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Use of fluorescence methods to monitor unfolding transitions in β-lactoglobulin
Food Research International 35 (2002) 871–877 www.elsevier.com/locate/foodres
Use of fluorescence methods to monitor unfolding transitions in b-lactoglobulin Pablo Busti, Sonia Scarpeci, Carlos Gatti, Ne´stor Delorenzi* Area Fisicoquı´mica, Departamento de Quı´mica—Fı´sica, Facultad de Ciencias Bioquı´micas y Farmace´uticas, Universidad Nacional de Rosario, Suipacha 531, (2000) Rosario, Argentina Received 18 October 2001; accepted 28 February 2002
Abstract The degree of exposure of tryptophanyl residues (Trp) in beta-lactoglobulin (b-LG) molecules can be evaluated by following the external quenching of the intrinsic protein fluorescence by added acrylamide. Based on this technique, we propose a method to monitor b-LG equilibrium denaturation profile by urea. The results were analyzed by the dissociation coupled unfolding (DCU) model. This model takes into account the impact of dimerization on b-LG stability. The values of free energy change for denaturing b-LG (G DCU) obtained in this work were 63.3 ( 0.5) kJ mol1 at pH 6.8 and 73.4 ( 2.3) kJ mol1 at pH 2.5. These results are in good agreement with previous results reported by other authors, that monitored the denaturation process by ultraviolet difference spectrophotometry. In addition, the protein dependence of denaturation equilibrium profiles by urea followed by fluorescence polarization measurements suggests that b-LG denatures by 3-state DCU process at both pH 6.8 and pH 2.5, with the dissociation of dimers preceding the unfolding of the monomers. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Beta-lactoglobulin; Chemical unfolding; Fluorescence quenching; Fluorescence polarization
1. Introduction Beta-lactoglobulin (b-LG) is the most abundant globular protein in bovine milk. It is a globular protein with a monomer molecular weight of about 18 300 and exists in various oligomeric states (dimers and octamers) as a function of pH, temperature, concentration, ionic strength and genetic variant (Wong, Camirand, & Pavlath, 1996). Unfolding studies have promoted knowledge of the structure and stabilization of simple globular proteins (Pace, 1986, 1990; Tanford, 1968) and of multi-unit proteins (Jaenicke & Rudolph, 1989; Neet & Teem, 1994; Galani & Apenten, 1999). Dissociation may affect b-LG stability (Qi, Brownlow, Holt, & Sellers, 1995) and its gelation properties (Matsuura & Manning, 1994). In a recent work, Galani and Apenten (1999) hypothesized that b-LG denatures by 3-state dissociation coupled unfolding (DCU) process at acidic and neutral pH. In this model, a dimer (D2) begins with * Corresponding author. Fax: +54-341-4372704. E-mail address: [email protected] (N. Delorenzi).
dissociation. The monomer (N) then unfold forming the denatured state (U): ) 2N * ) 2U D2 *
ð1Þ
b-LG dimer dissociates in the presence of 3 M urea without changing its 3 structure. Urea concentrations above 3 M are necessary to unfold b-LG subunits. The Gibbs free energy change for denaturing b-LG (G DCU) obtained by Galani and Apenten (1999) was 60.0 ( 2.3) kJ mol1 at pH 7.0 and 72.0 ( 1.9) kJ mol1 at pH 2.6. By comparison, the dissociation free energy for b-LG dimer (GD) is 26.0 kJ mol1 at pH 7.0 and 22.6 kJ mol1 at pH 2.6. Hence, dimerization accounts for 43% (pH 7.0) and 32% (pH 2.6) of the stability of native b-LG. b-LG denaturation by urea has been studied using optical methods and differential scanning calorimetry (Alexander & Pace, 1971; Creamer, 1995; Cupo & Pace, 1983; Galani & Apenten, 1999; Kella & Kinsella, 1988; Lapanje & Poklar, 1989; Molinari et al., 1996; Pace & Tanford, 1968; Poklar, Vesnaver, & Lapanje, 1993; Relkin, 1994, 1996).
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P. Busti et al. / Food Research International 35 (2002) 871–877
Because of its high sensitivity to the conformational state of a macromolecule, fluorescence techniques are very useful tools to obtain thermodynamic and kinetic information about transitions of macromolecules, such as protein folding reactions (Eftink, 1994). The intrinsic fluorescence of tryptophanyl residues (Trp) are particularly sensitive to their micro-environments and provide appropriate methods to perform this kind of studies. Creamer (1995) and Busti, Scarpeci, Gatti, and Delorenzi (1999) reported a red shift in b-LG intrinsic fluorescence emission in accordance with the loss of the protein native structure promoted by urea denaturation. This red shift indicates that the Trp residues in b-LG moved from an apolar environment to a more polar region. The degree of exposure of Trp residues in b-LG molecules can also be evaluated by following the external quenching of the intrinsic protein fluorescence by added solutes (Busti, Gatti & Delorenzi, 1998; Creamer, 1995; Eftink & Ghiron, 1981; Palazolo, Rodrı´guez, Farruggia, Pico´, & Delorenzi, 2000). In a recent work, the surface hydrophobicity of whey protein concentrates, where b-LG is the major component, was evaluated by application of this method (Moro, Gatti, & Delorenzi, 2001). Steady state polarization measurements can also be useful in determining the unfolding profile of proteins. Besides this, polarization of intrinsic Trp fluorescence can be useful in discriminating between dissociation and denaturation in multimeric proteins (Royer, 1995). The objective of this work was to propose a novel method to monitor the b-LG equilibrium denaturation profiles by urea based on protein fluorescence quenching by acrylamide and to analyze the results using the new DCU model proposed by Galani and Apenten (1999). In order to confirm if dissociation and unfolding are separate events, fluorescence polarization measurements has been applied to the study of b-LG denaturation by urea.
2. Materials and methods 2.1. Materials b-LG AB and acrylamide were purchased from Sigma (St. Louis, MO) and used without further purification. The protein was more than 90% pure when examined via sodium dodecyl sulfate polyacrilamide gel electrophoresis. All other chemicals were of analytical grade.
into the cell of a Jasco FP- 770 spectrofluorometer and an aliquot of b-LG solution was added to give a final protein concentration of 20 mM. The emission spectrum (excitation at 295 nm) of each solution was run about 2-h after protein addition, and then it was scanned across the peak and lMAX measured. After this, the fluorescence intensity at 337 nm (excitation at 295 nm) was measured (FO). Aliquots of 5 M acrylamide were mixed into the cell content, and the fluorescence intensity was re-determined (F). Final acrylamide concentrations ranged from 0 to 0.8 M. A correction factor C was applied to F because of the absorbance of acrylamide at the exciting wavelength. C was calculated as: At 1 10Af C¼ ð2Þ Af 1 10At where Af and At are the absorbances at the exciting wavelength for the fluorophore and total solution, respectively (Lloyd, 1981). An appropriate form of the Stern-Volmer equation for the quenching of heterogenously fluorescent protein is: n X F0 fi ¼ F ð 1 þ K ½ Q ÞeVi ½Q i i¼1
!1 ð3Þ
where FO and F are the fluorescence in the absence and presence of quencher Q, respectively (Eftink & Ghiron, 1981). Ki and Vi are the dynamic and static quenching constants for fluorescent component i and fi is the fractional contribution of component i to the total fluorescence. Thus, FO/F measurements and estimation of fi values allowed us to determine the dynamic and static constants for the quenching process. 2.3. -LG denaturation according to the DCU model If the native (N) and the unfolded (U) species of a protein posses different values of an observed property (Y), at any given urea concentration, the fraction of protein denatured (Fden), the denaturation equilibrium constant (KDCU) and free energy for denaturation (GDCU) were determined from the following equations: Fden ¼
ðY YMIN Þ ðYMAX YMIN Þ
GDCU ¼ RT lnKDCU ¼ RT ln
ð4aÞ 4Pt ðFden Þ2 ð1 Fden Þ
ð4bÞ
2.2. Equilibrium unfolding of -LG A series of urea solutions (0–8 M) were made from weighed quantities of urea in phosphate buffer (50 mM, pH 6.8) or citrate buffer (50 mM, pH 2.5). A 2.5 ml aliquot of the various urea solutions was placed directly
where YMAX and YMIN are the observed property values in 0 M urea and 8 M urea, respectively. Values for YMAX and YMIN at other urea concentrations were obtained by interpolating the values from 0 M to 8 M urea. Pt is the total protein concentration (Galani &
P. Busti et al. / Food Research International 35 (2002) 871–877
Apenten, 1999). GDCU vary linearly with urea concentration with a slope m and an intercept G DCU. Therefore, G DCU is the free energy change for protein unfolding in the absence of urea. Another parameter of importance is [urea]1/2, the urea concentration necessary to denature 50% of protein molecules.
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3. Results and discussion Bovine b-LG contains two Trp residues, Trp-19 and Trp-61, which are in quite different environments. The lattice Z crystal structure (Qin, Creamer, Baker & Jameson, 1998; Qin, Bewley, Creamer, Baker & Jame-
2.4. Fluorescence polarization Samples of b-LG 1 and 2 mg/mL were made in a series of urea solutions (0–8 M), phosphate buffer (50 mM, pH 6.8) or citrate buffer (50 mM, pH 2.5). A 2.5 ml aliquot of the various urea solutions was placed directly into a cell and the corrected polarization of fluorescence values were obtained using the Jasco FP- 770 spectrofluorometer equipped with polarizers. All experiments were performed at 25 C and repeated two or three times. Statistical analysis were performed using Sigma-Plot (Version 4.0 for Windows 95).
Fig. 1. The urea denaturation profile for beta-lactoglobulin. (A) pH 6.8. (B) pH 2.5. (*) Fden determined from lMAX measurements. (*) Fden determined from fluorescence quenching measurements. Protein concentration: 20 mM. Temperature: 25 C.
Fig. 2. Stern–Volmer plots of beta-lactoglobulin fluorescence quenching by acrylamide at various urea concentrations. (A) pH 6.8. (B) pH 2.5. Urea concentrations: (*) 0 M, (*) 3.5 M, (&) 4M, (&) 4.5 M, (^) 5M, (^) 5.5 M, (~) 6 M, () 6.5 M, (!) 7 M, (!) 8 M. Protein concentration: 20 mM. Temperature: 25 C.
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son, 1998) shows that Trp-19 is in an apolar environment within the cavity of b-LG whereas Trp-61 protrudes beyond the surface of the molecule and is quite close to the Cys-66–Cys-160 disulfide bridge, which can be an effective Trp fluorescence quencher. The intrinsic fluorescence of the protein is almost exclusively attributed to Trp-19 (Cho, Batt, & Sawyer, 1994; Manderson, Hardman, & Creamer, 1999). The alteration in b-LG fluorescence quenching upon its denaturation with urea followed the trend observed for other globular proteins. In other words, there was a shift in b-LG intrinsic fluorescence emission from 337 nm to longer wavelengths (red shift) induced by the denaturation process due to the increased exposure of the two Trp residues to the aqueous environment. However, the quantum yield increases when b-LG is denatured in urea solution presumably because of the decrease in quenching of Trp-61, which becomes more distant from the Cys66–Cys-160 disulfide bond (Manderson et al., 1999). Figs. 1 A and 1 B show the effect of urea concentration on the conformational state of b-LG at pH 6.8 and at pH 2.5. These figures show Fden calculated from Eq. (4a) using lMAX as the observable property, Y. The major exposure of Trp residues to the aqueous solvent promotes an increase in the fluorescence quenching of denatured b-LG by acrylamide, as it can be seen in Figs. 2 A and 2 B. These figures show the Stern–Volmer plots for the quenching of b-LG by acrylamide at each urea concentration used and at both pH assayed. In order to obtain Fden from quenching data, some assumptions in the fitting of the Stern–Volmer plots were made: (a) Because the intrinsic fluorescence of the native protein is almost exclusively attributed to Trp-19 (Cho et al., 1994, Manderson et al., 1999, Palazolo et al., 2000), we have assumed that f19(N)=1 and f61(N)=0 in the Stern–Volmer equation for the untreated b-LG (0 M urea). In such case this equation predicts a linear plot of Fo/F vs. quencher concentration and the slope is the usual KSV [equal to K19(N)]: 1 F0 1 ¼ ¼ 1 þ KSV ½Q 1 þ K19 ðNÞ½Q F N
ð5Þ
The Stern–Volmer plots for the untreated b-LG observed in Figs. 2 A and 2 B reflect this particular behavior at both pH assayed. Therefore, fluorescence quenching data at 0 M urea were analyzed by a linear least squares regression analysis to yield KSV. (b) The unfolding with urea ‘‘normalizes’’ the environment and exposes the two Trp residues of b-LG. Thus a good fitting of the experimental Stern–Volmer plot for unfolded b-LG in the presence of 8 M urea solution was achieved considering equal contribution of each of the two Trp residues present in the denatured b-LG molecule (f19(U)=f61(U)=0.50): 0
11 0:5 B ð1 þ K19 ðUÞ½Q ÞeV19 ðUÞ½Q C B C F0 C ¼B B C F U @ A 0:5 þ V ð U Þ ½ Q 61 ð1 þ K61 ðUÞ½Q Þe
ð6Þ
the fluorescence quenching data at 8 M urea were analyzed by a non-linear least squares regression analysis to obtain the dynamic and static quenching parameters [K19(U), V19(U), K61(U), V61(U)]. Table 1 shows the quenching parameters values calculated from Eqs. (5) and (6). (c) Townend, Herskovits, and Timasheff (1969) showed that b-LG dissociates without altering the solvent exposure of either Trp residue. Our observation that the Stern–Volmer plots at urea concentrations between 0 and 3.5 M were almost overlapped (Figs. 2 A and 2 B), reinforces the supposition that the dissociation of b-LG dimers that occurs in this range (Creamer, 1995; O’Neill & Kinsella, 1987) does not produce modifications in the accessibility of Trp residues to the external quenching reagent. In conclusion, the dissociation reaction for b-LG will, therefore, not show up in the denaturation profiles. Thus, if there are no intermediates in the unfolding process of b-LG, the fluorescence quenching observed between urea concentrations higher than 0 M and smaller than 8 M may be considered
Table 1 Acrylamide quenching parameters for beta-lactoglobulin (b-LG)a System
K19(M)1
V19(M)1
K61(M1)
V61(M1)
f19
f61
b-LG, pH 6.8 (N) b-LG, pH 6.8, urea 8 M (U) b-LG,pH 2.5 (N) b-LG, pH 2.5, urea 8 M (U)
2.1930.023 6.2425.163 2.2160.018 6.0285.503
– 1.2551.037 – 1.2601.086
– 6.242 5.163 – 6.028 5.503
– 1.255 1.037 – 1.260 1.086
1 0.5 1 0.5
0 0.5 0 0.5
a
Temperature 25 C, lExc. 295 nm, lEm. 337 nm, protein concentration 20 mM.
P. Busti et al. / Food Research International 35 (2002) 871–877
as the sum of the contributions of native and unfolded b-LG fluorescence quenching: F0 F0 F0 ¼ ð1 Fden Þ þFden F F N F U
ð7Þ
The continuous curves observed in Figs. 2 A and 2 B were calculated by a non-linear least squares fit of fluorescence quenching data using Eq. (7). The obtained values of Fden were also represented as a function of urea concentration at pH 2.5 and 6.8 (Figs. 1 A and 1 B).
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ment with the results obtained by Galani and Apenten (1999) monitoring equilibrium denaturation profiles by ultraviolet difference spectrophotometry. This fact provides confidence in the fluorescence quenching method for estimating G DCU and reinforces the validity of the assumptions made in this work. The values of m and [urea]1/2 were slightly smaller and higher, respectively, than those values reported by Galani and Apenten (1999), probably due to differences in the protein samples used. The polarization of fluorescence of the Trp residues decreased as the urea concentration was increased from 3 to 8 M, at both pH assayed (Figs. 4 A and 4 B). The depolarization indicates an enhancement of the rotational
It can be seen in Figs. 1 A and 1 B that the denaturation profiles obtained by this fluorescence quenching method overlap with the profiles obtained by the red shift in fluorescence. This fact indicates that both methods reflect the same consequences caused by disruption of b-LG native structure during denaturation by urea and related with the major exposure of Trp residues to the aqueous solvent. The stability of b-LG towards DCU by urea was analyzed using Eqs. (4a) and (4b). G DCU was found by linear extrapolation (Fig. 3). Only data obtained at urea concentrations sufficient to unfold 10–90% of the available proteins were used in linear extrapolation. Stability parameters for DCU are reported in Table 2. G DCU was 63.3 ( 0.5) kJ mol1 at pH 6.8 and 73.4 ( 2.3) kJ mol1 at pH 2.5, indicating that b-LG protein structure is more stable to the overall denaturation process at acidic pH. These results are in good agree-
Fig. 3. Determination of the free energy change for beta-lactoglobulin denaturation via dissociation and unfolding at (*) pH 6.8, (*) pH 2.5.
Fig. 4. Fluorescence polarization of beta-lactoglobulin in the presence of increasing concentrations of urea. (A) pH 6.8. (B) pH 2.5. Protein concentration: (*;) 1 mg/ml, (*) 2 mg/ml. Temperature: 25 C.
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Table 2 The stability of beta-lactoglobulin (b-LG) determined with the dissociation-coupled unfolding model (DCU) Condition
m kJ/(mol M)
[urea] 12 (M)
G DCU (kJ/mol)
pH 6.8 pH 2.5
–7.30.1 –7.90.4
5.2 5.5
63.30.5 73.42.3
mobility of Trp residues exposed to the solvent caused by the loss of b-LG structural integrity in the presence of increasing concentrations of urea (Civera, Sevilla, Moreno, & Churchich, 1996; Royer, 1995). While the polarization seems to be concentration independent at urea concentrations higher than 3 M, below this value the polarization increased with increasing b-LG concentration, fact that is more pronounced at pH 6.8 (Fig. 4 A). Galani and Apenten (1999) estimated the percentage of b-LG dimer present over a wide range of protein concentrations. At neutral pH 63% of b-LG molecules were dimerized for Pt=1 mg/ml and 71% for Pt=2 mg/ml. At pH 2.6, 41% or 51% of b-LG was dimerized when Pt=1 or 2 mg/ml, respectively. Therefore, the reason for the concentration dependence of b-LG polarization fluorescence at low urea concentrations must be related with the number of dimers present in each solution. It is tempting to speculate that the freely rotation of Trp residues around the Ca–Cb bond is limited when two monomers interact to form a dimer. Hence, higher the dimer concentration is, the higher is the polarization measured. The protein concentration dependence below 3 M leads to the conclusion that this first phase of the equilibrium denaturation profiles correspond to the dissociation of the protein dimer, whereas the second phase at higher urea concentrations correspond to the denaturation of the monomer. These results suggest that b-LG denatures by 3-state DCU process at both pH 2.5 and pH 6.8, with the dissociation of dimers preceding the unfolding of the monomers [Eq. (1)].
References Alexander, S. S., & Pace, C. N. (1971). A comparison of the denaturation of bovine beta-lactoglobulins A and B and goat b-lactoglobulin. Biochemistry, 10, 2738–2743. Busti, P., Gatti, C. A., & Delorenzi, N. (1998). Some aspects of b-lactoglobulin studied by fluorescence quenching. International Journal of Biological Macromolecules, 23, 143–148. Busti, P., Scarpeci, S., Gatti, C., & Delorenzi, N. (1999). Interaction of alkylsulfonate ligands with b-lactoglobulin AB from bovine milk. Journal of Agriculture and Food Chemistry, 47, 3628–3631. Cho, Y., Batt, C. A., & Sawyer, L. (1994). Probing the retinol-binding site of bovine b-lactoglobulin. Journal of Biological Chemistry, 269, 11102–11107. Civera, C., Sevilla, P., Moreno, F., & Churchich, J. E. (1996). Structural changes of b-lactoglobulin B induced by urea. Evidence
of residual structure. Biochemistry and Molecular Biology International, 38, 773–781. Creamer, L. K. (1995). Effect of sodium dodecyl sulfate and palmitic acid on the equilibrium unfolding of b-lactoglobulin. Biochemistry, 34, 7170–7176. Cupo, J. F., & Pace, C. N. (1983). Conformational stability of mixed disulfide derivates of b-lactoglobulin-B. Biochemistry, 22, 2654– 2658. Eftink, M. R. (1994). The use of fluorescence methods to monitor unfolding transitions in proteins. Biophysical Journal, 66, 482–501. Eftink, M. R., & Ghiron, C. A. (1981). Fluorescence quenching studies with proteins. Analytical Biochemistry, 114, 199–227. Galani, D., & Apenten, R. K. O. (1999). Beta-lactoglobulin denaturation by dissociation-coupled unfolding. Food Research International, 32, 93–100. Jaenicke, R., & Rudolph, R. (1989). Folding proteins. In T. E. Creighton (Ed.), Protein structure: a practical approach (pp. 117–144). Oxford: IRL Press at Oxford University Press. Kella, N. D., & Kinsella, J. E. (1988). Enhanced thermodynamic stability of b-lactoglobulin. A possible mechanism. Biochemical Journal, 255, 113–118. Lapanje, S., & Poklar, N. (1989). Calorimetric and circular dichroic studies of the thermal denaturation of beta-lactoglobulin. Biophysical Chemistry, 34, 155–162. Lloyd, J. B. F. (1981). Standards in fluorescence spectrometry. New York: Chapman and Hall. Manderson, G. A., Hardman, M. J., & Creamer, L. K. (1999). Effect of heat treatment on bovine b-lactoglobulin A, B and C explored using thiol availability and fluorescence. Journal of Agriculture and Food Chemistry, 46, 3617–3627. Matsuura, J. E., & Manning, M. C. (1994). Heat-induced gel formation of beta-lactoglobulin. A study on the secondary and tertiary structure as followed by circular dichroism spectroscopy. Journal of Agriculture and Food Chemistry, 42, 1650–1656. Molinari, E., Ragona, L., Varani, L., Musco, G., Consonni, R., Zetta, L., & Monaco, H. L. (1996). Partially folded structure of monomeric bovine b-lactoglobulin. FEBS Letters, 381, 237–243. Moro, A. S., Gatti, C., & Delorenzi, N. (2001). Hydrophobicity of whey protein concentrates measured by fluorescence quenching and its relation with surface functional properties. Journal of Agriculture and Food Chemistry, 49, 4784–4789. Neet, K. E., & Timm, D. E. (1994). Conformational stability of dimeric proteins: qauntitative studies by equilibrium denaturation. Protein Science, 3, 2167–2174. O’Neill, T. E., & Kinsella, J. E. (1987). Binding of alkanone flavors to b-lactoglobulin: effects of conformational and chemical modification. Journal of Agriculture and Food Chemistry, 35, 770– 774. Pace, N. C. (1986). Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods in Enzymology, 131, 266–282. Pace, N. C. (1990). Conformational stability of globular proteins. Trends in Biochemical Science, 15, 14–17. Pace, N. C., & Tanford, C. (1968). Thermodynamics of the unfording of beta-lactoglobulin A in aqueous urea solutions between 5 and 50 C. Biochemistry, 7, 198–208. Palazolo, G., Rodrı´guez, F., Farruggia, B., Pico´, G., & Delorenzi, N. (2000). Heat treatment of b-lactoglobulin: structural changes studied by partitioning and fluorescence. Journal of Agriculture and Food Chemistry, 48, 3817–3822. Poklar, N., Vesnaver, G., & Lapanje, S. (1993). Studies by UV spectroscopy of thermal denaturation of b-lactoglobulin in urea and alkylurea solutions. Biophysical Chemistry, 47, 143–151. Qi, X. L., Brownlow, S., Holt, C., & Sellers, P. (1995). Thermal denaturation of beta-lactoglobulin—effect of protein concentration at pH-6.75 and pH-8.05. Biochimica et Biophysica Acta. (Protein Str. Mol. Enzymol.), 1248, 43–49.
P. Busti et al. / Food Research International 35 (2002) 871–877 Qin, B. Y., Bewley, M. C., Creamer, L. K., Baker, E. N., & Jameson, G. B. (1998b). Structural basis of the Tanford transition of bovine b-lactoglobulin. Biochemistry, 37, 14014–14021. Qin, B. Y., Creamer, L. K., Baker, E. N., & Jameson, G. B. (1998a). 12- Bromododecanoic acid binds inside the calyx of bovine b-lactoglobulin. FEBS Letters, 438, 272–278. Relkin, P. (1994). Differential scanning calorimetry: a useful tool for studying protein denaturation. Thermochimica Acta, 246, 371–386. Relkin, P. (1996). Thermal unfolding of b-lactoglobulin, a-lactalbumin and bovine serum albumin: a thermodynamic approach. Critical Review of Food Science and Nutrition, 36, 565–601.
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Royer, C. A. (1995). Fluorescence spectroscopy. In B. A. Shirley (Ed.), Protein stability and folding. Theory and practice (pp. 65–89). Totowa, NJ: Humana Press. Tanford, C. (1968). Protein denaturation. Advances in Protein Chemistry, 23, 122–282. Townend, R., Herskovits, T. T., & Timasheff, S. N. (1969). The state of amino acid residues in b-lactoglobulin. Archives of Biochemistry and Biophysics, 129, 567–580. Wong, D. W. S., Camirand, W. M., & Pavlath, A. E. (1996). Structures and functionalities of milk proteins. Critical Review of Food Science and Nutrition, 36, 807–844.
Use of fluorescence methods to monitor unfolding transitions in b-lactoglobulin Pablo Busti, Sonia Scarpeci, Carlos Gatti, Ne´stor Delorenzi* Area Fisicoquı´mica, Departamento de Quı´mica—Fı´sica, Facultad de Ciencias Bioquı´micas y Farmace´uticas, Universidad Nacional de Rosario, Suipacha 531, (2000) Rosario, Argentina Received 18 October 2001; accepted 28 February 2002
Abstract The degree of exposure of tryptophanyl residues (Trp) in beta-lactoglobulin (b-LG) molecules can be evaluated by following the external quenching of the intrinsic protein fluorescence by added acrylamide. Based on this technique, we propose a method to monitor b-LG equilibrium denaturation profile by urea. The results were analyzed by the dissociation coupled unfolding (DCU) model. This model takes into account the impact of dimerization on b-LG stability. The values of free energy change for denaturing b-LG (G DCU) obtained in this work were 63.3 ( 0.5) kJ mol1 at pH 6.8 and 73.4 ( 2.3) kJ mol1 at pH 2.5. These results are in good agreement with previous results reported by other authors, that monitored the denaturation process by ultraviolet difference spectrophotometry. In addition, the protein dependence of denaturation equilibrium profiles by urea followed by fluorescence polarization measurements suggests that b-LG denatures by 3-state DCU process at both pH 6.8 and pH 2.5, with the dissociation of dimers preceding the unfolding of the monomers. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Beta-lactoglobulin; Chemical unfolding; Fluorescence quenching; Fluorescence polarization
1. Introduction Beta-lactoglobulin (b-LG) is the most abundant globular protein in bovine milk. It is a globular protein with a monomer molecular weight of about 18 300 and exists in various oligomeric states (dimers and octamers) as a function of pH, temperature, concentration, ionic strength and genetic variant (Wong, Camirand, & Pavlath, 1996). Unfolding studies have promoted knowledge of the structure and stabilization of simple globular proteins (Pace, 1986, 1990; Tanford, 1968) and of multi-unit proteins (Jaenicke & Rudolph, 1989; Neet & Teem, 1994; Galani & Apenten, 1999). Dissociation may affect b-LG stability (Qi, Brownlow, Holt, & Sellers, 1995) and its gelation properties (Matsuura & Manning, 1994). In a recent work, Galani and Apenten (1999) hypothesized that b-LG denatures by 3-state dissociation coupled unfolding (DCU) process at acidic and neutral pH. In this model, a dimer (D2) begins with * Corresponding author. Fax: +54-341-4372704. E-mail address: [email protected] (N. Delorenzi).
dissociation. The monomer (N) then unfold forming the denatured state (U): ) 2N * ) 2U D2 *
ð1Þ
b-LG dimer dissociates in the presence of 3 M urea without changing its 3 structure. Urea concentrations above 3 M are necessary to unfold b-LG subunits. The Gibbs free energy change for denaturing b-LG (G DCU) obtained by Galani and Apenten (1999) was 60.0 ( 2.3) kJ mol1 at pH 7.0 and 72.0 ( 1.9) kJ mol1 at pH 2.6. By comparison, the dissociation free energy for b-LG dimer (GD) is 26.0 kJ mol1 at pH 7.0 and 22.6 kJ mol1 at pH 2.6. Hence, dimerization accounts for 43% (pH 7.0) and 32% (pH 2.6) of the stability of native b-LG. b-LG denaturation by urea has been studied using optical methods and differential scanning calorimetry (Alexander & Pace, 1971; Creamer, 1995; Cupo & Pace, 1983; Galani & Apenten, 1999; Kella & Kinsella, 1988; Lapanje & Poklar, 1989; Molinari et al., 1996; Pace & Tanford, 1968; Poklar, Vesnaver, & Lapanje, 1993; Relkin, 1994, 1996).
0963-9969/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0963-9969(02)00096-0
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Because of its high sensitivity to the conformational state of a macromolecule, fluorescence techniques are very useful tools to obtain thermodynamic and kinetic information about transitions of macromolecules, such as protein folding reactions (Eftink, 1994). The intrinsic fluorescence of tryptophanyl residues (Trp) are particularly sensitive to their micro-environments and provide appropriate methods to perform this kind of studies. Creamer (1995) and Busti, Scarpeci, Gatti, and Delorenzi (1999) reported a red shift in b-LG intrinsic fluorescence emission in accordance with the loss of the protein native structure promoted by urea denaturation. This red shift indicates that the Trp residues in b-LG moved from an apolar environment to a more polar region. The degree of exposure of Trp residues in b-LG molecules can also be evaluated by following the external quenching of the intrinsic protein fluorescence by added solutes (Busti, Gatti & Delorenzi, 1998; Creamer, 1995; Eftink & Ghiron, 1981; Palazolo, Rodrı´guez, Farruggia, Pico´, & Delorenzi, 2000). In a recent work, the surface hydrophobicity of whey protein concentrates, where b-LG is the major component, was evaluated by application of this method (Moro, Gatti, & Delorenzi, 2001). Steady state polarization measurements can also be useful in determining the unfolding profile of proteins. Besides this, polarization of intrinsic Trp fluorescence can be useful in discriminating between dissociation and denaturation in multimeric proteins (Royer, 1995). The objective of this work was to propose a novel method to monitor the b-LG equilibrium denaturation profiles by urea based on protein fluorescence quenching by acrylamide and to analyze the results using the new DCU model proposed by Galani and Apenten (1999). In order to confirm if dissociation and unfolding are separate events, fluorescence polarization measurements has been applied to the study of b-LG denaturation by urea.
2. Materials and methods 2.1. Materials b-LG AB and acrylamide were purchased from Sigma (St. Louis, MO) and used without further purification. The protein was more than 90% pure when examined via sodium dodecyl sulfate polyacrilamide gel electrophoresis. All other chemicals were of analytical grade.
into the cell of a Jasco FP- 770 spectrofluorometer and an aliquot of b-LG solution was added to give a final protein concentration of 20 mM. The emission spectrum (excitation at 295 nm) of each solution was run about 2-h after protein addition, and then it was scanned across the peak and lMAX measured. After this, the fluorescence intensity at 337 nm (excitation at 295 nm) was measured (FO). Aliquots of 5 M acrylamide were mixed into the cell content, and the fluorescence intensity was re-determined (F). Final acrylamide concentrations ranged from 0 to 0.8 M. A correction factor C was applied to F because of the absorbance of acrylamide at the exciting wavelength. C was calculated as: At 1 10Af C¼ ð2Þ Af 1 10At where Af and At are the absorbances at the exciting wavelength for the fluorophore and total solution, respectively (Lloyd, 1981). An appropriate form of the Stern-Volmer equation for the quenching of heterogenously fluorescent protein is: n X F0 fi ¼ F ð 1 þ K ½ Q ÞeVi ½Q i i¼1
!1 ð3Þ
where FO and F are the fluorescence in the absence and presence of quencher Q, respectively (Eftink & Ghiron, 1981). Ki and Vi are the dynamic and static quenching constants for fluorescent component i and fi is the fractional contribution of component i to the total fluorescence. Thus, FO/F measurements and estimation of fi values allowed us to determine the dynamic and static constants for the quenching process. 2.3. -LG denaturation according to the DCU model If the native (N) and the unfolded (U) species of a protein posses different values of an observed property (Y), at any given urea concentration, the fraction of protein denatured (Fden), the denaturation equilibrium constant (KDCU) and free energy for denaturation (GDCU) were determined from the following equations: Fden ¼
ðY YMIN Þ ðYMAX YMIN Þ
GDCU ¼ RT lnKDCU ¼ RT ln
ð4aÞ 4Pt ðFden Þ2 ð1 Fden Þ
ð4bÞ
2.2. Equilibrium unfolding of -LG A series of urea solutions (0–8 M) were made from weighed quantities of urea in phosphate buffer (50 mM, pH 6.8) or citrate buffer (50 mM, pH 2.5). A 2.5 ml aliquot of the various urea solutions was placed directly
where YMAX and YMIN are the observed property values in 0 M urea and 8 M urea, respectively. Values for YMAX and YMIN at other urea concentrations were obtained by interpolating the values from 0 M to 8 M urea. Pt is the total protein concentration (Galani &
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Apenten, 1999). GDCU vary linearly with urea concentration with a slope m and an intercept G DCU. Therefore, G DCU is the free energy change for protein unfolding in the absence of urea. Another parameter of importance is [urea]1/2, the urea concentration necessary to denature 50% of protein molecules.
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3. Results and discussion Bovine b-LG contains two Trp residues, Trp-19 and Trp-61, which are in quite different environments. The lattice Z crystal structure (Qin, Creamer, Baker & Jameson, 1998; Qin, Bewley, Creamer, Baker & Jame-
2.4. Fluorescence polarization Samples of b-LG 1 and 2 mg/mL were made in a series of urea solutions (0–8 M), phosphate buffer (50 mM, pH 6.8) or citrate buffer (50 mM, pH 2.5). A 2.5 ml aliquot of the various urea solutions was placed directly into a cell and the corrected polarization of fluorescence values were obtained using the Jasco FP- 770 spectrofluorometer equipped with polarizers. All experiments were performed at 25 C and repeated two or three times. Statistical analysis were performed using Sigma-Plot (Version 4.0 for Windows 95).
Fig. 1. The urea denaturation profile for beta-lactoglobulin. (A) pH 6.8. (B) pH 2.5. (*) Fden determined from lMAX measurements. (*) Fden determined from fluorescence quenching measurements. Protein concentration: 20 mM. Temperature: 25 C.
Fig. 2. Stern–Volmer plots of beta-lactoglobulin fluorescence quenching by acrylamide at various urea concentrations. (A) pH 6.8. (B) pH 2.5. Urea concentrations: (*) 0 M, (*) 3.5 M, (&) 4M, (&) 4.5 M, (^) 5M, (^) 5.5 M, (~) 6 M, () 6.5 M, (!) 7 M, (!) 8 M. Protein concentration: 20 mM. Temperature: 25 C.
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son, 1998) shows that Trp-19 is in an apolar environment within the cavity of b-LG whereas Trp-61 protrudes beyond the surface of the molecule and is quite close to the Cys-66–Cys-160 disulfide bridge, which can be an effective Trp fluorescence quencher. The intrinsic fluorescence of the protein is almost exclusively attributed to Trp-19 (Cho, Batt, & Sawyer, 1994; Manderson, Hardman, & Creamer, 1999). The alteration in b-LG fluorescence quenching upon its denaturation with urea followed the trend observed for other globular proteins. In other words, there was a shift in b-LG intrinsic fluorescence emission from 337 nm to longer wavelengths (red shift) induced by the denaturation process due to the increased exposure of the two Trp residues to the aqueous environment. However, the quantum yield increases when b-LG is denatured in urea solution presumably because of the decrease in quenching of Trp-61, which becomes more distant from the Cys66–Cys-160 disulfide bond (Manderson et al., 1999). Figs. 1 A and 1 B show the effect of urea concentration on the conformational state of b-LG at pH 6.8 and at pH 2.5. These figures show Fden calculated from Eq. (4a) using lMAX as the observable property, Y. The major exposure of Trp residues to the aqueous solvent promotes an increase in the fluorescence quenching of denatured b-LG by acrylamide, as it can be seen in Figs. 2 A and 2 B. These figures show the Stern–Volmer plots for the quenching of b-LG by acrylamide at each urea concentration used and at both pH assayed. In order to obtain Fden from quenching data, some assumptions in the fitting of the Stern–Volmer plots were made: (a) Because the intrinsic fluorescence of the native protein is almost exclusively attributed to Trp-19 (Cho et al., 1994, Manderson et al., 1999, Palazolo et al., 2000), we have assumed that f19(N)=1 and f61(N)=0 in the Stern–Volmer equation for the untreated b-LG (0 M urea). In such case this equation predicts a linear plot of Fo/F vs. quencher concentration and the slope is the usual KSV [equal to K19(N)]: 1 F0 1 ¼ ¼ 1 þ KSV ½Q 1 þ K19 ðNÞ½Q F N
ð5Þ
The Stern–Volmer plots for the untreated b-LG observed in Figs. 2 A and 2 B reflect this particular behavior at both pH assayed. Therefore, fluorescence quenching data at 0 M urea were analyzed by a linear least squares regression analysis to yield KSV. (b) The unfolding with urea ‘‘normalizes’’ the environment and exposes the two Trp residues of b-LG. Thus a good fitting of the experimental Stern–Volmer plot for unfolded b-LG in the presence of 8 M urea solution was achieved considering equal contribution of each of the two Trp residues present in the denatured b-LG molecule (f19(U)=f61(U)=0.50): 0
11 0:5 B ð1 þ K19 ðUÞ½Q ÞeV19 ðUÞ½Q C B C F0 C ¼B B C F U @ A 0:5 þ V ð U Þ ½ Q 61 ð1 þ K61 ðUÞ½Q Þe
ð6Þ
the fluorescence quenching data at 8 M urea were analyzed by a non-linear least squares regression analysis to obtain the dynamic and static quenching parameters [K19(U), V19(U), K61(U), V61(U)]. Table 1 shows the quenching parameters values calculated from Eqs. (5) and (6). (c) Townend, Herskovits, and Timasheff (1969) showed that b-LG dissociates without altering the solvent exposure of either Trp residue. Our observation that the Stern–Volmer plots at urea concentrations between 0 and 3.5 M were almost overlapped (Figs. 2 A and 2 B), reinforces the supposition that the dissociation of b-LG dimers that occurs in this range (Creamer, 1995; O’Neill & Kinsella, 1987) does not produce modifications in the accessibility of Trp residues to the external quenching reagent. In conclusion, the dissociation reaction for b-LG will, therefore, not show up in the denaturation profiles. Thus, if there are no intermediates in the unfolding process of b-LG, the fluorescence quenching observed between urea concentrations higher than 0 M and smaller than 8 M may be considered
Table 1 Acrylamide quenching parameters for beta-lactoglobulin (b-LG)a System
K19(M)1
V19(M)1
K61(M1)
V61(M1)
f19
f61
b-LG, pH 6.8 (N) b-LG, pH 6.8, urea 8 M (U) b-LG,pH 2.5 (N) b-LG, pH 2.5, urea 8 M (U)
2.1930.023 6.2425.163 2.2160.018 6.0285.503
– 1.2551.037 – 1.2601.086
– 6.242 5.163 – 6.028 5.503
– 1.255 1.037 – 1.260 1.086
1 0.5 1 0.5
0 0.5 0 0.5
a
Temperature 25 C, lExc. 295 nm, lEm. 337 nm, protein concentration 20 mM.
P. Busti et al. / Food Research International 35 (2002) 871–877
as the sum of the contributions of native and unfolded b-LG fluorescence quenching: F0 F0 F0 ¼ ð1 Fden Þ þFden F F N F U
ð7Þ
The continuous curves observed in Figs. 2 A and 2 B were calculated by a non-linear least squares fit of fluorescence quenching data using Eq. (7). The obtained values of Fden were also represented as a function of urea concentration at pH 2.5 and 6.8 (Figs. 1 A and 1 B).
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ment with the results obtained by Galani and Apenten (1999) monitoring equilibrium denaturation profiles by ultraviolet difference spectrophotometry. This fact provides confidence in the fluorescence quenching method for estimating G DCU and reinforces the validity of the assumptions made in this work. The values of m and [urea]1/2 were slightly smaller and higher, respectively, than those values reported by Galani and Apenten (1999), probably due to differences in the protein samples used. The polarization of fluorescence of the Trp residues decreased as the urea concentration was increased from 3 to 8 M, at both pH assayed (Figs. 4 A and 4 B). The depolarization indicates an enhancement of the rotational
It can be seen in Figs. 1 A and 1 B that the denaturation profiles obtained by this fluorescence quenching method overlap with the profiles obtained by the red shift in fluorescence. This fact indicates that both methods reflect the same consequences caused by disruption of b-LG native structure during denaturation by urea and related with the major exposure of Trp residues to the aqueous solvent. The stability of b-LG towards DCU by urea was analyzed using Eqs. (4a) and (4b). G DCU was found by linear extrapolation (Fig. 3). Only data obtained at urea concentrations sufficient to unfold 10–90% of the available proteins were used in linear extrapolation. Stability parameters for DCU are reported in Table 2. G DCU was 63.3 ( 0.5) kJ mol1 at pH 6.8 and 73.4 ( 2.3) kJ mol1 at pH 2.5, indicating that b-LG protein structure is more stable to the overall denaturation process at acidic pH. These results are in good agree-
Fig. 3. Determination of the free energy change for beta-lactoglobulin denaturation via dissociation and unfolding at (*) pH 6.8, (*) pH 2.5.
Fig. 4. Fluorescence polarization of beta-lactoglobulin in the presence of increasing concentrations of urea. (A) pH 6.8. (B) pH 2.5. Protein concentration: (*;) 1 mg/ml, (*) 2 mg/ml. Temperature: 25 C.
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Table 2 The stability of beta-lactoglobulin (b-LG) determined with the dissociation-coupled unfolding model (DCU) Condition
m kJ/(mol M)
[urea] 12 (M)
G DCU (kJ/mol)
pH 6.8 pH 2.5
–7.30.1 –7.90.4
5.2 5.5
63.30.5 73.42.3
mobility of Trp residues exposed to the solvent caused by the loss of b-LG structural integrity in the presence of increasing concentrations of urea (Civera, Sevilla, Moreno, & Churchich, 1996; Royer, 1995). While the polarization seems to be concentration independent at urea concentrations higher than 3 M, below this value the polarization increased with increasing b-LG concentration, fact that is more pronounced at pH 6.8 (Fig. 4 A). Galani and Apenten (1999) estimated the percentage of b-LG dimer present over a wide range of protein concentrations. At neutral pH 63% of b-LG molecules were dimerized for Pt=1 mg/ml and 71% for Pt=2 mg/ml. At pH 2.6, 41% or 51% of b-LG was dimerized when Pt=1 or 2 mg/ml, respectively. Therefore, the reason for the concentration dependence of b-LG polarization fluorescence at low urea concentrations must be related with the number of dimers present in each solution. It is tempting to speculate that the freely rotation of Trp residues around the Ca–Cb bond is limited when two monomers interact to form a dimer. Hence, higher the dimer concentration is, the higher is the polarization measured. The protein concentration dependence below 3 M leads to the conclusion that this first phase of the equilibrium denaturation profiles correspond to the dissociation of the protein dimer, whereas the second phase at higher urea concentrations correspond to the denaturation of the monomer. These results suggest that b-LG denatures by 3-state DCU process at both pH 2.5 and pH 6.8, with the dissociation of dimers preceding the unfolding of the monomers [Eq. (1)].
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