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Egg White Catalase: 1. Catalatic Reaction1,2
WATERING SYSTEMS ACKNOWLEDGEMENTS
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 Electric Co. for supplying the time clocks and timers; and Fox Products Co., supplier of the nipple waterer equipment. The assistance of Mrs. Zelpha Johnson in the statistical analysis is gratefully acknowledged. REFERENCES
F tests. Biometrics, 1 1 : 1-42. Eley, C. P., and E. Hoffman, 1949. Feed particle size as a factor in water consumption and elimination. 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 Edition, 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 chickens 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 activity 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 activity 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 University 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 bactericidial concentrations of H 2 0 2 could be added. Henderson and Robinson (1969) also found that there was considerable variation 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 activity but potassium cyanide did inhibit the reaction.
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Duncan, D. B., 19SS. Multiple range and multiple
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H. R. BALL, JR. AND O. J. COTTERILL
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The progress of the reaction of catalase studies conducted to characterize the catawith hydrogen peroxide (H 2 0 2 ) was de- latic activity of EW. scribed by Maehly and Chance (1954). MATERIALS AND METHODS Initially the reaction is very rapid and apparently is first-order. Within a short peMaterials. 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, Unison (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 bacteinvolving 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 reequation (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 2 magnetic stirrer and magnetic stirring bar. VM KM+(S) Solutions of beef-liver catalase were prethat 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) obAll chemicals used were analytical reagtained the currently accepted Km of 1.1 M ent grade unless noted differently. for catalase at pH 7, and 30°C. by deterCatalase 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 piWork 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 were determined by titrating with 0.1N ity. This paper will present the results of Na 2 S 2 0 3 prepared and standardized as de-
EGG WHITE CATALASE
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 characterize the reaction. Conditions were selected to give results that would be indicate
1
1
1
1
1
r
Reaction Time (min) FIG. 1. Progress of the catalatic reaction of egg white.
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. Hydrogen 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 consumption 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 determined at shorter intervals during the first minute the results indicated that a linear rate occurs only during the initial few seconds of the reaction. Concentrations of react ants. To determine 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-
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scribed by Skoog and West (1963). A microburet 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 velocities were wanted, reaction times of 5, 10, IS and 30 seconds were used. All other times were 45 minutes unless noted differently. The results of the assays were expressed as initial velocity, percent H 2 0 2 consumed, or as percent activity. Percent activity was the proportion of H 2 0 2 consumed by treated EW relative to the H 2 0 2 consumed by native EW.
437
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H.
R.
BALL, JR. AND 0.
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 constant (ki) can be calculated as shown in equation (4) .
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 indicated in Figure 3. The activity was proportional up to about 30 mg. H 2 0 2 /ml. of EW. At higher concentrations of H 2 0 2 the reaction slowed. Activity and kinetics. The equations used to calculate the catalatic activity were presented by Maehly and Chance (1954). The older expression of activity, Katalasefahigkeit (Kat.f.) as shown in equation (2) is less
00(50)
(2)
Kat.f. = — ^ ml.g.-imin.- 1
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 equations. Also included in Table 1 are estimates 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 between the two numbers probably represents substrate inhibition of the catalatic active component of EW. The initial velocities of the catalatic reaction were determined at various concentrations 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 catalatic 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-
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 concentration of the enzyme is not known. The relationship of Kat.f. to the more acceptable expression of activity, the over all rate constant (ki) was also described by Maehly
TABLE 1.—Catalatic activity of egg while* Expression of Activity Enzyme Source constant, ki Egg White Beef-liver Catalase a
(ml.gr1 min.-i) (M"1 sec."') 6.3 3X10' 1.28X10'
' (U ml.">) 5.44Xl(H30b 1.96X10
6.13X10*
Conditions: pH 8.8, temperature 27CC., initial concentrationb of H202 0f 0.16M. Calculated from initial velocity. c Calculated from H2O2 consumed in 45 minutes.
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FIG. 3. Catalatic activity of egg white with increasing proportions of hydrogen peroxide.
k/ = — M-isec.- 1 e
(4)
EGG WHITE CATALASE
5
T
0.5 ml Egg White pH 8 . 8 , 27 C
o
n i
o X
FIG. 4. Double-reciprocal plot of the modified Michaelis-Mention equation.
~t
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Stability Curve
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Activity (%)
100
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-
Activity Curve
I 6
.
I
. 8
1 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-activity and pH-stability curves are presented in Figure 5. The catalatic activity of EW was observed over a broad pH range with an optimum 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. Samples 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 to 5.5. Temperature of reaction. To determine the effect of temperature on the catalatic reaction of EW the assay procedure was carried 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 media and EW was allowed to equilibrate to the desired temperature. As soon as the reactants reached the desired temperature, the EW was added and the reactions were allowed to run their prescribed times. After the reactions were stopped, the contents were
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lated as usual. The constant Km was reported 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 catalatic activity component. No differences were noted in the catalatic activity of samples held for the different time periods be-
439
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H. R. BALL, JR. AND O. J. COTTERILL
40
H2O2 Consumed
30
20
10
30
Reaction Temperature (° C)
Fic. 6. Initial velocity and hydrogen peroxide consumed at various reaction temperatures.
adjusted to room temperature and the residual 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 expressions of activity was 20°C. The similarity 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 expressed in the other form. These results show the influence of temperature on the reaction. Q10 for the reaction over the temperature range of 10 to 20°C. was 1.1 and 1.5 when calculated from consumption of hydrogen peroxide and initial velocities, respectively. Energies of activation estimated from consumption of hydrogen peroxide and initial velocities from 5 to 20°C. were 4.8 and 4.0 kcal./M, respectively. Heat stability. In order to establish the time-temperature effect on the active component of EW the following experiment was conducted. Two ml. portions of EW were placed in 8 X 12S mm. thin wall, glass test tubes. Three tubes were prepared for each exposure time required and all tubes were placed in a test tube rack. An
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Time at Temperature (min) FIG.
7. Heat stability of the catalatic activity of egg white.
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10
extra tube containing EW and a thermometer was also included in the rack. The rack of tubes was then submerged in a preheated water bath. The thermometer was monitored 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 equilibrated with room temperature, its activity was assayed. Figure 7 indicates that the active component of egg white is relatively heat stable up to S0°C. for 15 minutes. Activity was lost as the temperature was increased 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 required to achieve 90% inactivation at the
Activity
1—'—'—'—I—'—'—'—T"
441
EGG WHITE CATALASE
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
80
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0.16MH 2 O 2 0.5 ml Egg White pH 8.8, 27'C
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60
2.303
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55 50 45 1 ' ' i | | i i i | i i i i | i
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E = 61.3 k c a l / m o l e a
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FIG. 8. Arrhenius plot for the heat inactivation of the catalatic activity of egg white.
40 "
NaN3 J
20
1
1
1 3
rr
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 effectiveness 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 natural contaminate of EW. Various concentrations of liquid yolk were added to EW to determine its effect on the catalatic reaction. 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. After correcting for the H 2 0 2 that reacted with non-catalatic active proteins, the decrease in activity appeared to be essentially linear up to 25% (wt./wt.) yolk. Activity of dialyzed egg white. The possibility of adventitious iron in EW causing the decomposition of hydrogen peroxide was studied. Native EW was dialyzed against distilled water for 48 hours. Several changes of water were made during that time. The precipitated globulins were solubilized by dialyzing against 0.85% (wt./
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a first-order rate constant with units of reciprocal 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 Arrhenius 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 classical catalase inhibitors on the catalatic activity 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 inhibitors, pH was adjusted back to 8.8 with 0.1N HC1. Dilutions were equalized by adding water. Small amounts of the inhibitors reduced
442
H. R. BALL, JR. AND 0. J. COTTERILL
i
I 10
i
i 20
1
1 30
u
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 assayed 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 determining the activity, the pH was adjusted to 8.8. Table 2 summarizes the results of the above experiments. Equal volumes of dialyzed EW had about a quarter of the activity of native EW and EW with added iron had 20% more activity. DISCUSSION The term catalatic reaction is used to describe the reaction between EW and hydrogen peroxide. That term, as defined by Nicholls 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 hydrogen peroxide is oxygen. The progress of
TABLE 2.—Activity of dialyzed egg white and egg white with added iron Egg White Preparation Native egg white Native egg white+iron* Dialyzed egg white
Solids (%) Activity (%) 11.37 11.37 5.83
100.0 120.0 23.5
* 20 jug./ml. of egg white added to give approximately 5 times iron content of native egg white (Everson et al., 1957).
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I
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 hydrogen peroxide as the proportion of EW was increased indicates that the concentration of the active components of the EW was the limiting factor. For enzymatic reactions, 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 adventitious materials. Catalase can be inhibited by its substrate. Literature descriptions 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 constant, k / , that are low compared to isolated beef-liver catalase. The activity is easily observed but is considered low. The small values for catalase activity are probably best interpreted as indicating that small amounts are present in EW. Another consideration would be that certain components of EW may be acting as inhibitors. The units per ml. of EW reflects the progress 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
EGG WHITE CATALASE
alases in general. However, determinations of these values with crude enzyme preparations 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 reaction times. The influence of pH on the catalatic reaction of EW gives some information about the nature of the reaction between the substrate 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 active component. Studies with other catalases 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-substrate 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-
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concentration of catalase and the firstorder 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 substance 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. Lineweaver et al. (1948) used a manometric method and followed the progress of the reaction over a 15 minute period of time. The long reaction time would allow substrate inhibition to effect the observed rate constants. Also it is generally accepted that manometric techniques yield consistantly lower measured activities for catalase (Nicholls 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, Lineweaver et al. (1948) and this study have confirmed earlier literature reporting EW as a poor source of catalase. The increased rate of reaction as substrate concentration was increased, is typical of enzymatic reactions. Finding that the kinetics of the activity of EW were described by Albers' (1933) equation (1) is significant. Since the equation was derived for catalase activity determined by methods 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
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H. R. BALL, JR. AND 0. J. COTTEEILL
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 activity 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 peroxide. The effect of classical catalase inhibitors on the catalatic activity of EW strongly indicates that the activity is due to a catalase. Although the hematin groups of catalase 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 cyanide 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 reducing the effective concentration of the inhibitors. Retention of activity after dialysis and the activity of EW with added iron indicates 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.
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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) observed this in their studies with Aspergillus and beef-liver catalases. Their interpretation of this effect was that the Q10 for the inactivation of catalase by hydrogen peroxide was greater than the Q10 for the destruction 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 before 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 results reported in this study and the claims of Lloyd and Harriman (1957) may be explained by differences in the amounts of hydrogen peroxide used. Their results indicated 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
EGG WHITE CATALASE
SUMMARY
The catalatic reaction of EW was found to proceed as the reactions of other catalases. An initial rapid phase followed by a slowing rate of reaction was observed. Variations of EW and substrate concentrations
produced results expected of enzyme reactions. 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 described by a modified Michaelis-Menton equation. The optimum temperature for the reaction was 20°C. The active component of EW was labile to heat inactivation at temperatures 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 inhibited the reaction. Additions of yolk to EW reduced the activity of EW as a dilution effect. REFERENCES Albers, H., 1933. 2. Physiol. Chem. 218, 113. Quoted by P. Nicholls and G. R. Schonbaum, 1963. Catalase. In The Enzymes, Vol. 8, Part B, eds. P. O. Boyer, H. Lardy and D. Myrback, Academic Press, New York, pp. 147-225. Baker, C. M., and C. Manwell, 1962. Molecular genetics of avain proteins. I. The egg white proteins of the domestic fowl. British Poultry Sci. 3 : 161-174. Bonnichsen, R. K., B. Chance and H. Theorell, 1947. Catalase activity. Acta Chem. Scand. 1: 685-709. Chance, B., 1951. Enzyme-substrate compounds. In Advances in Enzymology, Vol. XII, ed. E. F. Nord, Interscience Pub., New York, pp. 153190. Corbin, K. W., and A. H. Brush, 1966. Catalaselike activity of chicken egg white; A reconsideration. Anal. Biochem. 14: 95-99. Cotterill, O. J., 1968. Equivalent pasteurization temperatures to kill salmonellae in liquid egg white at various pH levels. Poultry Sci. 47: 354-365. Cunningham, F. E., and H. Lineweaver, 1965. Stabilization of egg-white proteins to pasteurization temperatures above 60° C. Food Technol. 19: 136-141.
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Additions of yolk to EW reduced its activity at a rate that appeared to be greater than a simple dilution effect. During studies 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 available hydrogen peroxide. Assuming that yolk solids would utilize about the same amount of hydrogen peroxide, correction factors can be calculated that would indicate 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 residual hydrogen peroxide were increasingly difficult as the proportions of yolk increased. However, it appears that yolk contributes 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 temperatures 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 recommendations 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.
445
446
H. R. BALL, JR. AND O. J. COTTERILL In The Enzymes, Vol. 8, Part B, eds. P. O. Boyer, H. Lardy and D. Myrback, Academic Press, New York, pp. 147-225. Ogura, Y., 1955. Catalase activity at high concentration of hydrogen peroxide. Arch. Biochem. Biophys. 57: 288-300. Pennington, M. E., and H. C. Robertson, Jr., 1912. A study of the enzymes of the egg of the common fowl. Circular No. 104. Bureau of Chemistry. U. S. Dept. Agri. Scott, D., and F. E. Hammer, 1960a. Assay of catalase for commercial use. Enzymologia, 22: 194-200. Scott, D., and F. E. Hammer, 1960b. Properties of Aspergillus Catalase. Enzymologia, 22: 229237. Sizer, I. W., 1943. Effects of temperature on enzyme kinetics. In Advances in Enzymology, eds. N. V. Nord and C. H. Werkman, pp. 3562. Interscience Pub., New York. Skoog, D. Q., and D. M. West, 1963. Fundamentals of Analytical Chemistry, Holt, Rinehart and Winston, New York, pp. 488-490. Stumbo, C. R., 1965. Thermobacteriology in Food Processing, Academic Press, New York, pp. 59. Winternitz, M. C , and W. B. Rogers, 1910. The catalytic activity of developing hen's egg. J. Exp. Med. 12: 12-18.
Egg White Catalase: 2. Active Component1'2 H. R. BALL, 3 JR. AND O. J. COTTERILL Department of Food Science and Nutrition, University of Columbia, Missouri 65201
Missouri-Columbia,
(Received for publication August 18, 1970)
T
HERE is current interest in using egg white (EW) enzymes to test the efficiency of EW pasteurization (Henderson and Robinson, 1969; and Donovan et al.,
1 Contribution from the Missouri Agricultural Experiment Station. Journal Series Number 6052 Approved by the Director. 2 From a dissertation submitted by the sen'or author to the Graduate Faculty of the University 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.
1970). Catalase or the catalase like activity of EW has been considered for such use. Earlier workers (Loew, 1901; Winternitz and Rogers, 1910; Pennington and Robertson, 1912; Lineweaver et al., 1948; and Lloyd and Harriman, 1957) assumed that catalase was a component of EW. Baker and Manwell (1962) have raised doubt about its being present in EW. They observed results indicating catalase activity after separating EW proteins by starch gel electrophoresis. The activity, however, was located on regions of electrophoretograms
Downloaded from http://ps.oxfordjournals.org/ at Tulane University on May 12, 2015
Everson, G. J., and H. Saunders, Jr., 1957. Composition and nutritive importance of eggs. J. Amer. Diet. Assoc. 33 : 1244-1254. Feinstein, R. N., G. A. Sacher, J. B. Howard and J. T. Braum, 1967. Comparative heat stability of blood catalase. Arch. Biochem. Biophys. 122 : 338-343. Henderson, A. E., and D. S. Robinson, 1969. Effects of heat pasteurization on some egg white enzymes. J. Sci. Fd. Agric. 20: 755-760. Lineweaver, H., H. J. Morris, L. Kline and R. S. Bean, 1948. Enzymes of fresh hen eggs. Arch. Biochem. 16: 443-472. Lloyd, W. E., and L. A. Harriman, 1957. Method of treating egg whites. U. S. Patent 2,776,214. Loew, O., 1901. Catalase, a new enzyme of general occurrence, with special reference to the tobacco plant. Report No. 68. U. S. Dept. Agri. Luck, H., 1963. Catalase. In Methods of Enzymatic Analysis, ed. N. U. Bergmeyer, Academic Press, New York, pp. 885-894. Lush, I. E., 1966. Discontinuous variation of catalase in the egg albumen of the Japanese quail. Life Sci. 5: 1537-1542. Maehly, A. C , and B. Chance, 1954. The assay catalases and peroxidases. In Methods of Biochemical Analysis, Vol. 1, ed. D. Glick, Interscience Pub., New York, pp. 357-424. Nicholls, P., and G. R. Schonbaum, 1963. Catalase.
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 Electric Co. for supplying the time clocks and timers; and Fox Products Co., supplier of the nipple waterer equipment. The assistance of Mrs. Zelpha Johnson in the statistical analysis is gratefully acknowledged. REFERENCES
F tests. Biometrics, 1 1 : 1-42. Eley, C. P., and E. Hoffman, 1949. Feed particle size as a factor in water consumption and elimination. 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 Edition, 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 chickens 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 activity 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 activity 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 University 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 bactericidial concentrations of H 2 0 2 could be added. Henderson and Robinson (1969) also found that there was considerable variation 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 activity but potassium cyanide did inhibit the reaction.
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Duncan, D. B., 19SS. Multiple range and multiple
435
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H. R. BALL, JR. AND O. J. COTTERILL
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The progress of the reaction of catalase studies conducted to characterize the catawith hydrogen peroxide (H 2 0 2 ) was de- latic activity of EW. scribed by Maehly and Chance (1954). MATERIALS AND METHODS Initially the reaction is very rapid and apparently is first-order. Within a short peMaterials. 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, Unison (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 bacteinvolving 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 reequation (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 2 magnetic stirrer and magnetic stirring bar. VM KM+(S) Solutions of beef-liver catalase were prethat 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) obAll chemicals used were analytical reagtained the currently accepted Km of 1.1 M ent grade unless noted differently. for catalase at pH 7, and 30°C. by deterCatalase 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 piWork 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 were determined by titrating with 0.1N ity. This paper will present the results of Na 2 S 2 0 3 prepared and standardized as de-
EGG WHITE CATALASE
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 characterize the reaction. Conditions were selected to give results that would be indicate
1
1
1
1
1
r
Reaction Time (min) FIG. 1. Progress of the catalatic reaction of egg white.
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. Hydrogen 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 consumption 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 determined at shorter intervals during the first minute the results indicated that a linear rate occurs only during the initial few seconds of the reaction. Concentrations of react ants. To determine 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-
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scribed by Skoog and West (1963). A microburet 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 velocities were wanted, reaction times of 5, 10, IS and 30 seconds were used. All other times were 45 minutes unless noted differently. The results of the assays were expressed as initial velocity, percent H 2 0 2 consumed, or as percent activity. Percent activity was the proportion of H 2 0 2 consumed by treated EW relative to the H 2 0 2 consumed by native EW.
437
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H.
R.
BALL, JR. AND 0.
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 constant (ki) can be calculated as shown in equation (4) .
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 indicated in Figure 3. The activity was proportional up to about 30 mg. H 2 0 2 /ml. of EW. At higher concentrations of H 2 0 2 the reaction slowed. Activity and kinetics. The equations used to calculate the catalatic activity were presented by Maehly and Chance (1954). The older expression of activity, Katalasefahigkeit (Kat.f.) as shown in equation (2) is less
00(50)
(2)
Kat.f. = — ^ ml.g.-imin.- 1
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 equations. Also included in Table 1 are estimates 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 between the two numbers probably represents substrate inhibition of the catalatic active component of EW. The initial velocities of the catalatic reaction were determined at various concentrations 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 catalatic 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-
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 concentration of the enzyme is not known. The relationship of Kat.f. to the more acceptable expression of activity, the over all rate constant (ki) was also described by Maehly
TABLE 1.—Catalatic activity of egg while* Expression of Activity Enzyme Source constant, ki Egg White Beef-liver Catalase a
(ml.gr1 min.-i) (M"1 sec."') 6.3 3X10' 1.28X10'
' (U ml.">) 5.44Xl(H30b 1.96X10
6.13X10*
Conditions: pH 8.8, temperature 27CC., initial concentrationb of H202 0f 0.16M. Calculated from initial velocity. c Calculated from H2O2 consumed in 45 minutes.
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FIG. 3. Catalatic activity of egg white with increasing proportions of hydrogen peroxide.
k/ = — M-isec.- 1 e
(4)
EGG WHITE CATALASE
5
T
0.5 ml Egg White pH 8 . 8 , 27 C
o
n i
o X
FIG. 4. Double-reciprocal plot of the modified Michaelis-Mention equation.
~t
1
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Stability Curve
.
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00
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o
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•. O*
Activity (%)
100
h40
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-
Activity Curve
I 6
.
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. 8
1 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-activity and pH-stability curves are presented in Figure 5. The catalatic activity of EW was observed over a broad pH range with an optimum 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. Samples 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 to 5.5. Temperature of reaction. To determine the effect of temperature on the catalatic reaction of EW the assay procedure was carried 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 media and EW was allowed to equilibrate to the desired temperature. As soon as the reactants reached the desired temperature, the EW was added and the reactions were allowed to run their prescribed times. After the reactions were stopped, the contents were
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lated as usual. The constant Km was reported 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 catalatic activity component. No differences were noted in the catalatic activity of samples held for the different time periods be-
439
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H. R. BALL, JR. AND O. J. COTTERILL
40
H2O2 Consumed
30
20
10
30
Reaction Temperature (° C)
Fic. 6. Initial velocity and hydrogen peroxide consumed at various reaction temperatures.
adjusted to room temperature and the residual 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 expressions of activity was 20°C. The similarity 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 expressed in the other form. These results show the influence of temperature on the reaction. Q10 for the reaction over the temperature range of 10 to 20°C. was 1.1 and 1.5 when calculated from consumption of hydrogen peroxide and initial velocities, respectively. Energies of activation estimated from consumption of hydrogen peroxide and initial velocities from 5 to 20°C. were 4.8 and 4.0 kcal./M, respectively. Heat stability. In order to establish the time-temperature effect on the active component of EW the following experiment was conducted. Two ml. portions of EW were placed in 8 X 12S mm. thin wall, glass test tubes. Three tubes were prepared for each exposure time required and all tubes were placed in a test tube rack. An
1
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.
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Time at Temperature (min) FIG.
7. Heat stability of the catalatic activity of egg white.
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10
extra tube containing EW and a thermometer was also included in the rack. The rack of tubes was then submerged in a preheated water bath. The thermometer was monitored 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 equilibrated with room temperature, its activity was assayed. Figure 7 indicates that the active component of egg white is relatively heat stable up to S0°C. for 15 minutes. Activity was lost as the temperature was increased 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 required to achieve 90% inactivation at the
Activity
1—'—'—'—I—'—'—'—T"
441
EGG WHITE CATALASE
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
80
__
1
!
I
-
"
1
0.16MH 2 O 2 0.5 ml Egg White pH 8.8, 27'C
"" \ \
60
2.303
k =
(5)
uu
.
\
.NaCN
" -
D
55 50 45 1 ' ' i | | i i i | i i i i | i
60
E = 61.3 k c a l / m o l e a
-0.4 -0.8
-
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-
-1.6
-
>v
~ >v
^v. \
-2.0
^
-2.4 -2.8
_
1 . , 3.00
3.05
3.10
-
3.15
1 / ! K X 103
FIG. 8. Arrhenius plot for the heat inactivation of the catalatic activity of egg white.
40 "
NaN3 J
20
1
1
1 3
rr
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 effectiveness 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 natural contaminate of EW. Various concentrations of liquid yolk were added to EW to determine its effect on the catalatic reaction. 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. After correcting for the H 2 0 2 that reacted with non-catalatic active proteins, the decrease in activity appeared to be essentially linear up to 25% (wt./wt.) yolk. Activity of dialyzed egg white. The possibility of adventitious iron in EW causing the decomposition of hydrogen peroxide was studied. Native EW was dialyzed against distilled water for 48 hours. Several changes of water were made during that time. The precipitated globulins were solubilized by dialyzing against 0.85% (wt./
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a first-order rate constant with units of reciprocal 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 Arrhenius 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 classical catalase inhibitors on the catalatic activity 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 inhibitors, pH was adjusted back to 8.8 with 0.1N HC1. Dilutions were equalized by adding water. Small amounts of the inhibitors reduced
442
H. R. BALL, JR. AND 0. J. COTTERILL
i
I 10
i
i 20
1
1 30
u
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 assayed 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 determining the activity, the pH was adjusted to 8.8. Table 2 summarizes the results of the above experiments. Equal volumes of dialyzed EW had about a quarter of the activity of native EW and EW with added iron had 20% more activity. DISCUSSION The term catalatic reaction is used to describe the reaction between EW and hydrogen peroxide. That term, as defined by Nicholls 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 hydrogen peroxide is oxygen. The progress of
TABLE 2.—Activity of dialyzed egg white and egg white with added iron Egg White Preparation Native egg white Native egg white+iron* Dialyzed egg white
Solids (%) Activity (%) 11.37 11.37 5.83
100.0 120.0 23.5
* 20 jug./ml. of egg white added to give approximately 5 times iron content of native egg white (Everson et al., 1957).
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I
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 hydrogen peroxide as the proportion of EW was increased indicates that the concentration of the active components of the EW was the limiting factor. For enzymatic reactions, 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 adventitious materials. Catalase can be inhibited by its substrate. Literature descriptions 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 constant, k / , that are low compared to isolated beef-liver catalase. The activity is easily observed but is considered low. The small values for catalase activity are probably best interpreted as indicating that small amounts are present in EW. Another consideration would be that certain components of EW may be acting as inhibitors. The units per ml. of EW reflects the progress 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
EGG WHITE CATALASE
alases in general. However, determinations of these values with crude enzyme preparations 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 reaction times. The influence of pH on the catalatic reaction of EW gives some information about the nature of the reaction between the substrate 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 active component. Studies with other catalases 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-substrate 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-
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concentration of catalase and the firstorder 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 substance 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. Lineweaver et al. (1948) used a manometric method and followed the progress of the reaction over a 15 minute period of time. The long reaction time would allow substrate inhibition to effect the observed rate constants. Also it is generally accepted that manometric techniques yield consistantly lower measured activities for catalase (Nicholls 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, Lineweaver et al. (1948) and this study have confirmed earlier literature reporting EW as a poor source of catalase. The increased rate of reaction as substrate concentration was increased, is typical of enzymatic reactions. Finding that the kinetics of the activity of EW were described by Albers' (1933) equation (1) is significant. Since the equation was derived for catalase activity determined by methods 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-
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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 activity 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 peroxide. The effect of classical catalase inhibitors on the catalatic activity of EW strongly indicates that the activity is due to a catalase. Although the hematin groups of catalase 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 cyanide 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 reducing the effective concentration of the inhibitors. Retention of activity after dialysis and the activity of EW with added iron indicates 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.
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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) observed this in their studies with Aspergillus and beef-liver catalases. Their interpretation of this effect was that the Q10 for the inactivation of catalase by hydrogen peroxide was greater than the Q10 for the destruction 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 before 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 results reported in this study and the claims of Lloyd and Harriman (1957) may be explained by differences in the amounts of hydrogen peroxide used. Their results indicated 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
EGG WHITE CATALASE
SUMMARY
The catalatic reaction of EW was found to proceed as the reactions of other catalases. An initial rapid phase followed by a slowing rate of reaction was observed. Variations of EW and substrate concentrations
produced results expected of enzyme reactions. 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 described by a modified Michaelis-Menton equation. The optimum temperature for the reaction was 20°C. The active component of EW was labile to heat inactivation at temperatures 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 inhibited the reaction. Additions of yolk to EW reduced the activity of EW as a dilution effect. REFERENCES Albers, H., 1933. 2. Physiol. Chem. 218, 113. Quoted by P. Nicholls and G. R. Schonbaum, 1963. Catalase. In The Enzymes, Vol. 8, Part B, eds. P. O. Boyer, H. Lardy and D. Myrback, Academic Press, New York, pp. 147-225. Baker, C. M., and C. Manwell, 1962. Molecular genetics of avain proteins. I. The egg white proteins of the domestic fowl. British Poultry Sci. 3 : 161-174. Bonnichsen, R. K., B. Chance and H. Theorell, 1947. Catalase activity. Acta Chem. Scand. 1: 685-709. Chance, B., 1951. Enzyme-substrate compounds. In Advances in Enzymology, Vol. XII, ed. E. F. Nord, Interscience Pub., New York, pp. 153190. Corbin, K. W., and A. H. Brush, 1966. Catalaselike activity of chicken egg white; A reconsideration. Anal. Biochem. 14: 95-99. Cotterill, O. J., 1968. Equivalent pasteurization temperatures to kill salmonellae in liquid egg white at various pH levels. Poultry Sci. 47: 354-365. Cunningham, F. E., and H. Lineweaver, 1965. Stabilization of egg-white proteins to pasteurization temperatures above 60° C. Food Technol. 19: 136-141.
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Additions of yolk to EW reduced its activity at a rate that appeared to be greater than a simple dilution effect. During studies 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 available hydrogen peroxide. Assuming that yolk solids would utilize about the same amount of hydrogen peroxide, correction factors can be calculated that would indicate 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 residual hydrogen peroxide were increasingly difficult as the proportions of yolk increased. However, it appears that yolk contributes 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 temperatures 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 recommendations 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.
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H. R. BALL, JR. AND O. J. COTTERILL In The Enzymes, Vol. 8, Part B, eds. P. O. Boyer, H. Lardy and D. Myrback, Academic Press, New York, pp. 147-225. Ogura, Y., 1955. Catalase activity at high concentration of hydrogen peroxide. Arch. Biochem. Biophys. 57: 288-300. Pennington, M. E., and H. C. Robertson, Jr., 1912. A study of the enzymes of the egg of the common fowl. Circular No. 104. Bureau of Chemistry. U. S. Dept. Agri. Scott, D., and F. E. Hammer, 1960a. Assay of catalase for commercial use. Enzymologia, 22: 194-200. Scott, D., and F. E. Hammer, 1960b. Properties of Aspergillus Catalase. Enzymologia, 22: 229237. Sizer, I. W., 1943. Effects of temperature on enzyme kinetics. In Advances in Enzymology, eds. N. V. Nord and C. H. Werkman, pp. 3562. Interscience Pub., New York. Skoog, D. Q., and D. M. West, 1963. Fundamentals of Analytical Chemistry, Holt, Rinehart and Winston, New York, pp. 488-490. Stumbo, C. R., 1965. Thermobacteriology in Food Processing, Academic Press, New York, pp. 59. Winternitz, M. C , and W. B. Rogers, 1910. The catalytic activity of developing hen's egg. J. Exp. Med. 12: 12-18.
Egg White Catalase: 2. Active Component1'2 H. R. BALL, 3 JR. AND O. J. COTTERILL Department of Food Science and Nutrition, University of Columbia, Missouri 65201
Missouri-Columbia,
(Received for publication August 18, 1970)
T
HERE is current interest in using egg white (EW) enzymes to test the efficiency of EW pasteurization (Henderson and Robinson, 1969; and Donovan et al.,
1 Contribution from the Missouri Agricultural Experiment Station. Journal Series Number 6052 Approved by the Director. 2 From a dissertation submitted by the sen'or author to the Graduate Faculty of the University 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.
1970). Catalase or the catalase like activity of EW has been considered for such use. Earlier workers (Loew, 1901; Winternitz and Rogers, 1910; Pennington and Robertson, 1912; Lineweaver et al., 1948; and Lloyd and Harriman, 1957) assumed that catalase was a component of EW. Baker and Manwell (1962) have raised doubt about its being present in EW. They observed results indicating catalase activity after separating EW proteins by starch gel electrophoresis. The activity, however, was located on regions of electrophoretograms
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Everson, G. J., and H. Saunders, Jr., 1957. Composition and nutritive importance of eggs. J. Amer. Diet. Assoc. 33 : 1244-1254. Feinstein, R. N., G. A. Sacher, J. B. Howard and J. T. Braum, 1967. Comparative heat stability of blood catalase. Arch. Biochem. Biophys. 122 : 338-343. Henderson, A. E., and D. S. Robinson, 1969. Effects of heat pasteurization on some egg white enzymes. J. Sci. Fd. Agric. 20: 755-760. Lineweaver, H., H. J. Morris, L. Kline and R. S. Bean, 1948. Enzymes of fresh hen eggs. Arch. Biochem. 16: 443-472. Lloyd, W. E., and L. A. Harriman, 1957. Method of treating egg whites. U. S. Patent 2,776,214. Loew, O., 1901. Catalase, a new enzyme of general occurrence, with special reference to the tobacco plant. Report No. 68. U. S. Dept. Agri. Luck, H., 1963. Catalase. In Methods of Enzymatic Analysis, ed. N. U. Bergmeyer, Academic Press, New York, pp. 885-894. Lush, I. E., 1966. Discontinuous variation of catalase in the egg albumen of the Japanese quail. Life Sci. 5: 1537-1542. Maehly, A. C , and B. Chance, 1954. The assay catalases and peroxidases. In Methods of Biochemical Analysis, Vol. 1, ed. D. Glick, Interscience Pub., New York, pp. 357-424. Nicholls, P., and G. R. Schonbaum, 1963. Catalase.