WATERING SYSTEMS 435

ACKNOWLEDGEMENTS F tests. Biometrics, 1 1 : 1-42.

Sincere appreciation is extended to Dr.
T. R. C. Rokeby for providing assistance in
design of the environmental facilities. The
authors would like to thank Paragon Elec-
tric Co. for supplying the time clocks and
timers; and Fox Products Co., supplier of
the nipple waterer equipment. The assis-
tance of Mrs. Zelpha Johnson in the statis-
tical analysis is gratefully acknowledged.
REFERENCES
Duncan, D. B., 19SS. Multiple range and multiple
Eley, C. P., and E. Hoffman, 1949. Feed particle
size as a factor in water consumption and elimi-
nation. Poultry Sci. 28: 715-722.
Kellerup, S. U., J. E. Parker and G. H. Arscott,
1965. Effect of restricted water consumption on
broiler chicks. Poultry Sci. 44: 78-83.
Maynard, L., 1951. Animal Nutrition. Third Edi-
tion, McGraw-Hill Book Co., Inc.
Steel, R. G., and J. H. Torrie, 1960. Principles and
Procedures of Statistics. McGraw-Hill Book
Co., Inc.
Weiss, H. S., 1960. Measures feed wasted by chick-
ens when drinking. New Jersey Agriculture
March-April, pp. 12-13.

Egg White Catalase: 1. Catalatic Reaction1'2

H. R. BALL3, JR. AND O. J. COTTERILL
Department of Food Science and Nutrition, University of Missouri-Columbia,
Columbia, Missouri 65201

(Received for publication August 18, 1970)

L OEW (1901) briefly looked at egg

white (EW) as a source of catalase.
He found what he believed was a catalatic
reaction and reported EW as a poor source
of catalase. The egg was used as a model
system by Winternitz and Rogers (1910)
to study the relationship of catalase activ-
ity to embryonic development. They found
that catalase activity of EW increased as
embryonic growth occurred. Pennington
and Robertson (1912) confirmed the above
studies. In addition, they noted that the ac-
tivity in EW did not decrease as the shell
egg or frozen EW was stored.
1
Contribution from the Missouri Agricultural
Experiment Station. Journal Series Number 6051
Approved by the Director.
2
From a dissertation submitted by the senior
author to the Graduate Faculty of the Univer-
sity of Missouri-Columbia in partial fulfillment
of the requirements for the Ph.D. degree.
3
Present address: Department of Food Science,
North Carolina State University at Raleigh,
Raleigh, North Carolina 27607.
Lineweaver et al. (1948) found that the
catalatic activity of EW could be destroyed
by heating EW to 63°C. or by holding EW
at pH 3. They also noted large egg to egg
variation in catalatic activity and reported
activities ranging from 7 X 10~3 to 69 X
10-3 Kat.f. They estimated that 85% of
the egg catalase would be found in the EW.
Lloyd and Harriman (1957) claimed that
heating EW to 54°C. for three minutes was
sufficient to inactivate catalase so that bac-
tericidial concentrations of H 2 0 2 could be
added. Henderson and Robinson (1969)
also found that there was considerable vari-
ation in the catalase activity from egg to
egg. They found that the catalatic activity
was decreased by heating and reported an
activation enthalpy of 39.8 kcal. mole-1.
The activity was shown to be proportional
to the concentration of H 2 0 2 up to 3mg. H2
0 2 /ml. Above that concentration activity
decreased. Dialysis did not reduce the ac-
tivity but potassium cyanide did inhibit the
reaction.
436 H. R. BALL, JR. AND O. J. COTTERILL

The progress of the reaction of catalase studies conducted to characterize the cata-
with hydrogen peroxide (H 2 0 2 ) was de- latic activity of EW.
scribed by Maehly and Chance (1954).
Initially the reaction is very rapid and ap- MATERIALS AND METHODS
parently is first-order. Within a short pe- Materials. The egg white (EW) used in
riod of time (one minute) depending on H2 this study was prepared as described by
0 2 concentration, the rate of the reaction Cotterill (1968) from eggs produced by the
begins to decrease. Henderson and Robin- Department of Poultry Husbandry, Uni-
son (1969) found that the catalatic reac- versity of Missouri. Characteristics of the
tion of EW was first-order. EW were normal for fresh EW, i.e., pH
Since Chance (1951) was able to demon- 8.8, 11.37% solids, 9.95% protein, and a
strate a reaction mechanism for catalase normal electrophoretic pattern. The bacte-
involving an enzyme-substrate complex, it rial population of the EW was less than
would appear that the Michaelis-Menton 3000 per ml.
equation could be applied to characterize Yolk was prepared as required from day
the reaction in terms of Km and Vm. Albers old unwashed eggs. Yolks were separated
(1933) suggested a modified Michaelis from the EW, rolled on a damp cloth to re-
equation (1). The equation indicates move adhering EW, the membrane was
broken and the contents were collected.
v (S)2 Pooled yolk material was mixed by using a
(1) = L-i
VM KM+(S)2 magnetic stirrer and magnetic stirring bar.
Solutions of beef-liver catalase were pre-
that two moles of H 2 0 2 would have to be pared from lyophilized beef-liver catalase
involved in an enzyme-substrate complex. (Nutritional Biochemical Corporation).
Bonnichsen et al. (1947) showed that The solutions were prepared in pH 8.8, 0.1
earlier determinations of Km were incorrect M TRIS-HC1 buffer (8.5 ml. 0.1 M HC1
due to inhibition of catalase at high con- per50ml.0.1MTRIS).
centrations of H 2 0 2 . Ogura (1955) ob- All chemicals used were analytical reag-
tained the currently accepted Km of 1.1 M ent grade unless noted differently.
for catalase at pH 7, and 30°C. by deter- Catalase assay. The catalase assay used
mining reaction rates during the first half was similar to those described by Luck
second of reaction. (1963) and Scott and Hammer (1960a).
Early observations of the decomposition The procedure differed in that the EW or
of hydrogen peroxide by EW was taken as an enzyme preparation was allowed to re-
"a priori" evidence that catalase was a act with H 2 0 2 in a 16 X 95 mm. test tube
component of EW. More recent work has and that the residual H 2 0 2 was determined
indicated that the catalatic activity of EW in the same tube. Briefly, 0.5 ml. EW or
may not be the action of a true catalase. enzyme preparation was blown from a pi-
Work by Baker and Manwell (1962), Cor- pet into the substrate solution so that the
bin and Brush (1966) and Lush (1966) final concentration of H 2 0 2 was ca 0.16 M
support the latter point of view. Those in a reaction volume of 1.5 ml. Several
workers did little to characterize the nature tubes of substrate were prepared at one
of the reaction and did not offer a satisfac- time using freshly prepared H 2 0 2 solutions.
tory explanation of the source of the activ- Initial and final substrate concentrations
ity. were determined by titrating with 0.1N
This paper will present the results of Na 2 S 2 0 3 prepared and standardized as de-
 EGG WHITE CATALASE
scribed by Skoog and West (1963). A mi-
croburet was used to deliver the sodium
thiosulfate and a 13 mm. stirring bar was
used to agitate the solution. One drop of
1% ammonium molybdate was used as a
catalyst as described by Scott and Hammer
(1960a).
Reaction times used were dependent on
the data required. When initial reaction ve-
locities were wanted, reaction times of 5,
10, IS and 30 seconds were used. All other
times were 45 minutes unless noted differ-
ently. The results of the assays were ex-
pressed as initial velocity, percent H 2 0 2
consumed, or as percent activity. Percent
activity was the proportion of H 2 0 2 con-
sumed by treated EW relative to the H 2 0 2
consumed by native EW.
EXPERIMENTS AND RESULTS
The decomposition of hydrogen peroxide
by egg white has been observed for many
years, but only limited information exists
to describe the reaction. The experiments
presented below were designed to charac-
terize the reaction. Conditions were se-
lected to give results that would be indica-
te 1 1 1 1 1 r
Reaction Time (min)
FIG. 1. Progress of the catalatic reaction of
egg white.
437
Egg White (ml)
FIG. 2. Decomposition of hydrogen peroxide with
increasing proportions of egg white.
tive of the reaction of native egg white.
Progress of the catalatic reaction. The
decomposition of hydrogen peroxide by egg
white with time was determined by using
the assay procedure described above. Hy-
drogen peroxide was destroyed at a slowing
rate as time increased. Figure 1 shows the
progress of the reaction up to 60 minutes.
Approximately one third of the consump-
tion of hydrogen peroxide occurred in the
first minute and approximately a third of
the available substrate was decomposed in
60 minutes. The reaction appears to have a
linear rate over the first minute. However,
when the progress of the reaction was de-
termined at shorter intervals during the
first minute the results indicated that a lin-
ear rate occurs only during the initial few
seconds of the reaction.
Concentrations of react ants. To deter-
mine the effect of increasing amounts of
EW on the catalatic reaction, the volume of
EW added to the substrate was varied from
0.1 ml. to 1.5 ml. A linear relationship was
found between volume of egg white and
percent hydrogen peroxide consumed in a
45 minute reaction time as indicated in
Figure 2.
The results presented in Figure 1 and
Figure 2 indicate that the concentration of
hydrogen peroxide used in the assay proce-
438 H. R. BALL, JR. AND 0.
. (4)
FIG. 3. Catalatic activity of egg white with in-
creasing proportions of hydrogen peroxide.
dure was sufficient to saturate the catalatic
active component of egg white.
Increasing the proportion of H 2 0 2 also
resulted in an increase in activity as indi-
cated in Figure 3. The activity was propor-
tional up to about 30 mg. H 2 0 2 /ml. of EW.
At higher concentrations of H 2 0 2 the reac-
tion slowed.
Activity and kinetics. The equations
used to calculate the catalatic activity were
presented by Maehly and Chance (1954).
The older expression of activity, Katalase-
fahigkeit (Kat.f.) as shown in equation (2)
is less
00(50)
(2) Kat.f. = — ^ ml.g.-imin.- 1
0)
where,
k = first-order rate constant
50 = reaction volume by definition
co = dry weight of enzyme
acceptable in current literature. It does
have an advantage when the molar concen-
tration of the enzyme is not known. The re-
lationship of Kat.f. to the more acceptable
expression of activity, the over all rate con-
stant (ki) was also described by Maehly
J. COTTERILL
and Chance (1954) and is as shown in
equation (3) where mol W is the molecu-
(3) Kat.f. = k/(520/mol.W)M- 1 sec.- 1
lar weight of catalase (250,000) and 520 is
a conversion factor. The over-all rate con-
stant (ki) can be calculated as shown in
equation (4)
k/ = — M-isec.- 1
e
where k is the first-order rate constant and
e is the concentration of the enzyme.
Table 1 presents the various expressions
of activity calculated from the above equa-
tions. Also included in Table 1 are esti-
mates of the units (U) of catalatic active
component found in the EW used in this
study. The U being defined as the amount
of enzyme which will catalyze one mole of
substrate per minute. The differences be-
tween the two numbers probably represents
substrate inhibition of the catalatic active
component of EW.
The initial velocities of the catalatic re-
action were determined at various concen-
trations of H 2 0 2 . These data were plotted
as the double-reciprocal form of Albers'
(1933) equation (Figure 4). It appears
that his equation does describe the catala-
tic reaction of EW. The values of Vm and
Km were 1.8 X 10"3M sec.-1 and 0.2 8M,
respectively. The constant Vm was calcu-
TABLE 1.—Catalatic activity of egg while*
Expression of Activity
Enzyme Source
constant, ki '
(ml.gr1 min.-i) (M"1 sec."') (U ml.">)
Egg White 6.3 3X10' 5.44Xl(H30b
1.96X10
Beef-liver Catalase 1.28X10' 6.13X10*
a
Conditions: pH 8.8, temperature 27CC., initial concentra-
tionb of H202 0f 0.16M.
Calculated from initial velocity.
c
Calculated from H2O2 consumed in 45 minutes.
 EGG WHITE CATALASE
lated as usual. The constant Km was re-
ported as the square root of the reciprocal
of the intercept, — 1/Km.
pH. This experiment was designed to
give a pH-activity curve and to determine
the pH-stability of the active component.
The albumen used in this experiment was
titrated with 0.1N HC1 or 0.1N NaOH to
obtain the desired pH. All samples were
corrected to the same dilution by adding
distilled water. Portions of the pH adjusted
egg white were removed and their activities
were determined. The remaining EW was
held from one half to three hours at room
temperature. After those times, the pH was
adjusted back to pH 8.8 and activity of the
samples was determined. The activity after
exposures to various pH conditions was
used to indicate the pH-stability of the cat-
alatic activity component. No differences
were noted in the catalatic activity of sam-
ples held for the different time periods be-
5 T
0.5 ml Egg White
pH 8 . 8 , 27 C
o
<u
tn
n
i
o to 5.5.
X
FIG. 4. Double-reciprocal plot of the modified
Michaelis-Mention equation.
439
1 ~t i • i • I_
100
o
Activity (%)
00
1
Stability Curve
.
•.
1
O*
o
h- Activity Curve -
40
« I . I . 1
6 8 10
pH
Fie. 5. The pH-activity curve and pH-stability
curve for the catalatic activity of egg white.
fore adjusting back to pH 8.8, therefore
those results were combined. The pH-activ-
ity and pH-stability curves are presented in
Figure 5.
The catalatic activity of EW was ob-
served over a broad pH range with an opti-
mum near pH 8. The decrease in activity
below pH 7 and above pH 9 was probably
due to protein denaturation. This view is
supported by the pH-stability curve. Sam-
ples held at pH values close to the native
pH tend to retain their activity. Some
recovery of activity on adjusting to native
pH was noted for samples held at pH 4.0
Temperature of reaction. To determine
the effect of temperature on the catalatic re-
action of EW the assay procedure was car-
ried out in a thermoregulated water bath.
Bath temperature was adjusted and a test
tube rack containing tubes of substrate was
suspended in the water. The substrate me-
dia and EW was allowed to equilibrate to
the desired temperature. As soon as the re-
actants reached the desired temperature, the
EW was added and the reactions were al-
lowed to run their prescribed times. After
the reactions were stopped, the contents were
440 H. R. BALL, JR. AND O. J. COTTERILL

1—'—'—'—I—'—'—'—T"
40
H2O2 Consumed
30
20
10
10 30
Reaction Temperature (° C)
Fic. 6. Initial velocity and hydrogen peroxide
consumed at various reaction temperatures.
adjusted to room temperature and the resid-
ual hydrogen peroxide was determined. Both
initial velocity and over-all consumption of
hydrogen peroxide were determined. The
results are presented in Figure 6.
The optimum temperature by both ex-
pressions of activity was 20°C. The simi-
larity of the two curves despite the wide
differences in reaction times would indicate
that expressions of activity in one manner
would reflect the trend in activity ex-
pressed in the other form. These results
show the influence of temperature on the
reaction. Q10 for the reaction over the tem-
perature range of 10 to 20°C. was 1.1 and
1.5 when calculated from consumption of
hydrogen peroxide and initial velocities, re-
spectively. Energies of activation estimated
Activity
extra tube containing EW and a thermome-
ter was also included in the rack. The rack
of tubes was then submerged in a preheated
water bath. The thermometer was moni-
tored and when it registered 0.1 °C. below
the bath temperature a stopwatch was
started. At predetermined intervals of time
a set of three tubes were withdrawn and
plunged into an ice water bath and held
there for one minute. After cooling, the
contents of the three tubes were combined
in a large tube and mixed with a magnet
and magnetic stirrer.
After the heated and cooled EW equili-
brated with room temperature, its activity
was assayed. Figure 7 indicates that the ac-
tive component of egg white is relatively
heat stable up to S0°C. for 15 minutes. Ac-
tivity was lost as the temperature was in-
creased to 60°C. Heating at 55°C. for
seven minutes or at 60°C. for one minute
reduced catalatic activity 50%.
An estimate of the activation energy to
inactivate the catalatic activity (E a ) was
made from the above results. The time re-
quired to achieve 90% inactivation at the
1 ' 1 1—
""I ' 1 _
45* a
a"^
80 —0"
50"
£
60

from consumption of hydrogen peroxide •V -

and initial velocities from 5 to 20°C. were >^A
4.8 and 4.0 kcal./M, respectively. •40 \ ^ —
Heat stability. In order to establish the 55 •

time-temperature effect on the active com-

ponent of EW the following experiment 20 60'
was conducted. Two ml. portions of EW 1 • • 1 .
were placed in 8 X 12S mm. thin wall, 2 6 10 14
glass test tubes. Three tubes were prepared Time at Temperature (min)
for each exposure time required and all FIG. 7. Heat stability of the catalatic activity of
tubes were placed in a test tube rack. An egg white.

temperatures studied were obtained by
plotting the log of activity against time.
The time, known as D by bacteriologists,
was used in equation (5) presented by
Stumbo (1965), where k is
2.303
(5) k =
D
a first-order rate constant with units of re-
ciprocal minutes, D is time required to
achieve 90% reduction in activity at a
given temperature, and 2.303 is a constant.
The log of k was plotted against reciprocal
temperature in the form of the classical Ar-
rhenius plot as shown in Figure 8.
The activation energy, 61.3 kcal./M,
was calculated from the slope of the line.
The value obtained is in the range expected
for denaturation of proteins.
Catalase inhibitors. The effect of classi-
cal catalase inhibitors on the catalatic ac-
tivity of EW was studied. Small volumes of
1.0M NaCN and 1.0M NaN 3 were added
to 10 ml. of EW. After addition of the in-
hibitors, pH was adjusted back to 8.8 with
0.1N HC1. Dilutions were equalized by
adding water.
Small amounts of the inhibitors reduced
60 55 50 45
1 ' ' i | | i i i | i i i i | i
-0.4 E = 61.3 k c a l / m o l e
a
-0.8 - >v
-1.2 - >v
-1.6 - ^v.
-2.0
-2.4
-2.8
1 . ,
3.00 3.05 3.10
1 / ! K X 103
FIG. 8. Arrhenius plot for the heat inactivation of
the catalatic activity of egg white.
EGG WHITE CATALASE 441
uu
__ ! 1 I -
" 1
0.16MH 2 O 2
-
0.5 ml Egg White
80
"" \ \ pH 8.8, 27'C
"
. .NaCN
60 \ -
40 " NaN3
J
20 1 1 3 rr
1
5 10 15 20
li mole Inhibitor/ml of Egg White
FIG. 9. Inactivation of the catalatic activity of
egg white with classical catalase inhibitors.
the activity as shown in Figure 9. The ef-
fectiveness of added inhibitors decreased as
inhibitor concentrations increased above 10
[/.mole/ml. of EW. Catalase activity could
be completely inhibited by concentrations
of 20 mmole/ml. of EW.
Yolk contamination. Yolk may be a nat-
ural contaminate of EW. Various concen-
trations of liquid yolk were added to EW
to determine its effect on the catalatic reac-
tion. Liquid yolk was weighed into 50 ml.
beakers and EW was added to give a final
weight of 10 gm. The yolk contaminated
EW was stirred on a magnetic stirrer for 20
minutes. After mixing, the pH was adjusted
to 8.8 with 0.1N NaOH. Dilutions were
equalized by adding water.
_ Additions of yolk decreased the catalatic
reaction of EW as shown in Figure 10. Af-
~ ter correcting for the H 2 0 2 that reacted
with non-catalatic active proteins, the de-
crease in activity appeared to be essentially
linear up to 25% (wt./wt.) yolk.
\ Activity of dialyzed egg white. The pos-
^ - sibility of adventitious iron in EW causing
the decomposition of hydrogen peroxide
was studied. Native EW was dialyzed
3.15 against distilled water for 48 hours. Several
changes of water were made during that
time. The precipitated globulins were solu-
bilized by dialyzing against 0.85% (wt./
442 H. R. BALL, JR. AND 0. J. COTTERILL
i I i i 1 1
I
10 20 30
Yolk Added (%)
FIG. 10. Catalatic activity of egg white with
added yolk.
vol.) NaCl for 24 hours. The pH of the
dialyzed EW was adjusted to 8.8 and as-
sayed with a reaction time of 45 minutes.
Two other samples of native egg white,
0.2 mg. of iron (iron wire was dissolved in
H 2 S0 4 and diluted with distilled water to
give a final concentration of 1 mg./ml.)
was added to 10 ml. of EW. Before deter-
mining the activity, the pH was adjusted to
8.8.
Table 2 summarizes the results of the
above experiments. Equal volumes of dia-
lyzed EW had about a quarter of the activ-
ity of native EW and EW with added iron
had 20% more activity.
DISCUSSION
The term catalatic reaction is used to de-
scribe the reaction between EW and hydro-
gen peroxide. That term, as defined by Nic-
holls and Schonbaum (1963), involves the
catalyzed reaction between two molecules
of hydrogen peroxide to yield water and
oxygen. It is assumed that the gas evolved
from the vigorous reaction of EW and hy-
drogen peroxide is oxygen. The progress of
the reaction, involving the decomposition
of hydrogen peroxide, indicates that only
one of the reactants is a limiting factor.
The linear increase in decomposition of hy-
drogen peroxide as the proportion of EW
was increased indicates that the concentra-
tion of the active components of the EW
was the limiting factor. For enzymatic re-
actions, the rate of the reaction or the total
amount of substrate altered in a given time
are functions of enzyme concentrations
when substrate concentration is in excess.
Also, enzymes can be inhibited by their
substrate, their reaction products, or by ad-
u
ventitious materials. Catalase can be inhib-
ited by its substrate. Literature descrip-
tions of substrate inhibition and reaction
progress (Maehly and Chance, 1954) are
similar to the results observed in this
study.
Determinations of catalase activity of
EW yields Kat.f. and an over-all rate con-
stant, k / , that are low compared to iso-
lated beef-liver catalase. The activity is
easily observed but is considered low. The
small values for catalase activity are prob-
ably best interpreted as indicating that
small amounts are present in EW. Another
consideration would be that certain com-
ponents of EW may be acting as inhibitors.
The units per ml. of EW reflects the prog-
ress of the reaction as indicated in Figures
1 and 3.
Maehly and Chance (1954) suggest that
unknown concentrations of catalase can be
estimated if one knows that k / of a known
TABLE 2.—Activity of dialyzed egg white
and egg white with added iron
Egg White Preparation Solids (%) Activity (%)
Native egg white 11.37 100.0
Native egg white+iron* 11.37 120.0
Dialyzed egg white 5.83 23.5
* 20 jug./ml. of egg white added to give approx-
imately 5 times iron content of native egg white
(Everson et al., 1957).
 EGG WHITE CATALASE
concentration of catalase and the first-
order rate constant of the unknown enzyme
system. Lineweaver et al. (1948) used a
similar approach to arrive at an estimate
that catalase was 10~4% of the EW solids.
Assuming that the catalatic active sub-
stance of EW is a catalase and using the
approach of Maehly and Chance (1954),
the catalase concentration of EW was
estimated to be 1.95 X 10"11 mole/ml. of
EW. Using Lineweaver's et al. (1948)
figure one can calculate that their EW was
5 X 10-13 mole/ml. of EW.
The difference in the estimates reflect on
the method of measuring the activity of EW
since the assumptions were the same. Line-
weaver et al. (1948) used a manometric
method and followed the progress of the re-
action over a 15 minute period of time. The
long reaction time would allow substrate
inhibition to effect the observed rate con-
stants. Also it is generally accepted that
manometric techniques yield consistantly
lower measured activities for catalase (Nic-
holls and Schonbaum, 1963). His estimate
would necessarily be lower than those made
in this study, since the assay procedure
used is expected to yield data less effected
by substrate inhibition. They reported a
Kat.f. for fresh EW of 0.058 as compared
to 6.3 determined in this study. Regardless
of the differences in the estimates, Line-
weaver et al. (1948) and this study have
confirmed earlier literature reporting EW
as a poor source of catalase.
The increased rate of reaction as sub-
strate concentration was increased, is typi-
cal of enzymatic reactions. Finding that
the kinetics of the activity of EW were de-
scribed by Albers' (1933) equation (1) is
significant. Since the equation was derived
for catalase activity determined by meth-
ods similar to the assay procedure used in
this study, it is indicative that the activity
of EW is due to a catalase. The Km and
Vm determined for EW are low for cat-
443
alases in general. However, determinations
of these values with crude enzyme prepara-
tions are not expected to yield the best
values. In addition, Ogura's (1955) work
indicates that best estimates of Km of
catalase should be made at very short re-
action times.
The influence of pH on the catalatic re-
action of EW gives some information about
the nature of the reaction between the sub-
strate and the active component. The shape
of the pH-activity curve and the observed
optimum (pH 8) are similar to activity
curves and optimums of catalases from
other sources. The pH-stability curve
shows failure of the EW samples to fully
recover their activity after bringing pH
back to 8.8, indicating that the reaction
was not ionic. Those results also indicate
pH induced alterations of the catalatic ac-
tive component. Studies with other cata-
lases have shown that pH does not effect
the ability of the enzyme to complex with
its substrate, but that pH effects the protein
moiety (Ogura, 1955).
Low pH environments could cause acid
cleavage of the hematin groups of catalase.
The slight recovery of activity when pH
was raised to 8.8 indicates that hydrolysis
of hematin groups would not account for
the loss of activity by exposure to low pH.
Precipitating EW proteins at pH 4 through
6 could have interfered with enzyme-sub-
strate complexing. The active component in
EW may have been trapped or coated by
insoluble proteins.
The influence of temperature on the
catalatic reactions of EW was similar to
effects of temperature on other catalases.
The values of Q10 and E a for the catalatic
reaction of EW are in close agreement with
values published by Maehly and Chance
(1954). The differences between the Q10's
(1.1 and 1.5) and Ea's (4.8 and 4.0 kcal./
M) estimated for the catalatic reaction of
EW indicate the effect of substrate inhibi-
444 H. R. BALL, JR. AND 0. J. COTTEEILL
tion. Reaction times used in determining
initial velocities would minimize substrate
inhibition. Therefore, calculations made
with initial velocities would give a higher
Qio and a lower E a .
When the catalatic reaction of EW was
carried out at temperatures above 20°C,
the active component appeared to be more
heat labile than indicated by the calculated
energy of activation for inactivation, 61.3
kcal./M. Scott and Hammer (1960b) ob-
served this in their studies with Aspergillus
and beef-liver catalases. Their interpreta-
tion of this effect was that the Q10 for the
inactivation of catalase by hydrogen per-
oxide was greater than the Q10 for the de-
struction of hydrogen peroxide by catalase.
It appears that their explanation would fit
the results observed in this study.
Catalatic activity of EW was sensitive to
heat when held at higher temperatures be-
fore assaying its activity. The trend of the
results obtained agree with those reported
by Henderson and Robinson (1969). The
activity observed in this study appeared to
be more sensitive to heat. The estimated
activation energy for inactivation, 61.3
kcal./M, was intermediate to values of 45
and 88 kcal./M reported by Sizer (1943)
and Feinstein et al. (1967) for beef-liver
and guinea pig-blood catalase respectively.
This thermodynamic data supports the
view held by Henderson and Robinson
(1969) that a natural undenatured protein
is required for the catalatic activity of EW.
The apparent deviation between the re-
sults reported in this study and the claims
of Lloyd and Harriman (1957) may be ex-
plained by differences in the amounts of
hydrogen peroxide used. Their results indi-
cated that a temperature as low as 49°C.
for one to five minutes would inhibit the
catalatic activity of EW. However they
were interested only in adding hydrogen
peroxide to give final concentrations of
0.075 to 0.3%. The hydrogen peroxide
used in this study was 1.5% and was five
times the largest amount that they added
to egg white. Reduced concentrations of
hydrogen peroxide would reduce the activ-
ity as indicated in Figure 3. In addition the
more severe effect of temperature during
the reaction could result in reduced activity
since they held EW at 49 °C. for two to five
minutes after addition of hydrogen perox-
ide.
The effect of classical catalase inhibitors
on the catalatic activity of EW strongly in-
dicates that the activity is due to a cata-
lase. Although the hematin groups of cata-
lase are believed to react independently of
each other, the lines in Figure 9 suggest
that not all of the hematins were available.
Considering the pH of HCN (8.68) and
the pH (S.8) of the EW, most of the cyan-
ide was probably in the form of hydrogen
cyanide. The amount of HCN required to
achieve 50% inhibition would be 5.7 //.mole
per ml. of EW. That amount of HCN
would indicate approximately 3 ^mole of
catalase per ml. of EW assuming that the
catalase had four hematins per mole and
that all of the hematins had to complex
HCN for complete inactivation. Experience
obtained in estimating activity of beef-liver
catalase makes it unlikely that EW carries
that much catalase. Similar calculations
with the amount of azide indicates a lower
concentration of catalase of 1.4 X 10~9
mole/ml. of EW. That value also seems
high. Other components are probably re-
ducing the effective concentration of the in-
hibitors.
Retention of activity after dialysis and
the activity of EW with added iron indi-
cates that the catalatic active component of
EW is a large molecule. Since additions of
iron, to a level approximately five times
that expected in native EW, only increased
the activity by 20%, it is unlikely that the
normal iron content would account for any
significant activity.
 EGG WHITE CATALASE
Additions of yolk to EW reduced its ac-
tivity at a rate that appeared to be greater
than a simple dilution effect. During stud-
ies with catalase inhibitors it was noted
that approximately 3 % of the hydrogen
peroxide would be consumed by albumen
proteins of EW inactivated by cyanide. On
the basis of solids content, every 0.055 g. of
EW solids would consume 3 % of the avail-
able hydrogen peroxide. Assuming that
yolk solids would utilize about the same
amount of hydrogen peroxide, correction
factors can be calculated that would indi-
cate that yolk reduced the activity of EW
essentially as a dilution effect. Also, it
would be expected that, if a component of
yolk interfered with active component, the
activity would decrease rapidly because of
the higher solids content of yolk. Because
of the color of yolk, determinations of re-
sidual hydrogen peroxide were increasingly
difficult as the proportions of yolk in-
creased. However, it appears that yolk con-
tributes very little to the activity.
The catalatic activity of EW could be
used to indicate efficiency of pasteurization
by the Cunningham-Lineweaver method
(Cunningham and Lineweaver, 1965). The
smaller reductions of activity at lower tem-
peratures and the inclusion of H 2 0 2 in the
EW would preclude the use of the catalatic
activity to test the efficiency of other EW
pasteurization processes. The recommen-
dations of Henderson and Robinson
(1969) to run assays of activity before and
after pasteurization should be followed to
compensate for variations in the catalatic
activity found in EW.
SUMMARY
The catalatic reaction of EW was found
to proceed as the reactions of other cata-
lases. An initial rapid phase followed by a
slowing rate of reaction was observed. Vari-
ations of EW and substrate concentrations
445
produced results expected of enzyme reac-
tions.
The activity, expressed in terms used to
express activity of catalases, was 6.3 Kat.f.
or 3 X 103 M^sec- 1 at pH 8.8, 27°C.
and at an initial concentration of 0.16 M of
H 2 0 2 . The activity of the reaction was de-
scribed by a modified Michaelis-Menton
equation.
The optimum temperature for the reac-
tion was 20°C. The active component of
EW was labile to heat inactivation at tem-
peratures above 50°C. Heating EW at 55°
C. for seven minutes or at 60°C. for one
minute reduced the catalatic activity by
50%. Activity was present over a broad pH
range with an optimum near pH 8. Small
concentrations of NaCN and NaN 3 inhib-
ited the reaction. Additions of yolk to EW
reduced the activity of EW as a dilution
effect.
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Egg White Catalase: 2. Active Component1'2

H. R. BALL, 3 JR. AND O. J. COTTERILL
Department of Food Science and Nutrition, University of Missouri-Columbia,
Columbia, Missouri 65201

(Received for publication August 18, 1970)

T HERE is current interest in using egg

white (EW) enzymes to test the effi-
ciency of EW pasteurization (Henderson
1970). Catalase or the catalase like activity
of EW has been considered for such use.
Earlier workers (Loew, 1901; Winternitz
and Robinson, 1969; and Donovan et al., and Rogers, 1910; Pennington and Robert-
1
son, 1912; Lineweaver et al., 1948; and
Contribution from the Missouri Agricultural
Experiment Station. Journal Series Number 6052
Lloyd and Harriman, 1957) assumed that
Approved by the Director. catalase was a component of EW. Baker
2
From a dissertation submitted by the sen'or and Manwell (1962) have raised doubt
author to the Graduate Faculty of the University of about its being present in EW. They ob-
Missouri-Columbia in partial fulfillment of the re- served results indicating catalase activity
quirements for the Ph.D. degree.
3
Present address: Department of Food Science,
after separating EW proteins by starch gel
North Carolina State University at Raleigh, Raleigh, electrophoresis. The activity, however, was
North Carolina 27607. located on regions of electrophoretograms