Biochem. J.

(1986) 238, 671-675 (Printed in Great Britain)

671

Riboflavin-binding protein
Concentration and fractional saturation in chicken eggs as a function of dietary riboflavin

Har'old B. WHITE III,* John ARMSTRONG and Colin C. WHITEHEAD

AFRC Poultry Research Centre, Roslin, Midlothian EH25 9PS, Scotland, U.K.

The concentration of riboflavin and riboflavin-binding protein were determined in the plasma, egg yolk and
albumen from hens fed a riboflavin-deficient diet (1.2 mg/kg) supplemented with 0, 1, 2, 3, 10 and 40 mg
of riboflavin/kg. We observed that the deposition of riboflavin in egg yolk and albumen is dependent on
dietary riboflavin and reaches half-maximal values at about 2 mg of supplemental riboflavin/kg. The
maximal amount of riboflavin deposited in the yolk is limited stoichiometrically by the amount of
riboflavin-binding protein, whereas the maximum amount of riboflavin deposited in albumen is limited by
other factors before saturation occurs. The amount of riboflavin-binding protein in yolk and albumen is
independent of dietary riboflavin. If there is a specific oocyte receptor for riboflavin-binding protein, it
cannot distinguish between the apo and holo forms of the protein. Riboflavin-binding protein is about six
times more concentrated in yolk than in plasma.
INTRODUCTION
The hen's egg is perhaps the most complete single food
(Williams et al., 1971). It contains all the nutrients for the
21-day development of a chick embryo. The yolk, which
contains most of the nutrients, is derived from the plasma
by endocytosis (Griffin et al., 1984). Our interest is in the
deposition of riboflavin in the egg and the role of
riboflavin-binding protein in this process.
Petersen et al. (1947) showed that up to 30 % of
ingested riboflavin can be transferred to the egg by a
laying hen. They also showed that the riboflavin content
of eggs is proportional to dietary riboflavin over the
range 0-5 mg of riboflavin/kg of feed and more-or-less
independent of dietary riboflavin above that range. This
saturation phenomenon is attributable to a specific
riboflavin-binding protein (Rhodes et al., 1959) to which
all of the riboflavin in an egg is bound (Blum, 1967).
Hens genetically unable to synthesize a functional
riboflavin-binding protein deposit insignificant amounts
of riboflavin in their eggs (Maw, 1954; Winter et al.,
1967; Farrell et al., 1970). Thus the amount of
riboflavin-binding protein appears to set an upper limit
on the amount of riboflavin that can be deposited in an
egg. On the basis of these observations and others, the
presence of a specific receptor that recognizes the
binding protein, but not the vitamin, was postulated
(Muniyappa & Adiga, 1979; Miller et al., 198 la). There
is indirect evidence both for and against this hypothesis.
Experiments by Miller and others have shown that
modification of protein-bound carbohydrate (Miller
et al., 198 1a,b, 1982a) or phosphate (Miller et al., 1982b),
or derivatization of amino acid side chains (Miller
et al., 1982b; Hammer et al., 1971), can drastically reduce
the uptake of the radiolabelled protein by the
oocyte. Because not all of these modifications result in
rapid clearance of riboflavin-binding protein from the
plasma by the liver or other tissues, an oocyte receptor
that distinguishes between the native and modified
*

protein seems likely. Furthermore, specific receptors for

vitellogenin (Yusko et al., 1981), immunoglobulins
(Roth et al., 1976), and low-density lipoproteins
(Krumins & Roth, 1981; Perry et al., 1984) have been
demonstrated in the hen's oocyte plasma membrane.
Despite these precedents for protein-specific receptors in
this system, continuing efforts to demonstrate directly a
receptor for riboflavin-binding protein have been
unsuccessful (Benore-Parsons, 1986).
Because the primary function of riboflavin-binding
protein is to deposit riboflavin in the egg, it would seem
likely that a specific receptor on the oocyte plasma
membrane would be able to discriminate between apo
and holo forms of the protein. However, Benore-Parsons
et al. (1985) reported that deposition of the protein
occurs in the absence of bound riboflavin. In order to
verify this observation, we fed laying hens diets differing
in their riboflavin content and monitored the amount of
riboflavin-binding protein and its fractional saturation in
the plasma, yolk and albumen. Both apo and holo forms
of the protein are transferred to the egg. We conclude
that, if a receptor exists, it does not distinguish between
these two forms of the protein.
EXPERIMENTAL
Hens and diets
The birds (ISA Brown) were obtained at 1 day old
from a commercial hatchery. From 18 weeks of age they
were housed in single-bird battery-cage units and had
free access to the standard layer's diet and water.
Individual egg production was recorded daily. The
experimental period started when the birds were 26 weeks
old.
Diet compositions are given in Table 1. The standard
layer's diet was found by analysis to contain 6.5 mg of
riboflavin/kg, an amount that exceeded the minimum
requirement of 2.2 mg/kg for egg-laying in hens

Permanent address and address for correspondence and reprint requests: Department of Chemistry, University of Delaware, Newark, DE 19716,

U.S.A.

Vol. 238

672

H. B. White III, J. Armstrong and C. C. Whitehead

Table 1. Composition of diets

Composition (g/kg)
Standard

Riboflavindeficient

Barley
255
Wheat
193
300
Maize
242
Maize starch
348
Isolated soybean
170
protein
Soybean meal
104
(extracted)
Herring meal
48
Meat and bone meal
19
Grass meal
50
Limestone flour
68
80
CaHPO4,2H20
14
40
Salt
2
4
Vegetable oil
50
Mineral supplement*
2.5
2.5
Vitamin supplement At
2.5
Supplement Bt
5.5
* Supplied (mg/kg of diet): copper,
3.5; iodide, 0.4; iron, 80;
magnesium, 300; manganese, 100; zinc, 50.
t Supplied (per kg of diet): retinol, 2.0 mg; cholecalciferol,
20,g; a-tocopherol, 17 mg; riboflavin, 4 mg; nicotinic acid,
28 mg; pantothenic acid, 10 mg.
t Supplied (per kg of diet): retinol, 3.0 mg; cholecalciferol,
50 jg; a-tocopherol, 10 mg; menadione, 1.2 mg; thiamin,
1.5 mg; folic acid, 0.4 mg; pyridoxine, 4 mg; nicotinic acid,
20 mg; panthothenic acid, 20 mg; cyanocobalamin, 10 jug;
choline chloride, 0.5 g; DL-methionine, 3 g, L-tryptophan, 0.6 g.

(National Research Council, 1984). The riboflavindeficient diet was found to contain 1.2 mg of riboflavin/
kg. Analysis of supplemented diets confirmed that the
appropriate amounts of riboflavin had been added to
each.
Experimental design
A total of 12 hens, all with a high rate of egg-laying,
were allocated to each of eight experimental groups.
Hens were housed in individual battery cages. At the
start of the experiment, six groups were transferred to the
riboflavin-deficient diet supplemented with 0, 1, 2, 3, 10,
or 40 mg of riboflavin/kg. The remaining two groups
were maintained on the standard layer diet as controls.
Plasma samples from each bird were taken at weekly
intervals and pooled by groups. Individual egg production
was monitored, and eggs laid on days 0, 7, 14, and 21
were collected for analysis of riboflavin and riboflavinbinding protein. If an egg was not available from a hen
on a particular day, the egg laid by that bird on the
previous day was taken, if available.
Preparation of samples
A 1 ml sample of blood was taken from a wing vein of
each bird and pooled with blood samples from the other
hens in the group in a chilled plastic tube containing
15 jug of a 15 mg/ml solution of heparin (Sigma
Chemical Co., St. Louis, MO, U.S.A.). Red blood cells
were removed by centrifugation (10 min at 1000 g) and
the plasma was stored frozen at -20 'C.

Egg yolk and albumen were separated. Albumen

adhering to yolks was removed by gently rolling yolks on
paper towels. The yolk and albumen from each egg was
weighed. The separately pooled albumen and yolk
samples from each group were homogenized with a
motor-driven propeller. Albumen samples were stored
frozen at -20 C without dilution. Yolk samples were
diluted (25% w/w) and homogenized with 25 mmTris/HCl, pH 7.5, before being frozen.
Radioligand binding assay for riboflavin-binding protein
The assay for riboflavin-binding protein (Lotter et al.,
1982; White, 1986) is based on the protein's ability to
bind riboflavin specifically with high affinity
(KD 2 nM). D-[2-14C]Riboflavin (52.4 mCi/mmol;
Amersham International) was incubated with diluted
samples at room temperature for 10 min by which time
added radioactive riboflavin had come to isotope
equilibrium with endogenous unlabelled riboflavin.
Bound and free riboflavin were separated by DEAEcellulose chromatography and the bound radioactivity
was determined by liquid-scintillation counting. The
concentration of riboflavin-binding sites and the fractional saturation by endogenous riboflavin was determined graphically. Although a fractional saturation of
less than zero is impossible, values less than zero can be
obtained by graphical analysis on samples that contain
little or no endogenous riboflavin. This method assumes
that binding is reversible, that isotope equilibrium is
attained during the incubation period, that free and
bound riboflavin are completely separated and that there
is a total recovery of bound riboflavin. In order to
correct for any losses, a sample of homogeneous
apo-(riboflavin-binding protein) was used as a standard
in each set of analyses. It was observed that the
concentration of the binding protein based on the
specific radioactivity of the bound [14C]riboflavin
underestimated the actual protein concentration by 40 %.
Fluorescence assay for riboflavin
Because virtually all the flavin in egg and plasma from
laying hens is riboflavin bound non-covalently to
riboflavin-binding protein, there was no need to
acid-hydrolyse these samples before analysis. The
following procedure was adapted from that described by
Koziol (1971). Plasma, albumen and yolk samples were
thawed and diluted 10-, 20- and 40-fold (w/v) respectively
with 25 mM-Tris/HCl, pH 7.5. A 2 ml portion of diluted
sample was mixed with an equal volume of 20% (w/v)
trichloroacetic acid. After 10 min, the precipitated
protein was removed by centrifugation. To 3.0 ml of the
supernatant were added 0.75 ml of 4 M-KH2PO4 and the
fluorescence was measured at 530 nm with excitation at
450 nm in a Perkin-Elmer 3000 fluorimeter. All transfers
and incubations were conducted in the dark or in
subdued light.
Riboflavin was recrystallized twice from acetic acid
and its concentration in stock solutions was determined
spectrophotometrically at 260 nm, 375 nm and 450 nm
(Yagi, 1962). Internal and external standards were used
to determine riboflavin concentrations in the samples.
Light-scattering caused by turbidity was a problem in
albumen samples. The light-scattering contribution to
the measurements on these samples was determined by
re-analysing the samples after photolysing the riboflavin
1986

Deposition of riboflavin in eggs

673

Table 2. Egg production and riboflavin content of plama, yolk and albumen from hens fed different amounts of riboflavin

Treated

Supplemental riboflavin

(mg/kg)...

10

40

Control

Egg production (% hen day)

Pretreatment week
90.0 + 6.9*
Week 3
75, 85t
32
81
74
76
80
83
Riboflavin content of:
Plasma (sg/ml)
Pretreatment
0.88 + 0.04*
Day 7
0.18
0.38
0.57
0.70
0.80
0.87
Day 14
0.04
0.17
0.37
0.42
0.68
0.53
Day 21
0.04
0.18
0.31
0.44
0.70
0.89
0.87, 0.75t
Yolk (jug/g)
Pretreatment
4.94+0.31*
Day 7
2.38
3.38
1.61
3.77
4.61
4.56
Day 14
0.85
1.64
2.66
3.74
4.72
4.64
Day 21
0.77
1.18
2.28
2.90
4.74
4.51
5.08,. 5.62t
Albumen (,ug/g)$
Pretreatment
3.90+0.16
2.47
Day 7
0.58
1.54
2.75
3.47
3.47
Day 14
0.49
0.96
2.05
3.37
4.07
4.42
Day 21
0.58
0.68
1.59
2.55
3.98
4.22
4.05, 3.96
* Mean + S.D. for the six groups immediately before the treatment period.
t Values for two groups of hens maintained on the control diet throughout the experimental period.
t All values are corrected for light-scattering.
'% Hen * day' is a convenient unit used in poultry research to express the rate of egg laying for a group of hens. Its value is given
by the equation: % hen -day = a/bc x 100, where a is the number of eggs laid by b hens in c days.

in sunlight for 1 h. Destruction of riboflavin is complete

under these conditions.
Riboflavin in the standard and riboflavin-deficient
diets was determined as described above, but after acid
hydrolysis (Koziol, 1971). There was a significant
amount of fluorescent material in the riboflavin-deficient
feed samples that was not destroyed by photolysis. The
portion that was destroyed by sunlight (- 40%) was
considered to be riboflavin. Because other fluorescent
compounds may also be destroyed, the riboflavin
estimate for the riboflavin-deficient sample is an upper
limit.
RESULTS
Riboflavin content of plasma, yolk and albumen
The concentration of riboflavin in plasma, yolk and
albumen from laying hens displays saturation behaviour
in relation to the amount of riboflavin in the diet (Table
2). Half-maximal deposition in yolk and albumen occurs
on diets containing a total of about 3 mg of riboflavin/kg
(the riboflavin-deficient diet supplemented with 2 mg of
riboflavin/kg). There is little, if any, additional deposition
of riboflavin when the dietary riboflavin is increased
from 10 to 40 mg/kg.
The transfer of hens from the control diet to diets
containing 3 mg of supplemental riboflavin/kg or less
caused a marked decrease in the riboflavin content of
plasma, yolk and albumen. It would appear from the
kinetics of these changes that internal sources of
riboflavin turn over and equilibrate with dietary
riboflavin within 2 weeks.
Vol. 238

Egg production by the riboflavin-deficient- group was

drastically decreased, because several hens ceased laying
and those that continued to lay produced fewer eggs
(Table 2). Analyses from this group were made on eight
eggs at day 14 and six eggs at day 21, whereas a
minimum of ten, but usually 11 or 12 eggs, were
represented in the samples from the other groups.
Because plasma samples were taken from all birds in a
group regardless of their laying status and pooled, the
riboflavin-deficient plasma samples come from a heterogeneous population and are not directly comparable with
corresponding plasma samples from the other groups.
Riboflavin-binding-protein content of plasma, yolk and
albumen
The concentration of riboflavin-binding protein in
plasma, yolk and albumen is independent of dietary
riboflavin (Table 3). The values for samples taken on
successive weeks were so similar that they were averaged
by groups. The concentration of riboflavin-binding
protein in yolk is about six times greater than that in the
plasma from which it is derived (6.08 + 0.48 for all but the
riboflavin-deficient group). This value in turn is similar
to the factor by which riboflavin is concentrated in yolk
relative to plasma (6.75 + 1.35). In other words, the
concentration ratio is virtually the same for the vitamin
and its binding protein and independent of dietary

riboflavin.

Thepercentagesaturationofriboflavin-bindingprotein,

determined by the graphical method of Lotter et al.

(1982) and tabulated in Table 3 or by comparing the
concentrations of riboflavin in Table 2 with the

H. B. White III, J. Armstrong and C. C. Whitehead

674

Table 3. Concentration and saturation of riboflavin-binding proteins in the plasma, yolk and albumen from hens fed different amounts
of riboflavin
Treated

Supplemental riboflavin

(mg/kg) ...

10

40

Control

[Riboflavin-binding protein]* in:

Plasma (mg/l)
Pretreatment (n = 6)
64+7
64+14
59+19
Treatment
91, 92t
67+12
85+27
65+9
-:
Yolk (mg/kg)
Pretreatment (n = 6)
568+ 119
390+35
407+24
504+ 118 427+43
Treatment
314+41
405+ 15
501,515t
Albumen (mg/kg)
905+75
Pretreatment (n = 6)
757+33
820+148
685+19
811,800t
728+53
786+112 782+13
Treatment
Percentage saturation* in:
Plasma
109+28
_
Pretreatment
90+12
104+50
48+30
65+ 10
97,103t
23+ 15
Treatment
Yolk
78+40
_
_
_
Pretreatment
118+40
11 + 19
64+48
85+ 15
108+25
113,103t
31 +6
Treatment
Albumen
42+12
Pretreatment
21+26
30+12
21+17
17+11
40,37t
-16+16 -9+17
Treatment
* Values are means + S.D. for samples taken on day 0 for the pretreatment group and on days 7, 14 and 21 for the treatment groups;
1 mg of riboflavin-binding protein binds 12.5 ,g of riboflavin.
t Values at day 21 for two groups of hens maintained on the control diet throughout the experimental period.
t Values declined through the experimental period as more hens ceased to lay.
Saturation values were determined graphically. Negative values are 'physically impossible', but here provide an indication of the
error in the method.
-

riboflavin-binding capacity derived from the data in

Table 3, show that riboflavin-binding protein is saturated
with riboflavin only when dietary riboflavin is high
(> 10 mg/kg). There is little evidence for significant
amounts of unbound riboflavin in any of the samples.
DISCUSSION
Riboflavin deposition in eggs
Our results for the deposition of riboflavin in the yolk
and albumen of chicken eggs in response to dietary
riboflavin agree with those of Stamberg et al. (1946) and
Petersen et al. (1947). At the time of their work, it was
not known that the transfer of riboflavin to the egg was
dependent upon riboflavin-binding protein. The data
presented here demonstrate that the amount of riboflavin
in an egg is limited by the amount of riboflavin-binding
protein and that, even at high riboflavin intake, little, if
any, unbound riboflavin appears in the egg.
Whereas the deposition of riboflavin in yolk involves
the direct transfer of the vitamin-protein complex from
plasma, the deposition of riboflavin in albumen is not
understood. Chickens are atypical birds in that more
than half of the riboflavin in their eggs is in the albumen
(Feeney & Allison, 1969). As a consequence, their
albumin has a definite yellow colour compared with the
colourless albumen of most other birds. The fact that all
bird eggs examined have riboflavin-binding protein in
their albumen (Feeney & Allison, 1969) that is
synthesized in the oviduct (Mandeles & Ducay, 1962),
implies that the oviduct in the chicken has the capacity
to accumulate riboflavin from the blood. It has been

assumed that this occurs by the uptake and lysosomal

destruction of plasma riboflavin-binding protein (Miller
et al., 1982a). This releases riboflavin to be bound by the
newly synthesized binding protein. The distinctive
post-translational glycosylations of plasma and albumenderived riboflavin-binding protein (Miller et al., 1982a;
Hamazume et al., 1984; Norioka et al., 1985) seem to
preclude a direct transfer from the plasma to the
albumen. Our observation that riboflavin-binding protein
in chicken albumen remains less than half-saturated
implies that it is the capacity of the plasma-to-oviduct
transfer process rather than the amount of binding
protein that limits the amount of riboflavin deposited in
albumen.
Deposition of riboflavin-binding protein in the egg
The results of Benore-Parsons et al. (1985), which
suggest that apo-(riboflavin-binding protein) is deposited
in yolk, have been confirmed. This indicates, that, if there
is a receptor for this protein on the oocyte plasma
membrane, it is unable to exclude the apoprotein.
Furthermore, the fact that the concentration ratio of
riboflavin between yolk and plasma is indistinguishable
from that of riboflavin-binding protein suggests there is
no discrimination between apo and holo forms during
yolk deposition. This is consistent with the physicochemical behaviour of phospho groups on riboflavinbinding protein.
Miller et al. (1982b) showed that removal of phospho
groupsfrom riboflavin-bindingproteindrastically reduced
the deposition of the 125I-labelled protein in oocytes. It
was presumed that a receptor on the oocyte plasma
1986

Deposition of riboflavin in eggs

membrane was recognizing these phospho groups and

that the conformation of the highly phosphorylated
region of the sequence (Fenselau et al., 1985) would
change upon binding riboflavin. However, the 31P-n.m.r.
spectra of apo- and holo-proteins were indistinguishable,
despite the presence of well-dispersed resonances (Miller
et al., 1984). Our observations imply that the conformational differences between apo- and holo-protein observed
with c.d.. and o.r.d. by Zak et al. (1972) also are not
recognized by a receptor, if such exists. Although these
results are contrary to our expectations, they may relate
to a more generalized and less discriminating process
associated with the endocytosis of yolk proteins.
The results observed for riboflavin and riboflavinbinding protein here are in contrast with those for biotin
and its two binding proteins in laying hens (White &
Whitehead, 1985). There only the saturated proteins are
found in plasma and yolk, and the production of the
proteins is in some way controlled by the availability of
biotin. Here the production and deposition of riboflavinbinding protein is unaffected by the availability of
riboflavin.
Dynamics of riboflavin-binding protein in plasma
There have been a number of reports on the half-life
of labelled riboflavin-binding protein in laying hens and
the effects of various modifications (Hammer et al., 1971;
Miller et al., 198 1a,b, 1982a,b). The observed values of 2 h
or less are in contrast with the 6-10 h for immature birds
without functioning ovaries (Murthy & Adiga, 1977). In
the studies with laying hens, only 10-12% of the injected
labelled protein was deposited in yolk. Thus the
observed half-lives may reflect atypical clearance of
damaged protein rather than typical metabolism of the
native protein.
If all riboflavin-binding protein in the plasma were
destined for the yolk with a half-life of 2 h, the total
amount of the circulating protein should be about 1 mg.
This is considerably less than the value of 4 mg that we
calculate in an estimated blood volume of 105 ml
(Newell & Shaffner, 1950). It seems apparent under
normal physiological conditions that either a relatively
small proportion of the circulating riboflavin-binding
protein is destined for the yolk or the plasma half-lives
are considerably longer than estimated with labelled
riboflavin-binding protein.
We thank Ms. Christine Murnin for her expert technical
assistance. H. B. W. was supported by National Institutes of
Health grants AM27873 and AM34445, and the AFRCUnderwood Fund during a sabbatical leave at the Poultry
Research Centre.

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Received 6 January 1986/9 May 1986; accepted 21 May 1986

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