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Kinetics of thermal inactivation of avidin
Food Research International
25 (1992) 89-92
Kinetics of thermal inactivation of avidin T. D. Durance & N. S. Wong Department
of Food Science, University of British Columbia, 6650 NW Marine Drive, Vancouver, BC, Canada V6T I W5
Thermal destruction of the biotin-binding activity of avidin was estimated by thermal destruction time (TDT) experiments between 73.3”C and 1256°C. Decimal reduction time at 121.1’C was 24.5 min while the response to temperature change was described by a z value of 33 Co. Heat stability of the biotin-binding site of avidin was shown to be much greater than previously inferred from DSC protein denaturation studies of avidin: Keywords: avidin, thermal, denaturation, protein, biotin
INTRODUCTION
inactivation,
TDT, DSC, egg white,
studies. However, the authors examined protein denaturation only and did not measure the loss of biotin binding activity of avidin. They stated that it was a ‘natural assumption that the binding site of avidin is destroyed on heat denaturation’ (Donovan & Ross, 1973) and other authors have accepted this assumption (Green, 1975). In a recent study in this laboratory, thermal stability of avidin in egg white was greater than suggested by the DSC study and substantial activity was detected in various cooked egg products (Durance, 1991). Thus a study was undertaken to define the heat stability of the biotin-binding activity of avidin, in terms of the thermal destruction time (TDT) parameters, D and z.
Avidin is a tetrameric glycoprotein which binds the vitamin biotin and renders it unavailable for animal or microbial growth. The thermal stability of avidin and its complex with biotin have been the subject of several investigations (Eakin et al., 1941; Wei & Wright, 1964; Green, 1966; Donovan & Ross, 1973). The subject was of interest both theoretically, because avidin’s stability is unusually high, and practically, because r&din-is an antinutrient in human food. Essentially complete destruction of avidin activity in cooked egg white was assumed in the past (Parkinson, 1966) although some evidence to the contrary existed. Wei and Wright (1964) found that avidin at pH 6 was only 29% inactivated after 60 min at 100°C. Three min at 90°C had no detectable effect on activity (Eakin et al., 1941). A differential scanning calorimetry (DSC) study of the denaturation of avidin was published some years ago (Donovan & Ross, 1973). They described avidin denaturation in terms of a transition temperature at 85°C and thermal denaturation rate constants (k). Their DSC study suggested that avidin was considerably less stable than indicated by the earlier
MATERIALS
AND METHODS
Reagents 2-(4’-Hydroxyazobenzene) benzoic acid (HABA), carboxy-methyl cellulose (CMC) (Whatman CM-52) and ion exchange purified avidin standard were purchased from Sigma Chemical Co. (St. Louis, MO). Avidin for thermal destruction time experiments was purified by CMC ion exchange as previously described (Durance 8z Nakai, 1988).
Food Research International 0963-9969/92l.%X.o0 0 1992 Canadian Institute of Food Science and Technology 89
T. D. Durance, N. S. Wong
90 Avidin activity
Table 1. Thermal destruction of avidm in @02M phate buffer at pH 7-3
Avidin activity was assayed by the HABA-standard curve method (Durance, 1991). Protein concentration was estimated by absorbance at 280 nm with Et% = 15.4 for avidin.
Container
Glass ampules
Heat treatment of avidin
Avidin solution (0.6 mg/ml in 0.02~ sodium phosphate, pH 7.3) was sealed into 2 ml Wheaton glass ampules, 12 mm o.d., 35 mm high and 11 mm i.d. (Millville, NJ) for the temperature range of 7393°C and into 1.8 ml stainless steel tubes (126 mm x 6.7 mm o.d. X 4.5 mm i.d.) closed at both ends with Swagelock tube fittings for the temperature range 103-126°C. T-type thermocouples (copper/ constantan) were placed in the center of one container of each type. Samples were equilibrated to an initial temperature of 0°C before being heated to the required time and temperature in an agitated oil bath. Upon removal from the oil bath, ampules and tubes were immediately submerged in a water/ ice bath. One or three containers per type were treated at each time-temperature combination; glass ampules were used below 100°C but metal TDT tubes were used above 100°C for safety reasons. Avidin activity in each container was assayed in triplicate. Destruction kinetics were calculated based on firstorder reaction kinetics. The slope of the linear regression of log avidin activity versus time at a particular destructive temperature was used to calculate D at that temperature. logA=logA,+mt
(1)
D = -l/m
(2)
where A is mean avidin activity, A, is intercept avidin activity, m is gradient and t is time at a destructive temperature T. The phantom thermal death time concept was applied to determine the so-called decimal reduction temperature, z, and D121.10C of avidin.
Metal TDT tubes
D value
Temperature (“C)
sodium phos-
r2a
The z value may also be calculated Arrhenius relationship: E, = 2.303 RTIT2/z
491 357 207 183 118
0.99 0.99 0.95 0.99 0.97
33.2
103.3 108.9 115.6 118.9 125.6
88 56 34 30 18
0.92 0.97 0.98 0.99 0.99
32.9
or2 = coefficient of determination versus time.
of log avidin activity
temperature ranges are similar (Ramaswamy et al., 1989). D values may be converted to thermal destruction constants (k) at the same temperature by the following equation. D = 2.303/k
RESULTS
(5)
AND DISCUSSION
Glass ampules and metal TDT tubes both heated quickly to target temperatures. Glass ampules had a lag time of about 2 min and TDT tubes about 1.5 min. Metal TDT tubes were included when it was found that, contrary to expectations, most of the avidin activity remained after heating to 93°C for 15 min. Although lag time was a source of error, it did not substantially effect the D value estimates (Fig. 1, Table l), because of the large z value of avidin, which indicated the relative insensitivity of avidin to small temperature differences. A linear relationship was observed between 1 1
(3)
from the
(4)
where R is the gas constant, E, is the activation energy and T is in K. The two methods, although conceptually different, yield similar results when
value (CO)
73.3 77.8 82.8 88.3 93.3
’
log (D1/D2) = (T, - Tl)/z
z
(min)
8
1oa.3
F
+
108.9
%
.
115.6
F
.
118.9
%
l
196.6
F
0.1: 0
6
10 llma
Fig. 1.
16
hh)
Linear relationship between log avidin activity and incubation time at five destructive temperatures.
Thermal inactivation of avidin
the logarithm of biotin-binding activity of avidin and incubation time at various destructive temperatures (Fig. l), indicating essentially first-order thermal destruction reactions. Coefficients of determination (r2) were typically 0.97 or greater, although in one instance this figure was 0.92 (Table 1). Triplicate containers of avidin were analyzed at only seven out of a total of 50 time/temperature combinations, since limited avidin was available. Coefficients of variation between containers were 0.5-4.9%, with a mean of 3.7%. Coefficients of variation of triplicate assays within containers were 0.6-4.7%, with a mean of 2.6%. Since a major part of the variation was due to the assay rather than the heat treatment, triplicate heat treatments were not considered necessary at all experimental points. The time required to destroy 90% of the avidin activity at 121.1 “C, the reference decimal reduction time or D121.10c, was 24.5 min in the metal TDT tubes. The decimal reduction temperature or z value was 33 C”. In the glass ampules, the z was almost identical, but the extrapolated Dt21.10, was somewhat lower at 17.1 min. Coefficients of determination of the regression of log avidin activity on temperature (n = 5), from which z values were calculated in glass and metal containers, were 0.969 and O-998, respectively. Regressions were both significant (p I 0.01). Pooling of the glass and metal container D values to calculate z was not advisable. Comparisons of the two phantom TDT curves by appropriate t-tests indicated that although the slopes were not significantly different, the y-intercepts were different at p 5 0.01 (Ostle, 1954). Pooling of the data would therefore give an erroneous estimate of z. To summarize, the response of avidin to temperature change was consistent between 73~3°C and 125.6”C. The difference in D,21.10, may reflected error due to extrapolation of the glass vial data. The direct evaluation of thermal destruction of avidin activity reported here indicated very different stability than that inferred from DSC results by Donovan and Ross (1973). When k values reported in that DSC study were converted to D values, a Dt210c of 1.O X 10-d min was estimated, in 0.02~ potassium phosphate at pH 6.84. The z value, calculated from Eqn 4, E, and temperatures reported by Donovan and Ross (1973), was 6.6C”. These values are much lower than indicated by the present study. Although pH and ionic strength varied somewhat between the two studies, the differences were not sufficient to account for the
91
observed discrepancy. The z values reported here are consistent with the z of 33.3C” reported for trypsin inhibitor (Hackler et al., 1965) and 37.2C” reported for peroxidase (Adams & Yawger, 1961). Avidin data presently available are consistent with the hypothesis that, while denaturation of some portions of the molecule can be described by a transition temperature of 85°C as observed in the aforementioned DSC study, the biotin-binding sites are actually much more heat stable. Additional support for these conclusions was provided by the study of Wei and Wright (1964). They measured the loss of affinity of avidin for Cl4 labeled biotin in flowing steam. A Dl,eO, of 123 min was estimated from their published chromatographs. According to the present study, D 1000C = 107 min, while the data of Donovan and Ross (1973) requires a D,OOOCof 0.06 min.
CONCLUSIONS Thermal inactivation of the biotin-binding activity of avidin was described by Dt2t0, = 25 min and z = 33C”. Stability was thus shown to be much greater than suggested by DSC protein denaturation studies and the DSC data should not be used to estimate thermal destruction of avidin activity. The discrepancy between this and the previous study was not in calculation or determination of denaturation transitions of avidin, but in the assumption that a denaturation peak observed by DSC was equivalent to loss of the biotin-binding activity of avidin.
ACKNOWLEDGEMENT This study was funded in part by a Natural Sciences and Engineering Research Council of Canada operating grant.
REFERENCES Adams, H. W. & Yawger, E. S. (1961). Enzyme inactivation and color of processed peas. Food Technol., 15, 314. Donovan, J. W. & Ross, K. D. (1973). Increase in the stability of avidin produced by binding of biotin. A differential scanning calorimetric study of denaturation by heat. Biochem., 12, 512. Durance, T. D. (1991). Residual avidin activity in cooked egg white assayed with improved sensitivity. J. Food Sci., 56, 707.
T. D. Durance, N. S. Wong Durance, T. D. & Nakai, S. (1988). Purification of avidin by cation exchange, gel filtration, metal chelate interaction Can. Inst. and hydrophobic interaction chromatography. Food Sci. Technol. J., 21, 279. Eakin, R. E., Snell, E. E. & Williams, R. J. (1941). The concentration and assay of avidin, the injury-producing protein in raw egg white. J. Biol. Chem., 140, 535. Green, N. M. (1966). Thermodynamics of binding of biotin and some analogues by avidin. Biochem. J., 101, 774. Green, N. M. (1975). Avidin. Adv. in Protein Chem., 29, 85. Hackler, L. R., van Buren, J. P., Steinkraus, K. H., El Rawi, I. & Hand, D. B. (1965). Effect of heat treatment on the
nutritive value of soymilk protein fed to weanling rats. J. Food Sci., 30, 723. Ostle, B. (1954). Statistics in Research. Iowa State College Press, Ames, Iowa, pp. 127-35. Parkinson, T. L. (1966). The chemical composition of eggs. J. Sci. Food Agric., 17, 101. Ramaswamy, H. S., van de Voort, F. R. & Ghazala, S. (1989). An analysis of TDT and Arrhenius methods for handling process and kinetic data. J. Food Sci., 54, 1322. Wei, R. D. & Wright, L. D. (1964). Heat stability of avidin and avidin-biotin complex and influence of ionic strength on affinity of avidin for biotin. Proc. Sot. Exp. Biol. Med., 117, 341.
25 (1992) 89-92
Kinetics of thermal inactivation of avidin T. D. Durance & N. S. Wong Department
of Food Science, University of British Columbia, 6650 NW Marine Drive, Vancouver, BC, Canada V6T I W5
Thermal destruction of the biotin-binding activity of avidin was estimated by thermal destruction time (TDT) experiments between 73.3”C and 1256°C. Decimal reduction time at 121.1’C was 24.5 min while the response to temperature change was described by a z value of 33 Co. Heat stability of the biotin-binding site of avidin was shown to be much greater than previously inferred from DSC protein denaturation studies of avidin: Keywords: avidin, thermal, denaturation, protein, biotin
INTRODUCTION
inactivation,
TDT, DSC, egg white,
studies. However, the authors examined protein denaturation only and did not measure the loss of biotin binding activity of avidin. They stated that it was a ‘natural assumption that the binding site of avidin is destroyed on heat denaturation’ (Donovan & Ross, 1973) and other authors have accepted this assumption (Green, 1975). In a recent study in this laboratory, thermal stability of avidin in egg white was greater than suggested by the DSC study and substantial activity was detected in various cooked egg products (Durance, 1991). Thus a study was undertaken to define the heat stability of the biotin-binding activity of avidin, in terms of the thermal destruction time (TDT) parameters, D and z.
Avidin is a tetrameric glycoprotein which binds the vitamin biotin and renders it unavailable for animal or microbial growth. The thermal stability of avidin and its complex with biotin have been the subject of several investigations (Eakin et al., 1941; Wei & Wright, 1964; Green, 1966; Donovan & Ross, 1973). The subject was of interest both theoretically, because avidin’s stability is unusually high, and practically, because r&din-is an antinutrient in human food. Essentially complete destruction of avidin activity in cooked egg white was assumed in the past (Parkinson, 1966) although some evidence to the contrary existed. Wei and Wright (1964) found that avidin at pH 6 was only 29% inactivated after 60 min at 100°C. Three min at 90°C had no detectable effect on activity (Eakin et al., 1941). A differential scanning calorimetry (DSC) study of the denaturation of avidin was published some years ago (Donovan & Ross, 1973). They described avidin denaturation in terms of a transition temperature at 85°C and thermal denaturation rate constants (k). Their DSC study suggested that avidin was considerably less stable than indicated by the earlier
MATERIALS
AND METHODS
Reagents 2-(4’-Hydroxyazobenzene) benzoic acid (HABA), carboxy-methyl cellulose (CMC) (Whatman CM-52) and ion exchange purified avidin standard were purchased from Sigma Chemical Co. (St. Louis, MO). Avidin for thermal destruction time experiments was purified by CMC ion exchange as previously described (Durance 8z Nakai, 1988).
Food Research International 0963-9969/92l.%X.o0 0 1992 Canadian Institute of Food Science and Technology 89
T. D. Durance, N. S. Wong
90 Avidin activity
Table 1. Thermal destruction of avidm in @02M phate buffer at pH 7-3
Avidin activity was assayed by the HABA-standard curve method (Durance, 1991). Protein concentration was estimated by absorbance at 280 nm with Et% = 15.4 for avidin.
Container
Glass ampules
Heat treatment of avidin
Avidin solution (0.6 mg/ml in 0.02~ sodium phosphate, pH 7.3) was sealed into 2 ml Wheaton glass ampules, 12 mm o.d., 35 mm high and 11 mm i.d. (Millville, NJ) for the temperature range of 7393°C and into 1.8 ml stainless steel tubes (126 mm x 6.7 mm o.d. X 4.5 mm i.d.) closed at both ends with Swagelock tube fittings for the temperature range 103-126°C. T-type thermocouples (copper/ constantan) were placed in the center of one container of each type. Samples were equilibrated to an initial temperature of 0°C before being heated to the required time and temperature in an agitated oil bath. Upon removal from the oil bath, ampules and tubes were immediately submerged in a water/ ice bath. One or three containers per type were treated at each time-temperature combination; glass ampules were used below 100°C but metal TDT tubes were used above 100°C for safety reasons. Avidin activity in each container was assayed in triplicate. Destruction kinetics were calculated based on firstorder reaction kinetics. The slope of the linear regression of log avidin activity versus time at a particular destructive temperature was used to calculate D at that temperature. logA=logA,+mt
(1)
D = -l/m
(2)
where A is mean avidin activity, A, is intercept avidin activity, m is gradient and t is time at a destructive temperature T. The phantom thermal death time concept was applied to determine the so-called decimal reduction temperature, z, and D121.10C of avidin.
Metal TDT tubes
D value
Temperature (“C)
sodium phos-
r2a
The z value may also be calculated Arrhenius relationship: E, = 2.303 RTIT2/z
491 357 207 183 118
0.99 0.99 0.95 0.99 0.97
33.2
103.3 108.9 115.6 118.9 125.6
88 56 34 30 18
0.92 0.97 0.98 0.99 0.99
32.9
or2 = coefficient of determination versus time.
of log avidin activity
temperature ranges are similar (Ramaswamy et al., 1989). D values may be converted to thermal destruction constants (k) at the same temperature by the following equation. D = 2.303/k
RESULTS
(5)
AND DISCUSSION
Glass ampules and metal TDT tubes both heated quickly to target temperatures. Glass ampules had a lag time of about 2 min and TDT tubes about 1.5 min. Metal TDT tubes were included when it was found that, contrary to expectations, most of the avidin activity remained after heating to 93°C for 15 min. Although lag time was a source of error, it did not substantially effect the D value estimates (Fig. 1, Table l), because of the large z value of avidin, which indicated the relative insensitivity of avidin to small temperature differences. A linear relationship was observed between 1 1
(3)
from the
(4)
where R is the gas constant, E, is the activation energy and T is in K. The two methods, although conceptually different, yield similar results when
value (CO)
73.3 77.8 82.8 88.3 93.3
’
log (D1/D2) = (T, - Tl)/z
z
(min)
8
1oa.3
F
+
108.9
%
.
115.6
F
.
118.9
%
l
196.6
F
0.1: 0
6
10 llma
Fig. 1.
16
hh)
Linear relationship between log avidin activity and incubation time at five destructive temperatures.
Thermal inactivation of avidin
the logarithm of biotin-binding activity of avidin and incubation time at various destructive temperatures (Fig. l), indicating essentially first-order thermal destruction reactions. Coefficients of determination (r2) were typically 0.97 or greater, although in one instance this figure was 0.92 (Table 1). Triplicate containers of avidin were analyzed at only seven out of a total of 50 time/temperature combinations, since limited avidin was available. Coefficients of variation between containers were 0.5-4.9%, with a mean of 3.7%. Coefficients of variation of triplicate assays within containers were 0.6-4.7%, with a mean of 2.6%. Since a major part of the variation was due to the assay rather than the heat treatment, triplicate heat treatments were not considered necessary at all experimental points. The time required to destroy 90% of the avidin activity at 121.1 “C, the reference decimal reduction time or D121.10c, was 24.5 min in the metal TDT tubes. The decimal reduction temperature or z value was 33 C”. In the glass ampules, the z was almost identical, but the extrapolated Dt21.10, was somewhat lower at 17.1 min. Coefficients of determination of the regression of log avidin activity on temperature (n = 5), from which z values were calculated in glass and metal containers, were 0.969 and O-998, respectively. Regressions were both significant (p I 0.01). Pooling of the glass and metal container D values to calculate z was not advisable. Comparisons of the two phantom TDT curves by appropriate t-tests indicated that although the slopes were not significantly different, the y-intercepts were different at p 5 0.01 (Ostle, 1954). Pooling of the data would therefore give an erroneous estimate of z. To summarize, the response of avidin to temperature change was consistent between 73~3°C and 125.6”C. The difference in D,21.10, may reflected error due to extrapolation of the glass vial data. The direct evaluation of thermal destruction of avidin activity reported here indicated very different stability than that inferred from DSC results by Donovan and Ross (1973). When k values reported in that DSC study were converted to D values, a Dt210c of 1.O X 10-d min was estimated, in 0.02~ potassium phosphate at pH 6.84. The z value, calculated from Eqn 4, E, and temperatures reported by Donovan and Ross (1973), was 6.6C”. These values are much lower than indicated by the present study. Although pH and ionic strength varied somewhat between the two studies, the differences were not sufficient to account for the
91
observed discrepancy. The z values reported here are consistent with the z of 33.3C” reported for trypsin inhibitor (Hackler et al., 1965) and 37.2C” reported for peroxidase (Adams & Yawger, 1961). Avidin data presently available are consistent with the hypothesis that, while denaturation of some portions of the molecule can be described by a transition temperature of 85°C as observed in the aforementioned DSC study, the biotin-binding sites are actually much more heat stable. Additional support for these conclusions was provided by the study of Wei and Wright (1964). They measured the loss of affinity of avidin for Cl4 labeled biotin in flowing steam. A Dl,eO, of 123 min was estimated from their published chromatographs. According to the present study, D 1000C = 107 min, while the data of Donovan and Ross (1973) requires a D,OOOCof 0.06 min.
CONCLUSIONS Thermal inactivation of the biotin-binding activity of avidin was described by Dt2t0, = 25 min and z = 33C”. Stability was thus shown to be much greater than suggested by DSC protein denaturation studies and the DSC data should not be used to estimate thermal destruction of avidin activity. The discrepancy between this and the previous study was not in calculation or determination of denaturation transitions of avidin, but in the assumption that a denaturation peak observed by DSC was equivalent to loss of the biotin-binding activity of avidin.
ACKNOWLEDGEMENT This study was funded in part by a Natural Sciences and Engineering Research Council of Canada operating grant.
REFERENCES Adams, H. W. & Yawger, E. S. (1961). Enzyme inactivation and color of processed peas. Food Technol., 15, 314. Donovan, J. W. & Ross, K. D. (1973). Increase in the stability of avidin produced by binding of biotin. A differential scanning calorimetric study of denaturation by heat. Biochem., 12, 512. Durance, T. D. (1991). Residual avidin activity in cooked egg white assayed with improved sensitivity. J. Food Sci., 56, 707.
T. D. Durance, N. S. Wong Durance, T. D. & Nakai, S. (1988). Purification of avidin by cation exchange, gel filtration, metal chelate interaction Can. Inst. and hydrophobic interaction chromatography. Food Sci. Technol. J., 21, 279. Eakin, R. E., Snell, E. E. & Williams, R. J. (1941). The concentration and assay of avidin, the injury-producing protein in raw egg white. J. Biol. Chem., 140, 535. Green, N. M. (1966). Thermodynamics of binding of biotin and some analogues by avidin. Biochem. J., 101, 774. Green, N. M. (1975). Avidin. Adv. in Protein Chem., 29, 85. Hackler, L. R., van Buren, J. P., Steinkraus, K. H., El Rawi, I. & Hand, D. B. (1965). Effect of heat treatment on the
nutritive value of soymilk protein fed to weanling rats. J. Food Sci., 30, 723. Ostle, B. (1954). Statistics in Research. Iowa State College Press, Ames, Iowa, pp. 127-35. Parkinson, T. L. (1966). The chemical composition of eggs. J. Sci. Food Agric., 17, 101. Ramaswamy, H. S., van de Voort, F. R. & Ghazala, S. (1989). An analysis of TDT and Arrhenius methods for handling process and kinetic data. J. Food Sci., 54, 1322. Wei, R. D. & Wright, L. D. (1964). Heat stability of avidin and avidin-biotin complex and influence of ionic strength on affinity of avidin for biotin. Proc. Sot. Exp. Biol. Med., 117, 341.