| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Bioengineering, Faculty of Food Science and Engineering, Dunarea de Jos University, Domneasca 111, 800201 Galati, Romania
2 Laboratory of Food Technology, Department of Food and Microbial Technology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, B-3001 Heverlee, Belgium
Corresponding author: Marc Hendrickx; E-mail: Marc.Hendrickx{at}agr.kuleuven.ac.be.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
|---|
Key Words: ß-lactoglobulin • structural change • surface hydrophobicity • sulfhydryl/disulfide interchange reaction
Abbreviation key: ANS = 1-aniline 8-naphthalene sulfonate, DTNB = 5,5'-dithio-bis(2-nitrobenzoic acid), FI = fluorescence intensity
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
|---|
A kinetic study on the heat-induced structural changes in ß-LG should lead to a better understanding of the relationship between heat treatment and its effect on the functional properties of ß-LG, with the perspective of new applications of whey proteins in foods (de la Fuente et al., 2002). Additionally, a kinetic model for the thermal denaturation of ß-LG is important for optimizing heat treatment of milk products so that the desired functional properties are achieved.
The objective of this study was to follow the heat-induced changes in surface hydrophobicity, thiol availability, solubility, and turbidity of ß-LG solutions heated at pH 7.5. Kinetic studies were performed to quantitatively describe the contribution of hydrophobic and SH/SS interchange reactions to the thermal structural changes of ß-LG.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
|---|
Isothermal Treatment of ß-LG Solutions
ß-Lactoglobulin solutions (110 µL of 2.5 mg/mL in 0.02 M Tris-HCl buffer, pH 7.5) were heated in 1.5-mL flexible centrifuge tubes (Eppendorf, Eppendorf AG, Hamburg, Germany) in a thermostatically controlled water bath at constant temperatures between 67.5 and 82.5°C for 1 to 45 min. After heat treatment, samples were immediately transferred to ice-cold water to prevent further denaturation. Analysis of the heat-induced changes was performed exactly 2 min after thermal treatment.
Solubility
Diluted samples of treated and untreated ß-LG solutions were centrifuged for 15 min (Eppendorf 201 centrifuge, Eppendorf AG) at 19,900 x g and 4°C. Protein concentration in the supernatant was determined using Sigma procedure no. TPRO-562. This method of protein quantification is based on the reduction of Cu2+ by protein in an alkaline environment. Bicinchonininic acid forms a colored complex with the Cu+ formed. The absorbance of the complex was measured at 562 nm. Bovine serum albumin was used as a standard, and all samples were assayed in duplicate. Solubility was expressed as the percentage of protein content in the supernatant compared with the total protein content of the untreated sample.
Turbidity
Turbidity was determined spectrophotometrically after diluting the samples to 1 mL in 0.02 M Tris-HCl buffer, at a wavelength of 600 nm and 20°C. One hundred percent turbidity was defined as 0% transmission of light.
Surface Hydrophobicity
The surface hydrophobicity (S0) was determined spectrofluorometrically using 1-aniline 8-naphthalene sulfonate (ANS). A stock solution of ANS (8 mM) was prepared in 0.1 M phosphate buffer (pH 7.6). The treated and untreated protein solutions were diluted with the phosphate buffer (pH 7.6) to a final protein concentration in the range of 0.002 to 0.0125%. Excitation and emission wavelengths were fixed at 390 and 470 nm, respectively, with 5-nm slit widths. The relative fluorescence intensity (FI) of the dilutions with and without ANS was measured with a Cary-Eclipse spectrofluorimeter (Varian, Mulgrave, Victoria, Australia). The net relative FI for each sample was then calculated by subtracting the relative FI attributed to protein in buffer. The initial slope of the net relative FI vs. protein concentration plot was calculated by linear regression analysis and used as an index for protein surface hydrophobicity (Alizadeh-Pasdar and Li-Chan, 2000).
Analysis of Sulfhydryl Groups
The procedure using Ellman’s reagent [5,5'-dithio-bis (2-nitrobenzoic acid); DTNB] was applied to determine the sulfhydryl group content. A molar extinction coefficient of 13,600 M–1cm–1 was used to calculate the amount of SH groups, expressed in µmoles per gram of protein (Beveridge et al., 1974). Three procedures were followed to determine the amount of total and exposed (surface) SH groups and free SH121 groups (slow-reacting SH groups).
Determination of surface and total SH groups.
ß-Lactoglobuin solutions from all heat-treatment groups were diluted with 990 µL of standard buffer (0.086 M Tris, 0.09 M glycine, 4 mM Na2EDTA; pH = 8) for surface SH groups, or urea (8 M in standard buffer) for total SH groups. To these samples, 10 µL of DTNB (4 mg of DTNB/mL of standard buffer) was added. The absorbance at 412 nm was measured against a reagent blank after 2 min (total SH groups) or 15 min (surface SH groups) at 20°C (Ultrospec 2100 pro, Biochrom, Cambridge, UK).
Determination of slow reacting SH groups.
Content of slow reacting SH groups was determined spectrophotometrically, based on a method described by Shimada and Cheftel (1989). The absorbance at 412 nm was recorded during 30 min of reaction at 20°C. The content was obtained using the following pseudo-first-order equation:
 | ([1]) |
where SHt is the content of total SH groups obtained from the maximum absorbance value, SHr is the content of SH groups that reacts at time t, kSH is the rate constant for the reaction between DTNB and SH121, and SHs is the content in slow reacting SH groups.
Kinetic Data Analysis
Changes in surface hydrophobicity and thiol availability as a function of heating time could be described by a fractional conversion model (a modified first-order kinetic model):
 | ([2]) |
where X
is the equilibrium value of the response value X at infinite heating time, and Xi is the response value of the native protein at time t = 0.
The temperature dependence of the rate constant, k (/min) could be described by the Arrhenius equation:
 | ([3]) |
where T and Tref are the absolute and reference temperature (in degrees K), respectively, kref is the rate constant at Tref, Ea is the activation energy (kJ/mol), and R is the universal gas constant (8.314 J/mol per degree K). Kinetic parameters were estimated by nonlinear regression analysis (SAS Institute, 2001).
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
|---|
Heating at a temperature between 70 and 75°C results in a minimal loss of ß-LG solubility. A decrease in solubility of only 10 to 20% compared with the native protein was observed after 45 min of heating, probably because at lower temperatures, the intramolecular interchange reactions are favored. These results are in good agreement with the data concerning heat-induced changes in turbidity and surface hydrophobicity ß-LG solutions as a function of temperature, as can be seen in Figure 1
. The maximum extent of unfolding coincides for all these properties measured above 78°C. Therefore, partial protein denaturation and its solubility coincide to some extent and good solubility of ß-LG after thermal treatment under neutral conditions and at low ionic strength is theoretically expected (de Wit and Klarenbeek, 1983) and is observed. When too many hydrophobic sites are exposed due to thermal treatment, the hydrophobic interactions are enhanced, usually leading to a decrease in solubility. However, thermal treatment above 80°C results in protein aggregation, with a decrease in solubility of 60%. This observation indicates that thermal denaturation of ß-LG as measured by the changes in solubility involves 2 steps: an unfolding step (70 to 75°C) and an aggregation step (78 to 82.5°C), that mostly follows unfolding, leading to a major decrease in solubility.
|
A marked increase in surface hydrophobicity S0 upon heating of ß-LG can be observed in Figure 2a. This implies that during thermal treatment the molecules are unfolded leading to an exposure of the hydrophobic clusters, which can promote intra- and intermolecular interactions. Surface hydrophobicity seems to increase to a plateau value. The maximum value for S0 was 4.2 times higher than the initial values (78.4 ± 3.2), and was reached after 30 min of heating at 80°C. The increase in surface hydrophobicity due to heat treatment is considered a positive attribute for emulsifying and foaming capacities, as long as solubility is not lost (Moro et al., 2001). At 80 to 82.5°C, however, S0 appears to decrease after reaching this maximum value, as can be seen in Figure 2a. After prolonged heating, too many hydrophobic sites are exposed, increasing the probability of intermolecular hydrophobic interactions, and the protein aggregates. Monahan et al. (1995) and Relkin (1998) observed an increase in surface hydrophobicity for protein that was heated at temperatures between 60 and 85°C and 60 and 80°C, respectively.
|
). Increasing treatment time resulted in a higher level of SH groups until an equilibrium value was reached. The exposure of SH groups was more pronounced at higher temperatures. The maximum extent of thiol exposure, expressed as a percentage of the total SH groups of untreated ß-LG was reached after 45 min of heating at 80°C. In this condition, ~65% of the initial total SH groups was exposed. However, above 80°C, the formation of disulfide bonds seems to be favored, resulting in a lower level of surface-reactive SH groups after prolonged heating compared with the lower treatment temperatures. These new disulfide bonds are important due to their contribution to the functionality of ß-LG in different food systems (Monahan et al., 1995). Manderson et al. (1999) observed a maximum exposure of ~55% of total SH groups after heating different genetic variants of ß-LG at 85°C for 13.5 min.
Slow-reacting SH groups.
When heating ß-LG, SH-SS interchange reactions occur, as well as the exposure of buried SH groups. Theoretically, in heated protein solutions, every cysteine residue can participate in thiol-catalyzed disulfide bond interactions (Hoffman and van Mill, 1997). However, it was shown that blocking the free thiol groups of ß-LG gave a protein derivative that would not aggregate via SH/SS interchange reactions (Iametti et al., 1996; Hoffman and van Mill, 1997). The involvement of the free SH121 groups in the denaturation process of ß-LG has been shown in site-mutagenesis studies, in which Cys121 is substituted with serine. The absence of the SH121 group eliminates the irreversible heat-induced aggregation, confirming the role of SH121 in the early steps of ß-LG denaturation (Jayat et al., 2004).
On the other hand, some authors (McKenzie et al., 1972; Phelan and Malthouse, 1994) have suggested that equal amounts of free SH121 and SH119 are implicated in SH/SS interchange reactions with the Cys66–Cys160 bond. Shimada and Cheftel (1989) observed that the free SH121 group in native ß-LG reacts slowly with DTNB in urea when SDS is present. This may be explained by the reversible interaction between SDS and the hydrophobic region near the SH121 group, resulting in an inhibition of reaction between the SH group and DTNB. They also explained that the slow-reacting SH groups are mainly SH121 but may be SH groups in the 119-position. In the unfolded state, the environment near to the newly formed SH119 due to the SH/SS interchange reactions is likely to be similar to that near to the native SH121 groups. When these interactions occur between SH121 and the Cys66–Cys160 bond, the newly formed SH group is no longer surrounded by hydrophobic residues. Therefore, the reaction with DTNB in the presence of SDS will be faster compared with the reaction in the native protein. If ß-LG samples contain native and heat-induced displaced SH group, the reactions with DTNB will represent the sum of slow and fast reactions (the so-called slow-reacting SH groups).
The initial slow-reacting SH group content was found to be 45.46 ± 0.37 µmol/g of ß-LG, representing 90.5% of the initial total SH groups. This indicates that the SH groups in the commercial ß-LG are already exposed (as discussed earlier), and are partly implicated in SH/SS interchange reactions during preparations. The decrease in the amount of slow-reacting SH groups is depicted in Figure 2c, indicating SH/SS interchange reactions between a heat-modified monomer and a native, undenatured one, as described by the polymerization mechanism (Roefs and De Kruif, 1994). In this case, the newly SH group formed in position 66 or 160 was more available for interactions with DTNB even in the presence of SDS. Creighton (1984) argued that at low protein concentration (0.05 to 0.2%) and sufficient reactivity of thiol groups, the formation of intramolecular disulfide bonds would prevail over intermolecular SH/disulfide interchange reactions. Considering the minimal increase in turbidity of ß-LG solutions heated between 70 and 75°C, the intramolecular interchange reactions seem to be favored. Above 75°C, the intermolecular interchange reactions predominated, leading to the polymerization of heat-modified molecules, as can be observed in the increased turbidity of the samples (Figure 1).
The observed pseudo-first-order rate constant (kSH) for the reaction between DTNB and slow-reacting groups of ß-LG in the presence of SDS at neutral pH was found to decrease from 0.255 ± 0.001 (/min) for the native protein to 0.119 ± 0.014 (/min) for the heat-treated protein at 82.5°C for 35 min. These results contrast with those reported by Shimada and Cheftel (1989). They observed a constant rate for the reaction between DTNB and slow-reacting SH groups after heating of 0.1% ß-LG solutions at 85°C for 0 to 45 min. An explanation could be that at higher temperatures, the environment of SH121 groups is changing, leading to different reactivity of SH121 in the presence of SDS. However, the remaining amount of slow-reacting SH groups after 45 min of heating at 82.5°C was only 2.94 µmol/g of protein.
Total SH groups.
The total SH groups content for pure ß-LG is reported to be 54.6 µmol/g of protein (Brunner, 1977). In our study, a similar value of 50.9 ± 0.7 µmol/g of proteins was found for native protein. The formation of intermolecular S-S bonds due to SH oxidation during thermal treatment could be neglected, as shown by the marginal decrease in total SH groups, compared with the decrease in slow-reacting SH groups (Figure 3). However, after 45 min of heating at 82.5°C, the total SH group content was 41.87 ± 0.5 µmol/g, representing 82.3% of the total SH groups for untreated protein. An explanation for the decrease in the total SH group content could be that at higher temperatures, the denatured ß-LG molecules may be involved in thiol oxidation reactions as well as in intermolecular interchange reactions. These results are in agreement with the observation concerning the role of intermolecular oxidation and thiol/disulfide interchange reactions in the heat-aggregation process of ß-LG molecules, reported in the literature (Shimada and Cheftel, 1989; Monahan et al., 1995; Iametti et al., 1996; Relkin, 1998).
|
. The rate constants (k values) for this model are given in Table 1. Between 70 and 82.5°C, the temperature dependence of k could accurately be described by the Arrhenius equation (equation 3), resulting in activation energy of 233.9 ± 8.6 kJ/mol (r2 = 0.9945; Figure 4a). It should be noted that at 80 and 82.5°C, this model was applied only for the increase in surface hydrophobicity, without taking into account the decrease in S0 after prolonged heating time. Given the differences in experimental conditions and analytical methods, a comparison with data from literature was difficult. The present value for Ea was consistent with the Ea range for skimmed and whole milk (265 to 280 kJ/mol) in the temperature range of 75 to 95°C as reported by Dannenberg and Kessler (1988) and Anema and McKenna (1996), but it is much lower than the Ea value reported by Galani and Apenten (1997) for thermal denaturation of ß-LG in Tris-HCl buffer in the same temperature range (376.09 kJ/mol).
|
|
for the different thermal treatments. The decrease in slow-reacting SH groups content could be described by a first-order fractional conversion model. The kinetic parameters are summarized in Table 1. The temperature dependence of the k values in the temperature range of 70 to 82.5°C (Figure 4a) could be described by the Arrhenius equation (equation 3), resulting in an activation energy of 148.19 ± 6.7 kJ/mol (r2 = 0.991). Apenten and Chee (2004), who used a method based on 
-casein-DTNB complex to measure the activation of SH groups in ß-LG solution in the temperature range 25 to 55°C, reported an Ea of 114.1 kJ/mol. The present values for k (Table 1) are comparable with those reported by Koka et al. (1968) for heated skimmed milk.
In contrast with the temperature dependence of the k values describing heat-induced changes in surface hydrophobicity and slow reacting SH groups, kinetics describing the heat induced-changes in surface SH groups of ß-LG solutions showed a break in the Arrhenius plot around 80°C (Figure 4b), resulting in a clear distinction of Ea values in the 2 temperature ranges studied (Table 1). This may be due to the complexity of the irreversible thermal denaturation process of ß-LG, involving a number of successive reaction steps. A possible model for the thermal behavior of ß-LG in buffer involves 3 steps as reported by Roefs and De Kruif (1994). The early step is the dissociation of the dimer to monomer favored at neutral pH, followed by a heat-induced unfolding step with the exposure of the free reactive thiol group and hydrophobic residues. The last step involves the aggregation of the molecules, due to sulfhydryl/disulfide interchange and noncovalent reactions. The Arrhenius plot becomes nonlinear above the transition temperature, which may be due to the heat-induced aggregation of the protein and expressed in the second part of the curve. Theoretically, if heat-treated ß-LG contains a higher amount of free SH groups, the aggregation step will be favored. In this context, the Ea value should be lower, as explained by Anema and McKenna (1996). They suggest that in an aggregation process in which a few intermolecular bonds are formed and the state of order of the system is increased, Ea will be lower.
A break in the linear Arrhenius plots around 85°C has been reported in the literature (Dannenberg and Kessler, 1988; Anema and McKenna, 1996; Galani and Apenten, 1997; Claeys, 2003). This unusual behavior could be a consequence of different rate-determining steps involving the participation of 2 consecutive reactions in the denaturation process, characterized by 2 different Ea values, as explained by Anema and McKenna (1996).
By monitoring the kinetics of changes in protein surface hydrophobicity and slow reacting SH groups content, we observed that the rate constants had approximately the same magnitude in the temperature range 70 to 78°C (Table 1). It is possible that the denaturation process in this temperature range is a combination of SH/SS interchange reactions and noncovalent associations (and probably not only hydrophobic, but also van der Waals and ionic interactions) of the unfolded protein molecules, which can occur simultaneously or sequentially. Our results confirmed the hypothesis that in lower temperature ranges (67.5 to 78°C), the rate-determining step is the unfolding of the molecules, whereas at higher temperatures (78 to 82.5°C), the aggregation process involving unfolded molecules becomes rate determining. Our suggestion is that the hydrophobic interactions have a relatively higher contribution for the thermal sensitivity of ß-LG in buffer above 78°C, leading to the unfolding of the molecules, which is accompanied, probably immediately, by the intermolecular interchange reactions promoted by the free, highly reactive SH group. Galani and Apenten (1999) suggested that the noncovalent interactions (mainly hydrophobic) become more important to the denaturation mechanism of ß-LG at temperatures close to the break in the Arrhenius plot. These heat-induced modifications were found to affect protein solubility.
|
CONCLUSIONS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
|---|
The findings of this study suggest a first-order fractional conversion model in the temperature range of 70 to 82.5°C for surface hydrophobicity and slow reacting SH groups, as well the existence of a break in the Arrhenius plot showing the temperature dependence of the rate constant k, obtained by monitoring the exposure of SH groups.
|
ACKNOWLEDGEMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
|---|
Received for publication October 25, 2004. Accepted for publication January 27, 2005.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
|---|
-lactoglobulin and effects of urea. Biochemistry 24:4539–4547.
This article has been cited by other articles:
|
|
 |
|
  W. L. Chen, W. T. Liu, M. C. Yang, M. T. Hwang, J. H. Tsao, and S. J. T. Mao A novel conformation-dependent monoclonal antibody specific to the native structure of beta-lactoglobulin and its application. J Dairy Sci, March 1, 2006; 89(3): 912 - 921. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), turbidity (
), and surface hydrophobicity (
) of ß-lactoglobulin solutions heat-treated for 45 min at different temperatures.
= 72.5°C, 
= 75°C, 
= 78°C, 
