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J. Phys. Chem. 1996, 100, 1914-1917
Study of the Denaturation Process in Albumin-Urea Solutions by Means of the Thermally Stimulated Depolarization Currents Technique A. Vassilikou-Dova,*,† S. Grigorakakis,† P. Varotsos,† A. Kapetanaki,‡ and D. Koutsikos‡ Athens UniVersity, Physics Department, Section of Solid State Physics, Panepistimiopolis, 15 784 Zografos, Greece, and Athens UniVersity, Aretaieon Hospital, Department of Nephrology, 76 Vas. Sofias AVenue, 115-28 Athens, Greece ReceiVed: April 7, 1995; In Final Form: September 19, 1995X
The dielectric relaxation of bovine serum albumin (BSA) aqueous solution was studied in various stages of urea denaturation by means of the thermally stimulated depolarization currents (TSDC) technique. A large variation of urea concentration was utilized (0-4 M). Special attention was given for concentrations between 0.006 and 0.050 M, which are comparable to those measured in normal and uremic human serum. The TSDC spectrum consists of two main bands in the temperature range 100-300 K: (1) a broad low-temperature band (A) with two maxima at about 128 and 142 K, which are attributed to the (re)orientation mechanisms of bulk and hydration water molecules, respectively, and (2) an intense band (B), whose position varies in the range between 230 and 245 K and which is attributed to the (re)orientation of protein macromolecules. Two peaks of lower intensity (B1 and B2) are resolved in the rising part of band B and are most likely due to relaxation mechanisms of the side chains of BSA. The activation energies for the last three mechanisms depend on urea concentration, and this fact might be attributed to the conformational changes of BSA upon denaturation. It is of interest to note that the macromolecules’ activation energies depend drastically on the concentration of urea up to 0.05 M.
1. Introduction Native bovine serum albumin (BSA) is a globular protein which undergoes structural changes on the addition of denaturing agents such as urea1 and transforms into its unfolded or tertiary configuration. This secondary structure consists of hydrogenbonded R-helices and β-sheets and is called the large-scale structure. The transition between folded and unfolded states is sharp and unique and is encoded into the sequence of monomers. Urea is an end product of the metabolism of proteins, being made in the liver and removed from circulation in the kidneys. Thus changes in the dielectric behavior of BSA due to the presence of urea could contribute to a better understanding of biological mechanisms. The protein solutions’ dielectric relaxation studies carried out during the last fifty years have provided important information about the potential relaxation mechanisms.1-7 Several theoretical models have been proposed to relate the experimental findings to the dielectric behavior of globular proteins in solution (ref 8 and references therein). The dielectric decrement (I ) 0 - ∞/g) and the relaxation time of native BSA were found to increase on the addition of a high concentration of urea up to 8 M,7 although the dielectric properties of the denatured state were not drastically different from those of the native protein (thus indicating that urea-denatured BSA still maintains considerable amounts of the original folding of peptide chains). The dielectric relaxation in 1 and 2 M aqueous solutions of urea has also been measured in the frequency range between 1 MHz and 1 GHz.9 Different relaxation functions based on models of the solutions have been fitted to the spectra. The results show that, around urea, the number of molecules with a relaxation time different from that of pure water is usually small. The thermally stimulated depolarization currents (TSDC) * To whom all correspondence should be addressed. † Athens University, Physics Department, Section of Solid State Physics. ‡ Athens University, Aretaieon Hospital, Department of Nephrology. X Abstract published in AdVance ACS Abstracts, December 15, 1995.
0022-3654/96/20100-1914$12.00/0
technique recently has made a significant contribution to the updated understanding of relaxation phenomena in macromolecules10 and is currently considered to be superior11 than other dielectric techniques, mainly for three reasons: (1) It can resolve competing relaxation processes by applying suitable procedures (e.g. the usual “partial depolarization” or the “thermal sampling”). (2) It has a higher sensitivity and can detect a 100 times lower dipolar concentrationsin alkali halidessthan that detected by the dielectric loss method when applied in the temperature range 77-300 K. (3) It covers a wide range of corresponding frequencies from megahertz to gigahertz, at room temperature. Despite the large body of dielectric loss and conductivity studies in proteins accumulated during the last fifty years,12,13 very little information exists pertaining to the dielectric properties of their denatured states. We performed the present investigation in order to study systematically the (re)orientation mechanisms in various stages of BSA denaturation, by applying the TSDC technique. 2. Experimental Section Technique. The TSDC technique can be outlined as follows: (1) a constant electic field Ep is applied to a sample containing electric dipoles, for some time tp . τ(Tp), where τ(Tp) denotes the relaxation time. The electric dipole moments are oriented, and hence a saturation polarization P0 is reached which is given by the Langevin function P0 ) RNµ2Ep/kTp, where N is the concentration of dipoles, µ is the effective dipole moment, and R is a geometrical factor, which depends on the symmetry of the dipole moment’s surroundings (e.g. for cubic symmetry R is equal to 1/3). (2) The sample is cooled down to a low temperature T0 (e.g. liquid nitrogen temperature), where the relaxation time τ(T0) becomes very high, and then the external field is switched off. The polarization remains “frozen in”, and the sample is kept for some time at this low temperature in order for the fast electronic and atomic polarizations to decay © 1996 American Chemical Society
Denaturation Process in Albumin-Urea Solutions
J. Phys. Chem., Vol. 100, No. 5, 1996 1915
isothermally. (3) Then the sample is warmed up at a constant rate b ) dT/dt, and the relaxation time τ(T) gradually decreases. In the case of a single relaxation process obeying the Arrhenius equation
τ(T) ) τ0 exp(E/kT)
(1)
the rate of the decrease of the polarization as obtained from first-order kinetics results in a current density
JD(T) ) dP(t)/dt ) -P(t)/τ(t)
(2)
The depolarization current density J(T) is given by the equation
J(T) ) (P0/τ0) exp[(-E/kT) - (1/bτ0) exp(-E/kT′) dT′] (3) where τ(T) is the relaxation time, E is the activation enthalpy of the relaxation mechanism, τ0 is the pre-exponential factor, T is the absolute temperature, k is the Boltzmann’s constant, and P0 is the initial polarization. The depolarization current reaches its maximum value Im at a temperature Tm, for which the following thermodynamical relation holds:
bE/kTm ) Tm/τ(Tm)
(4)
Note that it has been recently shown14 that eq 4 is valid even if b is not constant. In cases where there are overlapping TSDC bands resulting from processes with distributed relaxation parameters, a variation of the TSDC technique known as the thermal sampling (TS) techinique is used.15,16 Material. A suitable amount of pure urea (20% w/w) was dissolved in BSA in order to prepare solutions with urea concentration in the range 0-4 M. The solutions were stored in a refrigerator for 24 h prior to their use, because the structural changes are nearly completed in this period of time. The pH of the solutions was around 5.5 for all urea concentrations. The above solutions were chosen to start from urea concentrations similar to those found for normal (0.006 M) and uremic human serum (0.050 M) but also to approach a high degree of protein denaturation (4 M). The sample consisted of a 100 µL droplet placed between parallel plate platinum electrodes (2.5 mm from each other). Methodology. The instrumentation for TSDC experiments has been described in detail elsewhere.17 The sample is polarized by applying 200 V DC in series with a resistor equal to 22 MΩ, and the whole system is kept in a nitrogen atmosphere. The 22 MΩ resitor is connected in order to decrease the current if an excess voltage is applied in the case of samples of low resistivity. The polarization temperature is equal to 300 K, and the polarization time tp is on the order of 1 min. The cooling rate should be rapid enough to avoid any increase of the initial equilibrium polarization. The measurements involve a temperature sweep in the range 77-300 K with a heating rate b ) 5 K/min using a temperature controller. The spectrum was monitored by a Keithley 617 Electrometer, and the data were collected and analyzed by a computer. Attempts with samples of higher than 4 M urea concentrations turned out to be unsuccessful due to the increase of the viscosity within a few days of sample storage, which led to the vitrification of the solutions. The TSDC results in the present work are considered to represent true bulk effects. We support this consideration because we have paid attention to excluding possible electrode effects and performing experiments as a function of test sample thickness (e.g. by using a MISM configuration, the temperature
Figure 1. Characteristic TSDC spectra for BSA-urea aqueous solutions with urea concentrations of 0.006-4 M. Band A1 is hardly visible due to its low intensity.
maximum Tm of the peaks was unchanged under the same experimental conditions; by changing the sample thickness and keeping constant the polarizing field Ep, the peak maximum intensity was unchanged, as expected, since the concentration N of dipoles remained constant). 3. Results and Discussion Characteristic TSDC spectra are depicted in Figure 1. For all urea concentrations two main bands are resolved in the temperature range 77-300 K: (1) the low-temperature broad band A with two maxima A1 and A2 at about 128 and 142 K (an increase of the urea concentration resulted in a decrease of the area under peak A2 and an increase of the area under peak A1 with a simultaneous shift of their maxima to lower temperatures) and (2) the intense band B. [In most of the experiments the exact temperature of the intensity maximum Tm could not be detected due to the very high current (six orders of magnitude higher), so it was only concluded by monitoring the rising and lowering parts of this band, which lie between 230 and 245 K.] (3) With increasing urea concentration, two secondary TSDC peaks B1 and B2 with maxima in the vicinity of 180 and 195 K were recorded, with decreasing intensities and a gradual shift of their intensity maxima to higher temperatures. For samples with a urea concentration higher than 4 M, B1 disappeared and B2 turned systematically down to negative values. (4) Additional peaks are occasionally resolved in the rising part of the HT band, but they are barely discernible in most of the spectra. The two low-temperature peaks, A1 and A2, are directly related to the hydration conditions of our samples. The bound water relaxation maximizing around 140 K is the one which is commonly observed in the megahertz region in a number of biomaterials such as muscle, living tissues, egg albumen, and biopolymers in aqueous solution.17 For BSA-urea aqueous solutions the intensity of the A1 peak increases while the intensity of the A2 peak decreases, and its maximum shifts to lower temperatures, with consequent lowering of the relaxation time, as the urea concentration increases. This result is expected if we consider that upon BSA denaturation the ordered structure
1916 J. Phys. Chem., Vol. 100, No. 5, 1996
Vassilikou-Dova et al.
Figure 2. Activation energy of bulk (2) and hydration (4) water as a function of urea concentration.
TABLE 1: Relaxation Parameters for Bulk and Hydration Water in BSA Aqueous Solution (20% w/w) for Various Urea Concentrations urea concentration (M) 0.000 0.006 0.046 1.000 2.000 3.000 4.000
peak
Im (pA)
Tm (K)
E (eV)
N (pCb)
A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2
4.7 62.7 6.3 43.4 4.8 17.6 5.5 8.5 9.9 7.1 8.9 3.8 4.3 7.3
128.9 151.0 132.2 147.3 125.3 145.2 123.7 143.3 125.8 142.6 122.5 141.3 126.3 141.3
0.26 0.36 0.18 0.42 0.20 0.39 0.20 0.43 0.24 0.38 0.22 0.26 0.19 0.37
855 10371 1260 4979 803 2912 1528 1286 1799 984 2009 876 1029 1070
of the hydration water molecules becomes loose, the portion of disordered molecules increases, the relaxation time becomes shorter,18 and the activation energy for reorientation approaches that of bulk water. The relaxation parameters were calculated by applying the so-called “whole band analysis” and are given in Table 1. These values agree well with data reported in the literature for the relaxation of bulk and hydration water in biopolymers. The difference between the start and the end values for the activation energies of hydration water (0.15 eV) is approximately equal to the energy of a hydrogen bond. In view of the fact that the hydrogen bond is cooperative, no suggestion of the exact number of water linkages can be made despite the value of 0.45 eV, beyond the minimum of the order of three hydrogen bonds proposed to be a chainlike structure around BSA. The hydration properties of urea have been extensively studied by dielectric relaxation in 1 and 2 M solutions by Kaatze et al.9 It was shown that most of the coordination water molecules have a reorientation time similar to that of the pure solvent. One reason for this outstanding feature could be the fact that hydrophobic parts are almost missing with these solute molecules. Besides the relaxation due to solvent water molecules, a weak dispersion in the gigahertz region was attributed to the reorientation of urea molecules with an effective dipole moment of 3.33 D.9 The relaxation time was found to depend increasingly on the molar volume φ )
Figure 3. Activation energy of various relaxation mechanisms of BSA macromolecules as a function of urea concentration.
TABLE 2: TSDC Parameters of the Different Relaxation Mechanisms of BSA Macromolecules in BSA Aqueous Solution (20% w/w) with Variable Urea Concentrations urea urea Im Tm E concentration Im Tm E concentration (M) peak (pA) (K) (eV) (M) peak (pA) (K) (eV) 0.000 0.006 0.046 1.000 2.000
B B1 B2 B B1 B2 B B1 B2 B B1 B2 B B1 B2
0.73 67 176 0.58
2.500
1.01 38 176 0.57 148 189 0.70 1.14 29 182 0.60 102 194 0.90 1.26 20 188 0.72 54 198 1.00 1.35 16 195 0.87 45 202 1.03
3.000 3.500 4.000
B B1 B2 B B1 B2 B B1 B2 B B1 B2
1.46 1.53 1.20 1.55 1.63
V/c of solute for the series of n-alkyl derivatives of urea. These results are in accord with a molecular dynamics calculation19 where it was found that urea can enter into water without appreciable distortion of the solvent structure. So in our case the variations in the low-temperature TSDC spectra are mainly due to BSA denaturation. The activation energy depends upon the potential barrier that has to be surmounted for the reorientation. Thus changes in the local environment of a dipole will affect its relaxation parameters. Within such a frame, fluctuations in the activation energy of bulk water can be understood. In Figure 2 the dependence of activation energies of bulk and hydration water versus urea concentration is depicted. The spread of the relaxation parameters for both A1 and A2 may reflect the disturbing action of the solute molecules. Another reason for that could be some contribution from the relaxation of urea molecules which cannot be resolved due to the low intensity or to rather similar dipolar moments to those of hydration water molecules. The high-temperature TSDC band B must be related to albumin relaxation mechanisms which give rise to one prominent and at least two weaker mechanisms. These processes are accomplished by breaking and creating hydrogen bonds, which gives rise to different energy barriers. The relaxation parameters for the B, B1, and B2 peaks are given in Table 2. The activation energy as a function of urea concentration is depicted in Figure
Denaturation Process in Albumin-Urea Solutions
J. Phys. Chem., Vol. 100, No. 5, 1996 1917 over the frequency ranges 0.01-10 MHz, 10 MHz to 1 GHz, and 1-100 GHz.20 The intermediate dispersion was suggested to be due to the rotation of polar chains projecting from the protein molecules.21 Protein-folding transitions in associative memory models are described by applying a molecular-level theory.22 The ratio R of effective dipole moments for hydration versus bulk water has been calculated from the total polarization of each band and is proportional to the concentration of hydration to bulk water. From Figure 4 it can be seen that the structural changes during urea denaturation are very well expressed by the dependence of the ratio R on urea concentration. Accordingly this ratio could be used as a measuring factor of the critical urea concentration of the unfolding of peptide groups. 4. Conclusions
Figure 4. Ratio R ) P0(A1)/P0(A2) of effective dipole moments for peaks A1 and A2 as calculated from the total polarization P0 versus urea concentration.
3. As far as band B is concerned, with increasing urea concentration up to 0.050 M the activation energy rapidly increases. This increase is beyond the experimental error if we consider that the error from the least square fitting in the determination of E is around 0.05 eV. The rate of this increase is (9 ( 2) eV L/mol. A much weaker increase with a rate of (0.13 ( 0.02) eV L/mol characterizes the remaining range of urea concentrations. It is of interest to note that the energy E versus urea concentration exhibits a sharp change at a concentration near 0.05 M, which fits with the upper limit of concentrations found in uremic patients. This may suggest a drastic change in BSA-structure-like hydrogen bonding with peptide groups or formation of clusters. Another reason for this behavior could be a sharp unfolding of hydrophobic parts. Unfortunately the existing study of the denaturation process on BSA aqueous solutions7 does not involve a detailed study in this range of low concentration of urea. The dielectric properties of macromolecules have been found to be very sensitive to interactions with small molecules, due to the alteration of the mobility of the macromolecule. The general behavior of the energies for B1 and B2 versus the urea concentration is similar to that for band B, but the drastic increase in the region of low concentrations is appreciably lower (note that this increase seems to be larger for band B1 than for band B2). A simultaneous shift of their maxima to lower temperatures and a lowering in their intensities should be noticed. At urea concentrations higher than 3 M the B2 peak turns down to negative values, and this behavior occurs systematically with the negative minimum located at the same temperature. One reason for that could be internal depolarizing electric fields which grow up with the unfolding of BSA molecules or more than one opposed relaxation mechanism. For such a complicated molecule with 99 titratable carboxyl groups and 57 amino groups, it is expected to relax with more than one mechanism. Measurements of the dielectric properties of globular proteins in aqueous solutions have shown the presence of three principal dispersions occurring
Dramatic conformational changes during BSA denaturation occur for very low urea concentrations (up to 0.050 M), as indicated from the changes in the activation energies of the relaxation process. The structural changes of denatured BSA are very well expressed by the ratio of the concentrations of “hydration to bulk” water. Thus this ratio could be used as a measuring factor of the unfolding of peptide groups. Acknowledgment. We gratefully acknowledge support of this research by a grant from the General Secretariat for Research and Technology between the Physics Department of Athens University, the Department of Nephrology of Aretaieon Hospital, and Ergo Medical Company. References and Notes (1) Timasheff, S.; Arakawa,T. In Protein Structure. A Practical Approach; Reighton, T. E., Ed.; IRL Press: 1989. (2) Oncleay, J. L. In Proteins, Amino Acids and Peptides; Cohn, E. J., Edsall, J. T., Eds.; Reinhold: New York, 1943: Chapter 22. (3) Lumry, R.; Yue, R. H.-S. J. Phys. Chem. 1965, 69, 1162. (4) South, G. P.; Grant, E. H. Proc. R. Soc. London 1972, A328, 371. (5) Grant, E. H.; Sheppard, R. J.; South, G. P. Dielectric behaViour of biological molecules in solution; Clarendon Press: Oxford, 1978. (6) Desnica, D. Biopolymers 1979, 18, 1685. (7) Takashima, S. Biochim. Biophys. Acta 1964, 79, 531. (8) Mofers, F. J. M.; Casteleijn, G.; Levine, Y. K. Biophys. Chem. 1982, 16, 9. (9) Kaatze, U.; Gerke, H.; Pottel, R. J. Phys. Chem. 1986, 90, 5464. (10) Spathis, G.; Kontou, E.; Kefalas, V.; Apekis, L.; Christodoulidis, C.; Pissis, P. J. Macromol. Sci., Phys. 1990, B29 (1), 31. (11) Varotsos, P. A.; Alexopoulos, K. D. Thermodynamics of point defects and their relation with bulk properties; North Holland Physics Publishing: Amsterdam, 1986; pp 132-147, 422. (12) Bone, S.; Pething, R. J. Mol. Biol. 1985, 181, 323. (13) Pething, R. Annu. ReV. Phys. Chem. 1992, 43, 177 and references therein. (14) Varotsos, P. A.; Bogris, N. G.; Kyritsis, A. J. Phys. Chem. Solids 1992, 53, 1007. (15) Hino, T. Jpn. J. Appl. Phys. 1973, 12, 611. (16) Zielinski, M.; Kryzewski, M. J. Electrost. 1977, 3, 69. (17) Mashimo, S.; Kuwabara, S.; Yagihara, S.; Higasi, K. J. Phys. Chem. 1987, 91, 6337. (18) Bone, S.; Eden, J.; Pethig, R. Int. J. Quantum Chem., Quantum Biol. Symp. 1981, 8, 307. (19) Tanaka, H.; Touhara, H.; Nakanishi, K.; Watanabe, N. J. Chem. Phys. 1984, 80, 5170. (20) Buchanan, T. J.; Hagis, G. H.; Hasted, J. B.; Robinson, B. G. Proc. R. Soc. London, Ser. A 1952, 213, 399. (21) Pennock, B.; Schwan, H. P. J. Phys. Chem. 1969, 73, 2600. (22) Sasai, M.; Wolynes, P. G. Phys. ReV. Lett. 1990, 65, 2740.
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