I
002,~.7, x:7Y:n701-065YSO2 CrnN
THE PLASMA ALBUMINS AND OTHER LIVETIN PROTEINS IN EGG YOLK OF THE DOMESTIC FOWL (CALLUS DOMESTZCUS) W. M. MCINDOE and J. CULBERT Agricultural Research Council’s Poultry Research Centre, King’s Buildings, West Mains Road. Edinburgh EH9 3JS. U.K. (Rewired
2 Jtrnuarr
1979)
Abstract-l. The heterogeneity of the water soluble proteins of egg yolk in the domestic fowl livetin fraction was examined using starch-gel electrophoresis at pH 8.6, 5.4 and 4.6. 2. Seven major components and five minor components were observed. Fractionation of the livetin with ammonium sulphate revealed that the component migrating with the mobility of plasma y-globulin with ammonium sulphate. Three of the major components were was precipitated by 40”,, saturation with ammonium sulphate and three remained soluble. One of the precipitated with 50’!,, saturation components in the latter fraction migrated with the same mobility as plasma albumin in all three buffer systems. In addition, it was soluble in acid-ethanol as is mammalian plasma albumin. 3. An observation that the genetically determined plasma albumin types occurring in Brown Leghorn fowl can be detected in the livetin fraction of the yolk of eggs from the same bird confirmed that plasma albumin is present in livetin. Two minor albumin variants accompanied each major albumin,
Prepurrrtion
INTRODUCTION The early work on the livetin fraction of egg yolk was reviewed by Warner (1954). It is known to be heterogeneous. Moving-boundary electrophoresis has revealed three components (Shepard & Hottle, 1949) which have been named a-, b- and Y-livetin, in order of decreasing electrophoretic mobility, by Martin et trl. (1957). The latter authors also demonstrated them by ultracentrifugal analysis. Separation by paper electrophoresis (McCully et ul., 1959) revealed three components which are not necessarily identical to those mentioned above. The above methods have a relatively limited resolving power and the subsequent work of Mandeles (1960) and Williams (1962a) indicated a greater heterogeneity than had been observed hitherto. In the present work starch-gel electrophoresis was used to investigate the heterogeneity of the watersoluble proteins (livetin) of egg yolk. In addition, the protein components were compared with those of the blood plasma of the laying hen to find out which occurred in both places and may, therefore, have been transferred from the blood during yolk formation in the ovary. MATERIALS AND METHODS The birds used were from the Poultry Research Centre’s flock of Brown Leghorns and were I-2yr old. They were from the inbred lines I and B and the first cross (IB) (Blyth. 1954). Plasma was obtained from blood taken lrom the wing vein using a heparinized syringe. Eggs were obtained on the day on which they were laid. In general. the livetin fraction of individual yolks was examined, since preliminary experiments indicated the possible presence of genetical variants in at least one of the proteins. When yolks were pooled those from birds of the same plasma albumin type (see Results. Mclndoe, 1962) were used.
of the liretin
from
eyg yolk
Yolks were removed from eggs, rinsed in 0.9% (w/v) NaCl and rolled on filter paper to remove adhering white. The vitelline membrane was pierced and the yolk allowed to flow into a dialysis bag (Visking). To ease the flow of yolk, the pressure surrounding the bag was slightly reduced: the membrane was retained on the filter paper. Yolks were dialysed individually for 48 hr against 1 I. of distilled water containing a little benzoic acid (25 mg/l) and then centrifuged at 20,OOOg (MSE High Speed 13) for I hr at 2 C. The clear supernatant was retained for electrophoretic analysis. Elecfrophorcsis
oJ plosmu
und !dk
Horizontal starch-gel electrophoresis (Smithies, 1955) was used. The “starch-hydrolysed” was obtained from the Connaught Medical Research Laboratories, Toronto. Canada. The following buffer systems modelled on the discontinuous system of Poulik (1957) were used: (I) pH 8.6; electron compartments. 0.182 M boric acid, 0.05 M NaOH: gel. 0.04 M Tris. 0.005 M citric acid. More frequently the gel was prepared in a mixed buffer (pH 8.6) consisting of 0.04 M Tris, 0.005 M citric acid (4 parts) and 0.0182 M boric acid, 0.005 NaOH (I part). (2) pH 5.4; electrode compartments, 0.0245 M citric acid, 0.05 M NaOH: gel. 0.00735 M Tris, 0.005 M succinic acid. (3) pH 4.6; electrode compartments. 0.0344 M citric acid, 0.05 M NaOH: gel. 0.00417 M Tris, 0.005 succinic acid. Electrophoresis of up to five samples was carried out in gels measuring 20 x IO x 0.6 cm for 5-6 hr with a potential gradient of 556v/cm across the gel. The proteins were stained with O.OS”;, nigrosine in methanol/acetic acid/water (5: 1:5 by vol) for I hr and destained in several changes of the dye solvent. Ammonium
sulphure
froctionrrriotl
The livetin fractions from two to four yolks were pooled and saturated (NH,),SO, solution was added to give a final concentration of40”,, saturation at 20 C. The precipitate was allowed to flocculate for l5min and then centrifuged at 3OOOg for 10 min at 20 C. The supernatant was 659
660
W. M. M~INDOE and J. CULRERT
with saturated (NH.,)2S0, adjusted lo 50”,, saturation solution and centrifuged as above after standing a further 15 min. The latter supernatant was saturated with solid (NH,)*SO., and centrihqed at 30009 [or 30 min at 20 C. All three precipitates were washed by resuspension in an (NH,)2S04 solution of the appropriate concentration. and recentrifuuged. They were then dissolved in the minimum of distilled water and dialysed against three changes of distilled water.
Mammalian plasma albumin is soluble in acidified ethanol (Korner & Debro. 1956; Swert. 1957). thus the livetin fraction of yolk was treated to obtain the proteins which were soluble in acid/ethanol. Trichloroacetic acid (4 ml: IO”,, w/v) was mixed with 4ml of the livetin fraction at 0 C and the precipitate was suspended in 2 ml cold ethanol and then centriruged at 20009 for 5 min at 0 C. The supernatant was dialysed against distilled water at 2 C overnight. Fractionation of plasma was not carried out and rlectrophoresis was performed using whole plasma or plasma. diluted I to I. 2 to I. 4 to I or 5 to I with distilled water. RESULTS Electrophoresis
of the livetin
ethanol. oC egg yolk
in starch
gel using the discountinuous buffer, pH 8.6 gave rise to the pattern shown in Fig. 1. in which it is compared to that of laying hen plasma. The minor band (band 1) having the greatest mobility. was followed by five heavily stained protein bands migrating very close to one another: the second of these (band 3) had the same electrophoretic mobility as plasma albumin. The large diffuse faintly-staining region (band 11) corresponded in position to plasma y-globulin. In addition four other minor bands were detected, one of which (band 9) corresponded to transferrin (1. E. Lush, personal communication). An improved separation of the major protein components was achieved if the mixed buffer system (pH 8.6) was used in the gel (Fig. 1). The major bands were more discrete and more effectively separated.
+ Mixed -
D I
This was particularly true of the protein migratmg at the same rate as plasma albumin (band 2) which now moved considerably faster than the other main bands. Minor components occurred in positions I. 3, 8 and 9. The slow diffuse band (IO) corresponded to y-globulin. The livetins were discretely separated into three fractions by precipitation with various strengths of ammonium sulphate solution. The first fraction which precipitated at 40”,, saturation consisted of y-livetin (Martin et trl., 1957) and each of the other fractions contained three major components. In the mixed buffer, pH 8.6 the protein which was ammonium sulphate insoluble in 40”,, saturated migrated as band IO. The precipitate obtained at 40-50”,, saturated ammonium sulphate consisted of the leading half of band 5, bands 6 and 7 and the minor band 8 etc. and band 9. The apparent heterogeneity of band 5 was a consistent observation and leads one to believe that it consists of two components. The protein corresponding to plasma albumin (band 2) was the only component soluble in acid-
buffer
An initial survey of the separation of the livetin proteins of egg yolk by electrophoresis in buffer sy’rterns of different pH showed that good separation could be achieved at pH 5.4. when all but one of the proteins migrated to the anode (Fig. 2). Five major protein bands (2, 6 to 9) were present, the leadmg one of which corresponded to plasma albumin in the sample of diluted plasma. The components I and 9 also had the same mobility as plasma components. Three minor components (3. 4 and 5) lay immediate11 behind the albumin position. The diffuse band migrating to the cathode, corresponded to a similar component in the diluted plasma sample. Undiluted plasma gave an unsatisfactory pattern due. mainly. to the large area of the albumin band. However. m undiluted plasma two or three faint bands were seen which migrated in the region of bands 5 and 6 of the yolk livetin fraction.
2 3 4 5 6 7 e
9 IO
II
Origin IO Livetin
Origin Plasma Plasma
Livetin Livetin
Fig. 1. Starch-gel electrophoresis ol’ plasma (I in 4 dilution) and livetin in buffer at pH 8.6 and in the mixed buffer
(pH 8.6).
Fig. 2. Starch-gel
PlClSmCl
electrophoresis of livetin in 5 dilution) at pH 5.4.
and plasma
II
661
Livetin heterogeneity Ammonium sulphate fractionation confirmed that band 10 was y-livetin. The proteins precipitated between 40 and SO:/, saturated ammonium sulphate were represented by minor bands 4 and 5 and by major bands 6 and 7. The proteins that were soluble in SO”
002,~.7, x:7Y:n701-065YSO2 CrnN
THE PLASMA ALBUMINS AND OTHER LIVETIN PROTEINS IN EGG YOLK OF THE DOMESTIC FOWL (CALLUS DOMESTZCUS) W. M. MCINDOE and J. CULBERT Agricultural Research Council’s Poultry Research Centre, King’s Buildings, West Mains Road. Edinburgh EH9 3JS. U.K. (Rewired
2 Jtrnuarr
1979)
Abstract-l. The heterogeneity of the water soluble proteins of egg yolk in the domestic fowl livetin fraction was examined using starch-gel electrophoresis at pH 8.6, 5.4 and 4.6. 2. Seven major components and five minor components were observed. Fractionation of the livetin with ammonium sulphate revealed that the component migrating with the mobility of plasma y-globulin with ammonium sulphate. Three of the major components were was precipitated by 40”,, saturation with ammonium sulphate and three remained soluble. One of the precipitated with 50’!,, saturation components in the latter fraction migrated with the same mobility as plasma albumin in all three buffer systems. In addition, it was soluble in acid-ethanol as is mammalian plasma albumin. 3. An observation that the genetically determined plasma albumin types occurring in Brown Leghorn fowl can be detected in the livetin fraction of the yolk of eggs from the same bird confirmed that plasma albumin is present in livetin. Two minor albumin variants accompanied each major albumin,
Prepurrrtion
INTRODUCTION The early work on the livetin fraction of egg yolk was reviewed by Warner (1954). It is known to be heterogeneous. Moving-boundary electrophoresis has revealed three components (Shepard & Hottle, 1949) which have been named a-, b- and Y-livetin, in order of decreasing electrophoretic mobility, by Martin et trl. (1957). The latter authors also demonstrated them by ultracentrifugal analysis. Separation by paper electrophoresis (McCully et ul., 1959) revealed three components which are not necessarily identical to those mentioned above. The above methods have a relatively limited resolving power and the subsequent work of Mandeles (1960) and Williams (1962a) indicated a greater heterogeneity than had been observed hitherto. In the present work starch-gel electrophoresis was used to investigate the heterogeneity of the watersoluble proteins (livetin) of egg yolk. In addition, the protein components were compared with those of the blood plasma of the laying hen to find out which occurred in both places and may, therefore, have been transferred from the blood during yolk formation in the ovary. MATERIALS AND METHODS The birds used were from the Poultry Research Centre’s flock of Brown Leghorns and were I-2yr old. They were from the inbred lines I and B and the first cross (IB) (Blyth. 1954). Plasma was obtained from blood taken lrom the wing vein using a heparinized syringe. Eggs were obtained on the day on which they were laid. In general. the livetin fraction of individual yolks was examined, since preliminary experiments indicated the possible presence of genetical variants in at least one of the proteins. When yolks were pooled those from birds of the same plasma albumin type (see Results. Mclndoe, 1962) were used.
of the liretin
from
eyg yolk
Yolks were removed from eggs, rinsed in 0.9% (w/v) NaCl and rolled on filter paper to remove adhering white. The vitelline membrane was pierced and the yolk allowed to flow into a dialysis bag (Visking). To ease the flow of yolk, the pressure surrounding the bag was slightly reduced: the membrane was retained on the filter paper. Yolks were dialysed individually for 48 hr against 1 I. of distilled water containing a little benzoic acid (25 mg/l) and then centrifuged at 20,OOOg (MSE High Speed 13) for I hr at 2 C. The clear supernatant was retained for electrophoretic analysis. Elecfrophorcsis
oJ plosmu
und !dk
Horizontal starch-gel electrophoresis (Smithies, 1955) was used. The “starch-hydrolysed” was obtained from the Connaught Medical Research Laboratories, Toronto. Canada. The following buffer systems modelled on the discontinuous system of Poulik (1957) were used: (I) pH 8.6; electron compartments. 0.182 M boric acid, 0.05 M NaOH: gel. 0.04 M Tris. 0.005 M citric acid. More frequently the gel was prepared in a mixed buffer (pH 8.6) consisting of 0.04 M Tris, 0.005 M citric acid (4 parts) and 0.0182 M boric acid, 0.005 NaOH (I part). (2) pH 5.4; electrode compartments, 0.0245 M citric acid, 0.05 M NaOH: gel. 0.00735 M Tris, 0.005 M succinic acid. (3) pH 4.6; electrode compartments. 0.0344 M citric acid, 0.05 M NaOH: gel. 0.00417 M Tris, 0.005 succinic acid. Electrophoresis of up to five samples was carried out in gels measuring 20 x IO x 0.6 cm for 5-6 hr with a potential gradient of 556v/cm across the gel. The proteins were stained with O.OS”;, nigrosine in methanol/acetic acid/water (5: 1:5 by vol) for I hr and destained in several changes of the dye solvent. Ammonium
sulphure
froctionrrriotl
The livetin fractions from two to four yolks were pooled and saturated (NH,),SO, solution was added to give a final concentration of40”,, saturation at 20 C. The precipitate was allowed to flocculate for l5min and then centrifuged at 3OOOg for 10 min at 20 C. The supernatant was 659
660
W. M. M~INDOE and J. CULRERT
with saturated (NH.,)2S0, adjusted lo 50”,, saturation solution and centrifuged as above after standing a further 15 min. The latter supernatant was saturated with solid (NH,)*SO., and centrihqed at 30009 [or 30 min at 20 C. All three precipitates were washed by resuspension in an (NH,)2S04 solution of the appropriate concentration. and recentrifuuged. They were then dissolved in the minimum of distilled water and dialysed against three changes of distilled water.
Mammalian plasma albumin is soluble in acidified ethanol (Korner & Debro. 1956; Swert. 1957). thus the livetin fraction of yolk was treated to obtain the proteins which were soluble in acid/ethanol. Trichloroacetic acid (4 ml: IO”,, w/v) was mixed with 4ml of the livetin fraction at 0 C and the precipitate was suspended in 2 ml cold ethanol and then centriruged at 20009 for 5 min at 0 C. The supernatant was dialysed against distilled water at 2 C overnight. Fractionation of plasma was not carried out and rlectrophoresis was performed using whole plasma or plasma. diluted I to I. 2 to I. 4 to I or 5 to I with distilled water. RESULTS Electrophoresis
of the livetin
ethanol. oC egg yolk
in starch
gel using the discountinuous buffer, pH 8.6 gave rise to the pattern shown in Fig. 1. in which it is compared to that of laying hen plasma. The minor band (band 1) having the greatest mobility. was followed by five heavily stained protein bands migrating very close to one another: the second of these (band 3) had the same electrophoretic mobility as plasma albumin. The large diffuse faintly-staining region (band 11) corresponded in position to plasma y-globulin. In addition four other minor bands were detected, one of which (band 9) corresponded to transferrin (1. E. Lush, personal communication). An improved separation of the major protein components was achieved if the mixed buffer system (pH 8.6) was used in the gel (Fig. 1). The major bands were more discrete and more effectively separated.
+ Mixed -
D I
This was particularly true of the protein migratmg at the same rate as plasma albumin (band 2) which now moved considerably faster than the other main bands. Minor components occurred in positions I. 3, 8 and 9. The slow diffuse band (IO) corresponded to y-globulin. The livetins were discretely separated into three fractions by precipitation with various strengths of ammonium sulphate solution. The first fraction which precipitated at 40”,, saturation consisted of y-livetin (Martin et trl., 1957) and each of the other fractions contained three major components. In the mixed buffer, pH 8.6 the protein which was ammonium sulphate insoluble in 40”,, saturated migrated as band IO. The precipitate obtained at 40-50”,, saturated ammonium sulphate consisted of the leading half of band 5, bands 6 and 7 and the minor band 8 etc. and band 9. The apparent heterogeneity of band 5 was a consistent observation and leads one to believe that it consists of two components. The protein corresponding to plasma albumin (band 2) was the only component soluble in acid-
buffer
An initial survey of the separation of the livetin proteins of egg yolk by electrophoresis in buffer sy’rterns of different pH showed that good separation could be achieved at pH 5.4. when all but one of the proteins migrated to the anode (Fig. 2). Five major protein bands (2, 6 to 9) were present, the leadmg one of which corresponded to plasma albumin in the sample of diluted plasma. The components I and 9 also had the same mobility as plasma components. Three minor components (3. 4 and 5) lay immediate11 behind the albumin position. The diffuse band migrating to the cathode, corresponded to a similar component in the diluted plasma sample. Undiluted plasma gave an unsatisfactory pattern due. mainly. to the large area of the albumin band. However. m undiluted plasma two or three faint bands were seen which migrated in the region of bands 5 and 6 of the yolk livetin fraction.
2 3 4 5 6 7 e
9 IO
II
Origin IO Livetin
Origin Plasma Plasma
Livetin Livetin
Fig. 1. Starch-gel electrophoresis ol’ plasma (I in 4 dilution) and livetin in buffer at pH 8.6 and in the mixed buffer
(pH 8.6).
Fig. 2. Starch-gel
PlClSmCl
electrophoresis of livetin in 5 dilution) at pH 5.4.
and plasma
II
661
Livetin heterogeneity Ammonium sulphate fractionation confirmed that band 10 was y-livetin. The proteins precipitated between 40 and SO:/, saturated ammonium sulphate were represented by minor bands 4 and 5 and by major bands 6 and 7. The proteins that were soluble in SO”
