSION Our results show that, within experimental error, the free amino acid content of the entire egg tends to remain constant under the condition of storage at 4° and 2S°C. This confirms previous reports (Balls and Hoover, 1940; Lineweaver et al., 1948, 1949) that very little, if any, proteolytic activity is present in infertile eggs and that the slow and progressive rise in concentration of free amino acids in egg white probably represents diffusion of amino acids from the yolk across the vitellin membrane in a manner similar to the diffusion of glucose and inorganic salts from yolk to white (Pucher, 1927; Orru, 1933). While it is likely that the slow rate of diffusion of free amino acids from yolk into white is due in part to the high viscosity of both phases, our observation that equilibration is greatly accelerated when white and yolk are separated by a cellophane membrane indicates that permeability of the vitellin membrane plays a major role in determinTABLE 5.—Equilibration of amino acids through cellophane membrane
y. moles amino acid CO2 per ml.
Total 11 moles amino acid CO2 in white and yolk of 5 eggs
in in white yolk 0.98 40.4
4,200
13.0
4,100
Before equilibration After equilibration 24 hours 15.6
Downloaded from http://ps.oxfordjournals.org/ at NERL on April 13, 2015
0 1 2 6 10 20 30
brane was more permeable than the intact system. Results shown in Table 5 demonstrate that comparatively rapid equilibration of the amino acids occurs between yolk and white separated by cellophane. Hence, the slow diffusion observed in the egg is due to the vitellin membrane and surrounding structure rather than to some binding of amino acids in the yolk.
834
E. D. DUCAY, L. KLINE AND S. MANDELES
white constituents in a masked form. The latter possibility is now being examined in our laboratory. SUMMARY At least 16 free amino acids have been found in both white and yolk of infertile eggs from hens. These amino acids include aspartic acid, serine, threonine, glutamic acid, glycine, alanine, proline, valine, methionine, leucine, isoleucine, tyrosine, histidine, lysine, arginine, and phenylalanine. A positive test for indole indicates the presence of tryptophane. Free cysteine, detected by nitroprusside test, is virtually absent from egg white and yolk. The total concentration of free amino acids in the yolk is approximately 40 [unoles/ml. In white, the initial concentration from 0.14 to 0.54 u.moles/ml. and increases to as much as 2.3 j^moles under various conditions of storage. This rise is apparently due to the slow diffusion of amino acids from yolk to white. A much more rapid equilibration occurs when the yolk is separated from the white by a cellophane membrane. REFERENCES Balls, A. K., and S. R. Hoover, 1940. Behavior of ovomucin in the liquefaction of egg white. Ind. Eng. Chem. 32: 594-596. Block, R. J., E. L. Durrum and G. Zweig, 1958. A Manual of Paper Chromatography and Paper Electrophoresis, pp. 128-139. Academic Press, Inc., New York. Cohen, P. P., 1939. Microdetermination of glutamic acid. Biochem. J. 33: 551-558. Fiegel, F., 1954. Spot Tests, Organic Applications, 4th Ed., Volume II, p. 164. Elsevier Publishing Co., New York. Folin, O., and H. Wu, 1919. A system of blood analysis. J. Biol. Chem. 38: 81-110. Hardy, T. L., D. O. Holland and H. H. C. Naylor, 1955. One-phase solvent mixtures for the separation of amino acids. Anal. Chem. 27: 971974. Hirs, C. H., S. Moore and W. H. Stein, 1954.
Downloaded from http://ps.oxfordjournals.org/ at NERL on April 13, 2015
ing rate of diffusion from yolk to white. The exact mechanism by which the vitellin membrane impedes rapid equilibration of amino acids between yolk and white remains obscure. The presence of 40 [/.moles/ml. total free amino acid found in infertile egg yolk is not surprising in view of the fact that the immediate source of the yolk material is the hen's blood (Romanoff and Romanoff, 1949). The blood contains free amino acids which presumably reflect the amino acid composition of the average body protein both with respect to concentration and identity. The initial low level of free amino acids in white may be explained on the basis of diffusion from the yolk while the egg was in the oviduct. Another possibility is that the amino acids found initially in white were originally present as constituents in the white secreted from the anterior oviduct cells. This would imply that amino acids (supplied by the blood to the oviduct cells) which had escaped incorporation into egg white protein, or other metabolic use within the oviduct cells, had been carried along, in diminished concentration, in the secreted egg white. Our results do not permit a conclusion to be drawn in favor of one or the other of these alternatives. The reaction of the egg yolk and egg white filtrates to the NaN 3 and nitroprusside tests indicate that if free cysteine is present, its concentration in intact fresh yolk and white is less than 2 X 10"5 M, the lower limit for the nitroprusside test for sulfhydryl. When this concentration range is compared with a value of 4 X 10~2M, which is the concentration of total amino acids in the yolk, it can be seen that the relatively low concentration of cysteine probably does not correspond to its occurrence in the average hen's egg or body protein. This virtual absence of free cysteine from egg yolk and white suggests that, if present at all, it may be bound to yolk and
AMINO ACID CONTENT OF EGGS
McFarren, E. F., 1951. Buffered filter paper chromatography of the amino acids. Anal. Chem. 23: 168-174. Needham, J,, 1931. The osmotic properties of the isolated vitelline membrane. J. Exptl. Biol. 8: 330-334. Orru, A., 1933. On the permeability of the vitelline membrane of chicken eggs. Sulla permeabilita della membrana vitellina dell'uovo di gallina. Boll. soc. ital. bid. sper. 8: 688-669. Pucher, G. W., 1927. Chemical analysis of incubated non-fertile eggs. Proc. Soc. Exptl. Biol. Med. 25: 72-73. Romanoff, A. L, and A. J. Romanoff, 1949. The Avian Egg, p. 209, John Wiley and Sons, Inc., New York. Straub, J., and M. J. Hoogerduyn, 1929. The difference in osmotic concentrations between egg yolk and white of egg. Rec. trav. chim. 48: 49-82. Toennies, G., and J. J. Kolb, 1951. Techniques and reagents for paper chromatography. Anal. Chem. 23: 823^826.
The Availability to the Chick of Zinc as the Sulfate, Oxide or Carbonate1'2 ROBERT H. ROBERSON3-4 AND PHILIP J. SCHAIBLE Department of Poultry Science, Michigan State University, East Lansing, Michigan (Received for publication September 28, 1959)
R
OBERSON and Schaible (1958a) re• ported that both the chloride and sulfate of zinc at a hundred ppm. in a glucosesoybean protein diet prevented zinc deficiency symptoms and permitted normal growth and feed conversion in the growing chick. Pensack et al. (1958) stated that the carbonate, oxide, chloride or proteinate of zinc were equally available to the chick 1 Journal article 2504, Michigan Agricultural Experiment Station. 2 Supported in part by a grant from Hardy Salt Co., St. Louis, Mo. "Presented by the senior author in partial fulfillment of the Ph.D. degree. 4 Present address: Department of Poultry Husbandry, New Mexico State University, University Park, New Mexico.
when added to a cerelose-casein-gelatin diet with variable calcium:phosphorus ratios. The purpose of this study was to determine the relative availabilities of the carbonate, oxide or sulfate of zinc when added at minimal levels to a purified, soybeantype ration of otherwise normal mineral content. From previous work it was assumed that twenty ppm. of zinc was adequate for the chick when the zinc was available (Roberson and Schaible, 1958a; O'Dell et ah, 1958; and Moeller and Scott, 1958). EXPERIMENTAL
Male White Rock chicks, one-day of age, were allotted on the basis of weight into
Downloaded from http://ps.oxfordjournals.org/ at NERL on April 13, 2015
The chromatography of amino acids on ion exchange resins. Use of volatile acids for elution. J. Am. Chem. Soc. 76: 6063-6065. Horn, M. J., and D. B. Jones, 194S. A rapid colorimetric method for the determination of tryptophane in proteins and foods. J. Biol. Chem. 157: 153-160. Hunter, I. R., D. F. Houston and E. B. Kester, 195S. Adsorption dialysis, an extraction technique used in recovery of amino acids. Anal. Chem. 27: 965-968. Lea, C. H., and D. N. Rhodes, 1953. Partition chromatography of egg-yolk phospholipids on cellulose. Biochem. J. 54: 467-469. Lineweaver, H., H. J. Morris, L. Kline and R. S. Bean, 1948. Enzymes of fresh hen eggs. Arch. Biochem. 16: 443-472. Lineweaver, H., H. Franekel-Conrat and R. S. Bean, 1949. Determination of trypsin in the presence of egg white trypsin inhibitor and demonstration of absence of trypsin from egg white. J. Biol. Chem. 177: 205-207.
835
Western Regional Research Laboratory? Albany, Calif. (Received for publication September 28, 1959)
T
MATERIALS AND METHODS Identification of Amino Acids. Proteinfree extracts were made from white and yolk by the absorption dialysis method of Hunter et al. (1955). In this method a *A laboratory of the Western Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture.
slurry of ion-exchange resin such as Dowex-50 is separated from the biological material by a cellophane membrane so that only diffusable cations are adsorbed on the resin. The adsorbed substances can be eluted from the resin by suitable solvents. In our study, extracts prepared were next chromatographed on Dowex-50 columns according to the directions by Hirs, Moore and Stein (1954). The presence and identity of specific amino acids indicated by this initial separation were confirmed by paper chromatography (Hardy et al., 1955; McFarren, 1951), and specific color tests (Block, Durrum and Zweig, 1958) where applicable. Determination of Total Content of Amino Acids. The chloramine-T method of Cohen (1939) was used for estimation of total amino acid content in protein-free filtrates prepared by the method of Folin and Wu (1919). The absorption dialysis method was not used for this purpose because the Folin and Wu method was faster and easier to use. RESULTS Identification of Amino Acids. The results of analysis of a typical extraction of white and yolk from fresh eggs stored at 4°C. for one week are shown in Table 1. It may be seen that in addition to the amino acids cited by Lea and Rhodes (1953), serine, proline, isoleucine, methionine, histidine, lysine, and phenylalanine were found. We were unable to detect cysteine, cystine, or cysteic acid on our paper chromatograms. We have found that extracts prepared by our method do not respond to the nitroprusside test for cysteine
831
Downloaded from http://ps.oxfordjournals.org/ at NERL on April 13, 2015
HE free amino acids in infertile chicken eggs have received comparatively little attention since the observation by Pucher in 1927 that egg white contains 3.3 mg. amino acid N/100 g. and yolk contains 49 mg. amino acid N/100 g. His data also indicated that during storage of infertile eggs at incubator temperature, presumably 37°C, the free amino acid content of white increased two-fold. These observations were reported in a short note with no information on methods used. More recently, Lea and Rhodes (1953), in a study of impurities in yolk phospholipid preparations, identified 11 free amino acids in yolk. They used paper chromatography and made no attempt to identify all of the amino acids in yolk nor to determine total concentration. As part of an investigation of basic composition of chicken eggs, we have examined fresh infertile eggs for content and distribution of free amino acids, and have identified at least 16 which occur free in both white and yolk. In addition, we have observed a large difference in total concentration of free amino acids initially between yolk and white and increased concentration in white during storage of shell eggs at various temperatures.
832
E. D. DUCAY, L. KLINE AND S. MANDELES TABLE 1.—Free amino acids in egg
TABLE 2.—Initial free amino acid content of
yolk and white
infertile eggs
Amino acid
+ + + + + + + + + +
This report
+ + + + + + + + + + + + + + + + *
Additional color tests (7) Sakaguchi
Pauly
Isatin Periodate: Nessler Periodate: Nessler a-nitroso-£-naphthoI
Determination
Breed
1 2 3 4
Inbred Inbred Inbred Inbred
1 2
White Leghorn White Leghorn
p. moles li. moles amino amino acid C0 2 acid CO2 per ml. per ml. egg white egg yolk
Cross Cross Cross Cross
0.23 0.20 0.14 0.42
40 41 38 40
0.54 0.53
38
Ehrlich
+
* Tryptophane indicated by positive test for indole. ** Cysteine concentration <10 _ 6 M.
and cystine as given by Toennies and Kolb (1951) but are positive to the more sensitive NaN 3 test (Feigl, 1954) for sulfhydryl. Tryptophane could not be detected in extracts of white and yolk prepared by the absorption dialysis method, most probably because of the low pH of these extracts. A positive test was obtained for the presence of indole with Ehrlich's reagent (Horn and Jones, 1945) when a Zn(OH) 2 filtrate was made of white and yolk, indicating that free tryptophane may well be a constituent. Estimation of Total Free Amino Acids. In order to estimate the free amino acid content of white and yolk, very fresh infertile eggs were obtained from commercial sources at different times of the year and were separated within one day of being laid. Analyses for total free amino acid concentration in composite samples of whites and yolks, usually separated from 36 eggs, are shown in Table 2. The free amino acid concentration in the yolk ranged from 38 to 41 [/.M/ml. and was much lower in the white, varying from 0.14 ort 0.54 jiM/ml. As shown in Table 3, a gradual increase in concentration of free amino acids in the
whites occurred during storage for 2 months or more. In the experiment represented here, clean fresh infertile eggs were obtained on day of lay from a local ranch and divided into 2 groups. One group was partially oiled by spraying an emulsion directly on the eggs in the filler flats. The other group remained untreated. Half of the eggs in each group were held at 1°C. while the other half was kept at 15°C. It can be seen from the results in Table 3 that there is a tenfold increase in concentration of free amino acids in the white over a period of 80 days. The eggs used for these TABLE 3.—Rise in amino acid content of egg
white in infertile eggs Days of storage
Untreated eggs 0 9 20 36 77 83 Oiled eggs 0 9 20 35 77
Storage tempera- Storage temperature=l°C. ture = 15°C. li moles amino acid CO: per ml. white*
/i moles amino acid CO2 per ml. white*
0.24
0.24 0.41
0.85 1.0
1.3 1.9
2.3 0.24 0.65 0.81
0.24 0.33 0.86 2.3
*L100 ml. of separated white used per each analy-
Downloaded from http://ps.oxfordjournals.org/ at NERL on April 13, 2015
Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine Tryptophane Cysteine Citrulline
Lea & Rhodes
833
AMINO ACID CONTENT OF EGGS TABLE 4.—Change in amino acid concentration in white and yolk during storage Temperature = 25° in days 0 1 2 6 10 20
acid CO2 per ml. white*
acid CO2 per ml. yolk**
0.42
39.5 38.5 39.0 39.1 41.7 39.9
0.53 0.80 0.88 1.34 Temperature = 4° 0.42 0.54 0.65 0.69 0.78 0.94 1.23
39.5 39.2 38.7 38.3 39.5 40.6 39.4
* 100 ml. of separated white used for each analysis. ** 75 ml. of separated yolk used for each analysis.
studies were found to be microbiologically sterile throughout the whole study. The concentration of free amino acids in yolk apparently remains constant during storage, as shown in Table 4. In the experiment represented here, very fresh eggs were divided into 2 groups, one of which was held at 4°C. and the other at 25°C. No special oiling procedures were applied to the eggs. Although the yolk may be the source of free amino acid increase in white, the concentration in yolk is so high compared to concentration in white that the corresponding decrease in concentration in yolk cannot be detected within the experimental error of our procedure. If a diffusion mechanism is operative, the slow diffusion of amino acids into the white was expected on the basis of the work of Straub and Hoogerduyn (1929), and Needham (1931), who showed that the vitellin membrane was less permeable than ordinary dialysis membranes then in use, even though the isolated vitellin mem-
DISCUSSION Our results show that, within experimental error, the free amino acid content of the entire egg tends to remain constant under the condition of storage at 4° and 2S°C. This confirms previous reports (Balls and Hoover, 1940; Lineweaver et al., 1948, 1949) that very little, if any, proteolytic activity is present in infertile eggs and that the slow and progressive rise in concentration of free amino acids in egg white probably represents diffusion of amino acids from the yolk across the vitellin membrane in a manner similar to the diffusion of glucose and inorganic salts from yolk to white (Pucher, 1927; Orru, 1933). While it is likely that the slow rate of diffusion of free amino acids from yolk into white is due in part to the high viscosity of both phases, our observation that equilibration is greatly accelerated when white and yolk are separated by a cellophane membrane indicates that permeability of the vitellin membrane plays a major role in determinTABLE 5.—Equilibration of amino acids through cellophane membrane
y. moles amino acid CO2 per ml.
Total 11 moles amino acid CO2 in white and yolk of 5 eggs
in in white yolk 0.98 40.4
4,200
13.0
4,100
Before equilibration After equilibration 24 hours 15.6
Downloaded from http://ps.oxfordjournals.org/ at NERL on April 13, 2015
0 1 2 6 10 20 30
brane was more permeable than the intact system. Results shown in Table 5 demonstrate that comparatively rapid equilibration of the amino acids occurs between yolk and white separated by cellophane. Hence, the slow diffusion observed in the egg is due to the vitellin membrane and surrounding structure rather than to some binding of amino acids in the yolk.
834
E. D. DUCAY, L. KLINE AND S. MANDELES
white constituents in a masked form. The latter possibility is now being examined in our laboratory. SUMMARY At least 16 free amino acids have been found in both white and yolk of infertile eggs from hens. These amino acids include aspartic acid, serine, threonine, glutamic acid, glycine, alanine, proline, valine, methionine, leucine, isoleucine, tyrosine, histidine, lysine, arginine, and phenylalanine. A positive test for indole indicates the presence of tryptophane. Free cysteine, detected by nitroprusside test, is virtually absent from egg white and yolk. The total concentration of free amino acids in the yolk is approximately 40 [unoles/ml. In white, the initial concentration from 0.14 to 0.54 u.moles/ml. and increases to as much as 2.3 j^moles under various conditions of storage. This rise is apparently due to the slow diffusion of amino acids from yolk to white. A much more rapid equilibration occurs when the yolk is separated from the white by a cellophane membrane. REFERENCES Balls, A. K., and S. R. Hoover, 1940. Behavior of ovomucin in the liquefaction of egg white. Ind. Eng. Chem. 32: 594-596. Block, R. J., E. L. Durrum and G. Zweig, 1958. A Manual of Paper Chromatography and Paper Electrophoresis, pp. 128-139. Academic Press, Inc., New York. Cohen, P. P., 1939. Microdetermination of glutamic acid. Biochem. J. 33: 551-558. Fiegel, F., 1954. Spot Tests, Organic Applications, 4th Ed., Volume II, p. 164. Elsevier Publishing Co., New York. Folin, O., and H. Wu, 1919. A system of blood analysis. J. Biol. Chem. 38: 81-110. Hardy, T. L., D. O. Holland and H. H. C. Naylor, 1955. One-phase solvent mixtures for the separation of amino acids. Anal. Chem. 27: 971974. Hirs, C. H., S. Moore and W. H. Stein, 1954.
Downloaded from http://ps.oxfordjournals.org/ at NERL on April 13, 2015
ing rate of diffusion from yolk to white. The exact mechanism by which the vitellin membrane impedes rapid equilibration of amino acids between yolk and white remains obscure. The presence of 40 [/.moles/ml. total free amino acid found in infertile egg yolk is not surprising in view of the fact that the immediate source of the yolk material is the hen's blood (Romanoff and Romanoff, 1949). The blood contains free amino acids which presumably reflect the amino acid composition of the average body protein both with respect to concentration and identity. The initial low level of free amino acids in white may be explained on the basis of diffusion from the yolk while the egg was in the oviduct. Another possibility is that the amino acids found initially in white were originally present as constituents in the white secreted from the anterior oviduct cells. This would imply that amino acids (supplied by the blood to the oviduct cells) which had escaped incorporation into egg white protein, or other metabolic use within the oviduct cells, had been carried along, in diminished concentration, in the secreted egg white. Our results do not permit a conclusion to be drawn in favor of one or the other of these alternatives. The reaction of the egg yolk and egg white filtrates to the NaN 3 and nitroprusside tests indicate that if free cysteine is present, its concentration in intact fresh yolk and white is less than 2 X 10"5 M, the lower limit for the nitroprusside test for sulfhydryl. When this concentration range is compared with a value of 4 X 10~2M, which is the concentration of total amino acids in the yolk, it can be seen that the relatively low concentration of cysteine probably does not correspond to its occurrence in the average hen's egg or body protein. This virtual absence of free cysteine from egg yolk and white suggests that, if present at all, it may be bound to yolk and
AMINO ACID CONTENT OF EGGS
McFarren, E. F., 1951. Buffered filter paper chromatography of the amino acids. Anal. Chem. 23: 168-174. Needham, J,, 1931. The osmotic properties of the isolated vitelline membrane. J. Exptl. Biol. 8: 330-334. Orru, A., 1933. On the permeability of the vitelline membrane of chicken eggs. Sulla permeabilita della membrana vitellina dell'uovo di gallina. Boll. soc. ital. bid. sper. 8: 688-669. Pucher, G. W., 1927. Chemical analysis of incubated non-fertile eggs. Proc. Soc. Exptl. Biol. Med. 25: 72-73. Romanoff, A. L, and A. J. Romanoff, 1949. The Avian Egg, p. 209, John Wiley and Sons, Inc., New York. Straub, J., and M. J. Hoogerduyn, 1929. The difference in osmotic concentrations between egg yolk and white of egg. Rec. trav. chim. 48: 49-82. Toennies, G., and J. J. Kolb, 1951. Techniques and reagents for paper chromatography. Anal. Chem. 23: 823^826.
The Availability to the Chick of Zinc as the Sulfate, Oxide or Carbonate1'2 ROBERT H. ROBERSON3-4 AND PHILIP J. SCHAIBLE Department of Poultry Science, Michigan State University, East Lansing, Michigan (Received for publication September 28, 1959)
R
OBERSON and Schaible (1958a) re• ported that both the chloride and sulfate of zinc at a hundred ppm. in a glucosesoybean protein diet prevented zinc deficiency symptoms and permitted normal growth and feed conversion in the growing chick. Pensack et al. (1958) stated that the carbonate, oxide, chloride or proteinate of zinc were equally available to the chick 1 Journal article 2504, Michigan Agricultural Experiment Station. 2 Supported in part by a grant from Hardy Salt Co., St. Louis, Mo. "Presented by the senior author in partial fulfillment of the Ph.D. degree. 4 Present address: Department of Poultry Husbandry, New Mexico State University, University Park, New Mexico.
when added to a cerelose-casein-gelatin diet with variable calcium:phosphorus ratios. The purpose of this study was to determine the relative availabilities of the carbonate, oxide or sulfate of zinc when added at minimal levels to a purified, soybeantype ration of otherwise normal mineral content. From previous work it was assumed that twenty ppm. of zinc was adequate for the chick when the zinc was available (Roberson and Schaible, 1958a; O'Dell et ah, 1958; and Moeller and Scott, 1958). EXPERIMENTAL
Male White Rock chicks, one-day of age, were allotted on the basis of weight into
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