EGG WHITE PROTEINS

II. AN ETHANOL FRACTIONATION SCHEME*

BY RICHARD H. FORSYTHE? AND JOSEPH F. FOSTER
(From the Department of Chemistry, Iowa State College, Ames)
(Received for publication, October 15, 1949)
Egg white as a protein system possesses much interest, both biochemical
and technological. Study of the egg white proteins has been handicapped
by the difficulty of preparation. The five variant system developed by
Cohn and coworkers for the fractionation of plasma offers many advan-
tages over the salting-out techniques (1). Low temperatures are em-
ployed to improve protein stability. Since low salt concentrations are
employed, prolonged dialyses are avoided. Ethanol can be removed easily
and rapidly by lyophilization. The ethanol fractionation procedures are
adaptable to commercial operations so that production of valuable by-
products is a possibility.
In this study, an attempt has been made to adapt the ethanol tech-
niques to the egg white system. Emphasis has been placed on the de-
velopment of an over-all scheme and not on the preparation of any single
fraction. Purity of individual fractions has been a secondary considera-
tion in order to obtain all the fractions in the highest possible yields.
EXPERIMENTAL

The procedures used in this study were similar to those described for
the plasma fractionation system (2). Fresh eggs were secured from the
Iowa State College Poultry Farm, the whites separated from the yolks,
and the chalazae removed. The white was next blended in a Waring
blendor equipped with a rheostat (to prevent excessive foaming). A com-
mercial grade of 95 per cent ethyl alcohol was diluted to 50 per cent (by
volume) and used for all alcohol additions. Acetate, phosphate, and car-
bonate buffers were used for pH adjustment and electrophoretic runs.
Preparation of acetate and phosphate buffers of known ionic strength was
facilitated by the use of the d’Ocagne (3) acetate nomogram and Green’s
(4) phosphate buffer chart. In all fractionation steps, temperatures were
* Journal Paper No. J-1699 of the Iowa Agricultural Experiment Station, Project
978. Supported in part by a grant from Swift and Company.
Taken from a thesis submitted by Richard H. Forsythe to the Graduate Faculty,
IowaState College, in partial fulfilment of the requirements for the Degree of Doctor
of Philosophy.
t Present address, Department of Poultry Husbandry, Iowa State College, Ames,
Iowa.
385

This is an Open Access article under the CC BY license.

386 EGG WHITE PROTEINS. II

maintained within l-2“ of the freezing point of the system. All pH meas-
urements were carried out at 25” with a glass electrode on diluted solutions.
The fractions were removed by centrifugation and the supernatant solu-
tions clarified by filtration when necessary. A Sharples continuous super-
centrifuge was employed when large volumes of solution with low foaming
tendencies were centrifuged.
Electrophoretic analysis of the fractions obtained was carried out as pre-
viously described (5). Routine analytical runs were made in phosphate-
chloride buffer of pH 7.7 to 7.8, I’/2 = 0.20 (NaCl = 0.15 M). Averages
of ascending and descending data were used in most instances. Nitrogen
was determined by a modified Pregl micro-Kjeldahl procedure with SeOC&
as a catalyst.
RESULTS AND DISCUSSION

Egg white has been separated into six fractions by application of the
ethanol fractionation procedures. The results of a typical fractionation
experiment are shown in Figs. 1 and 2 and the data summarized in Tables
I and II. 1 liter of egg white, of the following composition, was used as
the starting material:’ nitrogen 17.69, ovalbumin 11.50, ovomucoid 1.64,
globulin 1.54, conalbumin 2.41, and lysozyme 0.60 gm. In Figs. 1 and 2,
the conditions imposed on the system are given on the horizontal tie lines
where ~~ is the mole fraction of ethyl alcohol and N is the nitrogen in the
system in gm. per liter. The analytical data for each fraction are given
in the boxes where N is the total gm. of nitrogen in the fraction and A,
0, G, C, and L represent the gm. of ovalbumin, ovomucoid, globulin, con-
albumin, and lysozyme nitrogen, respectively. No attempt was made to
convert the nitrogen data to a protein basis.
The separation and purification of each fraction are discussed in the
order in which they were removed from the egg white.
Ovomucin Fraction-Egg white, blended in a Waring blendor, was sub-
jected to high speed centrifugation in a Sharples air-driven supercentrifuge
equipped with a batch type bowl. At a velocity of 45,000 r.p.m. (50,000
X g), a slimy, ropy precipitate characteristic of ovomucin was removed
after 15 minutes centrifugation. This fraction was designated Fraction I.
In several experiments, 1.1 per cent of the original protein (by N analysis)
was removed by this procedure. Attempts to remove ovomucin by the
classical dilution or salting out techniques resulted in the precipitation of
approximately 10 per cent of the protein, while the procedure used in this
study effected the separation of only 1.1 per cent of the original nitrogen.
This figure is probably more reliable than the higher figure (1.9 per cent)
1 Data obtained by electrophoretic analysis; ovomucin included only in the figure
for total nitrogen.
 R. R. FORSYTHE AND J. F. FOSTER 387

previously reported (6) because of the extreme difficulty of washing soluble

proteins from the classical ovomucin precipitate. That ovomucin can be
removed by centrifugation without any previous treatment supports the
view that this protein is present in egg white in the form of very large
(frbrillar) particles.

SUPERNATANT FROM
FRACTION I

pH=6.3, N:!=0.07, l-Z-6”

I V2=0.06 N=8.85
N = 4?32 gm.
FOR FRACTION
A0 f P 8,
” m
G l 0.51 ”
C-2.18 ”
LtO.65 ”

pHz5.5, r/2-, NZ= 0 , N = 12.O, T=I’

la- I
N = 1.05 gm.
A = 0.14 ::
0 = 0.10
g ; c&g I8
I,
L=o:13 ”
pH=6.3,w2=0.01,~~ =0, N=4.5, T=I* 1
I

pH=7.5, P/2’O,N,=O, T=l”

I
I rL-2-6-N-I I 1

FIG. 1. Separation and purification of the conalbumin-globulin fraction. For an

explanation of the symbols, see the text.

The precipitate as removed from the centrifuge rotor can be suspended

by means of the Waring blendor in 0.15 M (or higher) NaCl to yield rea-
sonably stable, though highly turbid, suspensions. Many other solvent
systems have been tried, including urea (6 M) and both anionic and cationic
detergents at pH levels as high as 12, none of them yielding anything ap-
388 EGG WHITE PROTEINS. II

proaching clear solutions. On the other hand, this precipitate can be

blended with egg white to give solutions practically as clear as the original
white. This may arise from the solvent action of the other proteins or
may be a refractive index effect.
Conalbumin-Globulin Fraction--Bain and Deutsch (7) described an eth-
anol fractionation scheme for the preparation of conalbumin from egg
white. A preparation of high purity was obtained, although in low yield.

SUPER NATANT FROM

FRACTION II

pH=4.6,r/2- 0, N2=0, T=I”

I
m- 2
; : ;:g?,“.
= 0.57”
: = 0.38 ”
c = 0.34;;
L= 0
pH=4.6,r.2+0, N2. =O.II, N= 11.7, T=-15O]

pH=4.6, P/2=0, N2 = 0, T= lo
I

FIG. 2. Separation and purification of the ovalbumin fraction. For an explana-

tion of the symbols, see the text.

In this investigation, it has not been possible to obtain satisfactory separa-

tion of either the conalbumin or globulin fraction individually. Several
attempts were made to remove the globulin prior to the conalbumin at
pH 5.4 to 5.6 and low ionic strengths. In all cases, the fraction was
highly contaminated with conalbumin and considerable quantities of ap-
parently denatured protein were obtained. This procedure was not con-
sidered theoretically sound because of the presence of oppositely charged
proteins, a condition which usually results in extensive interactions. At-
tempts to remove conalbumin at pH 6.0 to 7.0, I’/2 = 0.05 to 0.10, ~~ =
0.02 to 0.04, were unsuccessful because of large amounts of globulin im-
 R. H. FORSYTHE AND J. F. FOSTER 389

purities and incomplete precipitation of the conalbumin; 15 to 25 per cent

of the total globulin was precipitated under these conditions. The con-

TABLE I
Conditions for Separation of Egg White Proteins into Fractions
- -
I‘ Etha- N in N of
Fraction No. PH T nol, i-T.2 N kctior original
2
I, I __
T. t In. per 1 P.
I. . . . . . . . . . . . Supercentrifuge 17.69 0.20 1.1
II ....................... 6-3 0.06 -6 0.07 8.85 4.92 27.8
II-1 ..................... 5.5 -10 1 0 12.00 1.05 5.9
11-2-A. ................. 6.3 0.01 1 0 4.5 0.11 0.6
11-2-B-B. ................. 6.3 0.01 -6 0.07 0.85 4.8
11-2-B-a-l. .............. 7.5 1 0 0.95 5.4
11-2-B-a-2. .............. 7.5 1 0 1.65 9.3
III. ..................... 4.6 0.04 -16 0.11 3.4 9.69 54.6
III-1 .................... 4.6 -+O 1 0 0.86 4.8
111-2-A-a. ............... 4.6 +O 1 0 0.46 2.6
III-P-A+?. ................ 4.6 -+O 1 0 8.25 46.7
IV. ...................... Supernatant 1 0 0.54 3.1
- -____ --
Total yield ............. 84.3
-
No is the mole fraction of ethanol.

TABLE II
Distribution of Egg White Proteins into Fractions
Ovalbu- O:y- Globulin c”$p- Lysozyme
min
Fraction No. --P-F
(1)’ cot (1)
--------
(2) (1) (2) (1) (2) (1) (2)

Eggwhite.. _....,........._..... 64.9 9.2 8.7 13.8 3.4

11-l . . . . . . . . . . . . . . ..__._......_..... 13.4 1.2
9.5 6.161.041.5 2.9 1.212.421.7
II-2-B-(Y-1..........................14.7 1.2
2.1 1.217.911.041.016.219.020.0
II-2-B-or-2.......................... 19.4 02.8 0 5.4 5.875.852.0 0 0
111-2-A-B ,... .._....__...... 93.767.2 2.110.4 2.915.5 1.5 5.0 0 0
IV. .._..,,..., ,___.,.,,..,.._.,.... 11.1 0.588.929.3 0 0 0 0 0 0
~___ ~____
Total yield.. . .... .. . . .. 72.9 47.0 73.8 74.4 41.7

* (1), per cent of constituent in the fraction.

t (2), per cent of constituent, in the fraction, of that originally present in the
egg white.

albumin-globulin fraction was satisfactorily removed as Fraction II under

the conditions described in Fig. 1. There was no sharp pH optimum
390 EGG WHITE PROTEINS. II

between pH 6.0 to 7.0 for the removal of this fraction, but somewhat better
removal was effected in the more acid range. This fraction was removed
by supercentrifugation, and, although attempts were made to complete
the removal without excessive temperature increases, the effluent from the
centrifuge was usually 5-7” higher than that at which the fraction had
been precipitated. Upon cooling the solution to the precipitation tempera-
ture, a precipitate formed, indicating a high temperature coefficient of solu-
bility of the proteins in this medium. Repeated cooling and centrifuga-
tion, followed by filtration at the precipitation temperatures, apparently
effected complete removal of the fraction. Only about 50 per cent of the
globulin can be removed under these conditions, indicating the presence of
at least two globulin fractions with different solubility characteristics.
The globulin that remains in solution appears to be similar to the pseudo-
globulin reported by Kuchel and Bate-Smith (8). A partial separation of
the globulins was accomplished by these procedures.
All of the lysozyme present in the egg white is removed in this fraction
and can be crystallized by the procedure described by Alderton and Fevold
(9). Several fractionations have been carried out in which lysozyme has
been removed prior to the conalbumin-globulin fraction. Yields of lyso-
zyme are higher under these conditions, but the fractionation procedure is
prolonged by the extended crystallization period and all of the proteins are
subjected to high pH and salt concentrations. The removal of the salt
before further fractionation is a tedious and time-consuming process. For
best results in a comprehensive fractionation scheme, lysozyme should be
removed with the conalbumin-globulin fraction and then recovered by sub-
fractionation.
The globulin was separated from the conalbumin by lowering the pH
to 5.5 and dialyzing against water to lower the ionic strength. The degree
of separation obtained depends on the thoroughness of the washing of the
precipitate. All of the yellow color of the egg white was found in the
conalbumin (Fraction 11-2, Fig. 1). Further fractionation of Fraction II-2
resulted in two main fractions (Fractions 11-2-B-~-1 and 11-2-B-a-2). Al-
though Fraction 11-2-B-a-2 contained a considerably higher proportion of
conalbumin, Fraction II-2-B-a-1, insoluble in water but soluble in 5 per
cent NaCl, contained all of the yellow constituent originally present in the
fraction. This was unexpected, since it had been shown (7) that the ribo-
flavin is bound to the conalbumin and can be removed by dialysis only on
the acid side of the isoelectric point. It was concluded that either (1)
the riboflavin is not bound to the characteristic conalbumin constituent,
(2) the conalbumin does not behave as a typical albumin, or (3) the bound
riboflavin appreciably alters the solubility properties of conalbumin.
Ovalbumin Fraction-Following removal of the conalbumin-globulin frac-
 R. H. FORSYTHE AND J. F. FOSTER 391

tion, ovalbumin was removed as Fraction III by lowering the pH to 4.6

and increasing the ethanol concentration to mole fraction 0.11, as shown
in Fig. 2. All of the protein remaining after the removal of Fraction II,
except ovomucoid, was removed nearly quantitatively under these condi-
tions. Purification of the ovalbumin was carried out as indicated in Fig. 2.
Typical analytical data are shown in Table II. The ovalbumin exhibits a
high temperature coefficient of solubility in this medium, as do the con-
albumin and globulin. The solubility was lowered as the NaCl concentra-
tion was decreased in qualitative agreement with the observations of Ferry,
Cohn, and Newman (10). The initial removal of this fraction might be
improved by reducing the ionic strength to approximately 0.01, since pro-
tein-protein interactions would probably be of minor importance in this
case.
The ovalbumin from this fraction was readily crystallized by slight
modification of the classical ammonium sulfate procedures (11) and was
soluble in water at its isoelectric point. Attempts to crystallize the oval-
bumin from alcohol-water according to the procedures described by Cohn,
Hughes, and Weare (12) for serum albumin have resulted in small yields
of crystalline material, not yet demonstrated to be protein.
Ovomucoid Fraction-In the present procedure, the ovomucoid has been
concentrated in the supernatant remaining after the removal of the oval-
bumin fraction. As the purity of this fraction (IV), judged by electro-
phoretic analysis, has been quite high, no attempt has been made to pre-
cipitate ovomucoid from it. The volume of the supernatant containing
the ovomucoid has been about 3 to 4 times the volume of the initial egg
white. Concentration has been effected by pervaporation from large tub-
ular Visking membranes, alcohol and salts being subsequently removed
by dialysis and dry ovomucoid preparations obtained by lyophilization.
Yields of ovomucoid were usually higher than that given in Table II; in
several experiments, 80 to 85 per cent of the ovomucoid was recovered in
fractions which consisted of 75 to 80 per cent ovomucoid. The major
impurity in all cases was ovalbumin, which was readily removed by repeti-
tied of the ovalbumin precipitation. Ovomucoid preparations obtained
in this study have not been tested for antitryptic activity. In electro-
phoretic experiments, ovomucoid has exhibited reversible boundary spread-
ing as described by Longsworth et al. (13), indicating inhomogeneity.
SUMMARY

1. A method for the comprehensive fractionation of egg white in media

of low dielectric constant and low ionic strength is described. Several ad-
vantages are claimed for this method over those previously reported for
preparing egg white fractions.
392 EGG WHITE PROTEINS. II

Ovomucin has been removed by supercentrifugation with a minimum of

alteration and occlusion of other proteins.
The ethyl alcohol acts as a foam depressant so that the Sharples super-
centrifuge can be used for continuous operation.
The advantages previously described for the preparation of the plasma
proteins are applicable to the egg white fractionation.
2. The possibility that the riboflavin of egg white is not bound to the
main conalbumin component is suggested.
3. Evidence is given to support the view that two globulins, with differ-
ent solubility characteristics, are present in egg white.
4. Ovalbumin prepared by these procedures is readily crystallized by the
use of ammonium sulfate.
BIBLIOGRAPHY

1. Edsall, J. T., Advances in Protein Chem., 3, 383 (1947).

2. Cohn, E. J., Strong, L. E., Hughes, W. L., Jr., Mulford, D. J., Ashworth, J. N.,
Melin, M., and Taylor, H. L., J. Am. Chem. Sot., 68, 459 (1946).
3. Boyd, W. C., J. Am. Chem. SOL, 67, 1035 (1945).
4. Green, A. A., J. Am. Chem. Sot., 66, 2331 (1933).
5. Forsythe, R. H., and Foster, J. F., J. Biol. Chem., 184, 377 (1950).
6. Serensen, M., Biochem. Z., 269, 271 (1934).
7. Bain, J. A., and Deutsch, H. F., J. Biol. Chem., 172, 547 (1948).
8. Kuchel, C. C., and Bate-Smith, E. C., Great Britain Dept. of SC. and Ind. Res.,
Rep. Food Invest. Board., 1937, 25 (1938).
9. Alderton, G., and Fevold, H. L., J. Biol. Chem., 164,l (1946).
10. Ferry, R. M., Cohn, E. J., and Newman, E. S., J. Am. Chem. Sot., 68, 2370 (1936).
11. LaRosa, W., Chemist-Analyst, 16, 3 (1927).
12. Cohn, E. J., Hughes, W. L., Jr., and Weare, J. H., J. Am. Chem. Sot., 69, 1753
(1947).
13. Longsworth, L. G., Cannan, R. K., and MacInnes, D. A., J. Am. Chem. SOL,
62, 2580 (1940).