AlCl3 system

ELSEVIER

Preferential Interactions in the n20/Lysozyme/AICl3 System J. M. Socorro, R. Olmo, M. D. Blanco and J. M. Teij6n Departamento de Bioqulmica y Biologla Molecular, Facultad de Medicina, Universidad Complutense, Spain

ABSTRACT The preferential interactions of lysozyme with solvent components is studied in aqueous solutions of AIC13. The interaction parameter is negative at large salt concentrations, indicating that the interaction process of both salt and protein is thermodynamically favorable. The transfer free-energy parameter and the solubility data show that aluminum chloride is a salting-in agent for lysozyme. Moreover, these preferential interactions also are correlated with both protein solubility in the solvent medium and the influence of salt on the lysozyme structure. Viscometric and refractometric studies show that lysozyme can undergo a conformational change at 1 mM of salt, and spectrophotometric studies indicate a protein activity of approximately 75% at l0 mM of salt. Therefore, neither the interaction of AIC13 with the lysozyme nor the conformational change undergone directly affect the catalytic amino acid residues of the active site.

INTRODUCTION A large number of current studies focus on the interaction between macromolecules and other molecules. These embrace both physiological [1-3] and structural [4-6] views. A m o n g these, studies about interactions involving metals can be included. The majority of these studies are carried out on solutions. These macromolecules are in a folded state; thus it is highly important to control all the factors that can influence molecular stability. The conformation adopted by a biological macromolecule is a sensitive function of residue composition, the sequence of these residues, and solvent environment. Another highly important

Address reprint requests and correspondence to: Dr. Jos6 Maria Teij6n, Departamento de Bioqulmica y Biologla Molecular, Facultad de Medicina, Universidad Complutense, E-28040, Spain.

Journal of Inorganic Biochemistry, 57, 293-304 (1995) © 1995 Elsevier Science Inc., 655 Avenue of the Americas, NY, NY 10010

0162-0134/95/$9.50 SSDI 0162-0134(94)00044-B

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aspect in these studies is protein activity as a measure of the influence of structural changes on functionality [7, 8]. Ternary systems are formed by a macromolecule, a small molecule (generally a ligand), and a solvent. These systems are characterized by the preferential adsorption phenomena, due to the differences in both thermodynamic properties and size of solvent molecules [9]. In this context, preferential hydration, preferential interaction, and transfer free-energy parameters could explain all the changes that occur in the system [10-13]. These systems can be investigated in order to carry out complete structural and functional studies of interactions between different substances. It is well known that several salts can act as "salting-in" or "salting-out" agents. They can cause precipitation or solubilization of macromolecules due their interaction with the salts or with the solvent, thus frequently provoking conformational changes [14-18]. Lysozyme is a protein that takes part in the first barrier of defense. It is an enzyme, so it is possible to study its functionality through its lytic activity. It has a low molecular weight (14,000) and high stability. The combination of these factors make this enzyme an ideal macromolecule for studying the influence of preferential interactions on its activity [19, 20]. Aluminum is a trivalent metal, belonging to the third period. It is a very common metal with a large number of applications. It is usually present in an organism but cannot be considered as an oligoelement. It does not accumulate, but its elevated level in an organism can be related to a variety of diseases such as those related to encephalopathy [21].

MATERIALS AND METHODS Materials The and and was

lysozyme of hen egg white (E.C.3.2.1.17; Serva), was dialyzed against water then lyophilized. Aluminum chloride (AiCI3; Panreac), KHEPO 4 (Panreac), K z H P O 4 (Panreac) were used as received. Distilled and deionized water employed.

Solvent and Solutions The solvent was a binary mixture of water/metal chloride. Aluminum chloride concentrations between 1 and 10 mM were used. The protein solution was made gravimetrically: specific volumes of the binary mixtures (water/metal chloride) at 303 K were added to accurate weights of protein. The pH of the solution was 4.5. Potassium phosphate buffer (0.05 mol/1; pH 7.0) and Micrococcus luteus suspension were prepared for the spectrophotometric assays. Micrococcus luteus was triturated with phosphate buffer in an agate mortar and transferred to a volumetric flask. The suspension was diluted such that the absorbance was 0.750 _+ 0.050 at 450 nm (measured against air; d = 1 cm). Lysozyme was prepared immediately before measurement and was diluted 1:250 with repurifled water (control) or H:O(1)/A1CI3(3) mixture. The experiments were carried out in triplicate.

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Instrumentation and Techniques Viscometry. Viscosity measurements were carried out in a modified Ubbelohde hanging level viscometer, immersed in a thermostatically controlled bath at 303 K [22]. The temperature control was +0.01 K. Flow times were reproducible within _+0.03 s. The intrinsic viscosity -q was determined from Fuoss and Strauss [23]: C/~,~p = 1 / A + ( BC ~/2 )/,4

( 1)

where A and B are constants, T/sp is the specific viscosity, and C is the concentration of protein between 1 and 0.5% w/v. Straight lines are obtained when (C/rtsp) is plotted against C 1/2. When the extrapolation to C = 0 is made, 1 / A represents the intrinsic viscosity. Densimetry. The density of the solvents and solutions was measured using pycnometers with a volume between 4 and 5 ml. These were calibrated to measure accurate volumes with distilled water. They were syringe-filled and put in a thermostatically controlled bath at 303 K until temperature equilibrium (10 min). The filled pycnometers were weighed in a Sartorius balance, with 1/10,000 g precision. The partial specific volume of the protein (72) in the solution was determined from density values of solvent (O0) and solutions (p), using the equation [24] ~ = [1 - (ao/C2)T,,,,,,,~]/p,,

t2)

Here (c)p/OC2)T,P, m3 is the change in the density of the solution compared to the protein concentration (C z) at constant temperature (T), pressure (P), and mass of metal (m3). Six different concentrations of lysozyme between 0.2 and 1% w / v were used.

Equilibrium dialysis. Dialysis was carried out using dialysis tubing to obtain the density values of the solution at constant chemical potential of metals (/%), which are required to determine the preferential adsorption coefficient (A) [25]. This parameter was determined using the Kratochvill et al. [26] equation:

A = [(ap/OC2)T.p.~

- (ap/aC2)w.p.~,]/(ap/aC3)r,p,,,,~

-

(3)

Here (ap/OC2)r,p,~ is the change in density with the protein concentration at constant chemical potential (/z i) and (Op/aC~)r,p,,,,~ is the change in the density of the solutions with respect to the metal concentration at constant temperature (T), pressure (P), and mass of protein (m2), The Scatchard [27] and Stockmayer [28] notation has been followed. It is possible to consider that A can be given as A = ( a g 3 / a g 2 ) T ~,,~, where gi is the weight of component i in grams. For diluted solutions, the error is not significant [9].

296 J. M. Socorro et al.

The preferential hydration, (tggl/o~g2)T.~.~ ~ can be obtained from the equation [12, 29]

( 8gl/c9g2

)T, ~ , ~3 = -- ( 1 / g 3 ) (

8g3/Og2)T, ~,l,P'3

(4)

The preferential interaction (J/z2/8m 3)T, P, m ~ shows the relationship between the mutual perturbation of the chemical potentials of components 2 and 3

[30, 31]: (091d,2/PLm3)T,P,rn 2 = --(o~m3/Om2)T, tll,t~3(O~3/~m3)z,p,mz

(5)

the term (~m3/Om2)r,~,,,.3 can be determined from the expression [17]

( Om3/ Om2 )r,u,,~ 3 = ( Me/M3)( Og3/ Og2 )v,.,,u 3

(6)

where M/ and m i are the molecular weight and molarity of component i, respectively, and the self-interaction t e r m (Ot~3/3m3)T.e.m2 can be calculated from (cgl.£3/3m3)T,P, m2 = n R T / m 3 + n R T ( 3 In y__+/3m3)T,P,m2

(7)

where n = 4 for A I C 1 3 , R is the gas constant, and y + is the ionic activity coefficient of the salt. Starting from the preferential interaction parameter, it is possible to determine the transfer free energy A/~2 of the protein according to the equation [32, 33] m~[~2 =

/"~2, m 3 - - /'J~2,w =

fom3(O['l"2/cgm3)r,P,mz dm3

(8)

where /z reflects the relative affinities of salt and water for the protein at the given solvent composition and the subscript w refers to water.

Spectrophotometry. A 2.95-ml volume of Micrococcus luteus was pipetted into a cuvette. The reaction was started with 0.05 ml of lysozyme solution. Changes in absorbance were measured with a Unicam spectrophotometer at 303 K and the decrease in turbidity of the suspension was recorded at 450 nm. The parameter A A / m i n was calculated from the linear part of the curve. The activity was calculated using the equation [34] activity --- (1000/0.05)-(A A / m i n ) (units/ml lysozyme solution)

Solubility measurements. Protein solubility was determined by dissolving the protein in the desired solvent until the solution turned turbid or viscous. It was then dialyzed against several changes of the same solvent for approximately 24 h at 303 K. The solution plus precipitate was then removed from the dialysis bags and centrifuged for 10 min at 13,000 rpm in a Biofuge B (Heraeus) centrifuge. The protein concentration of the supernatant was measured spectrophotometrically by diluting it with the dialyzing solvent. This was defined as the solubility.

PREFERENTIAL INTERACTIONS

297

Refractometric measurements. The specific refractive index increment of lysozyme with concentration, dn/dc, was measured in a differential refractometer equipped with a Spectra Physics H e - N e laser (power = 0.5 mW) of 633 nm at 303 K. The temperature was regulated at _+0.1-K intervals with a thermostatically controlled bath. Fourteen measurements of An were recorded for four different lysozyme solutions (3, 5, 7 and 10 m g / m l ) at each metal concentration. RESULTS AND DISCUSSION The preferential interactions between salts and proteins in aqueous solutions often are accompanied by conformational changes of the macromolecular structures. These conformational alterations can be related to changes in the degree of solvation of the protein. In salt systems, the protein-solvent interactions are determined by different factors: thermodynamically unfavorable salt exclusion, most probably due to the increase in surface tension, which causes salting-out, and the thermodynamically favorable salting-in due to weak ion binding processes [35, 17]. Hence, the preferential solvation or preferential adsorption parameter A indicates the amount of water preferentially adsorbed in or excluded from the protein, giving a positive or negative parameter, respectively. In Figure 1, the solvation parameter for lysozyme in water/A1Cl 3 solvent mixture is shown. At the lower A1Cl 3 concentrations the parameter is negative, so the water is preferentially excluded from the inside part of the macromolecular coil. This

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298 J. M. Socorro et al.

could be due to the establishment of the first ionic interactions between the dissociated forms of the salts and the charged groups of the protein. At higher salt concentrations, the water is preferentially adsorbed inside the protein. The preferential term is related to the net balance of the water/salt ratio adsorbed inside the protein. At 3 mM of AIC13, the preferential solvation parameter is zero, which means that the amounts of both salt and water inside the protein are the same. These results are in accordance with the preferential hydration parameter (Fig. 2), calculated from Eq. (4), which gives insight into what is occurring in the immediate domain of the protein. This parameter reflects both the total protein hydration and total salt binding to the protein. At lower A1CI 3 concentrations, the preferential hydration exhibits positive values. This implies a preferential exclusion of the salt from the protein surroundings. A1C13 is therefore preferentially adsorbed inside the lysozyme, which is in accordance with solvation. This behavior also has been observed for other proteins [36, 37]. In addition to the solvation parameter, preferential hydration is also zero at 3 raM. This preferential hydration behavior is not usual for salting-out agents, which usually have a large positive value for this parameter. The preferential interaction (O/z2/Om 3) can be determined from the preferential solvation parameter using Eqs. (5) and (7). This parameter measures the thermodynamical quality of the solvent medium. Positive values of this parameter mean the salt-protein interaction is thermodynamically unfavorable, whereas negative values indicate favorable interactions. In Figure 3, the variation of this parameter for lysozyme in AICI 3 aqueous solutions is shown and the two

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PREFERENTIAL INTERACTIONS

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behaviors described previously are observed. The parameter is positive until a salt concentration of approximately 3 mM is reached, indicating a thermodynamically unfavorable protein-salt interaction. This could be due to the preferential adsorption of the salt inside the lysozyme (Figure 1). On the other hand, the preferential exclusion of salt is accompanied by negative values of the preferential interaction parameter, so the increase in salt concentration brings about a more favorable protein-salt interaction. A similar dependence has been observed for BSA and /3-1actoglobulin in MgCI 2 at pH 3 [13]. The different protein affinity for a specific solvent system and for pure water is reflected in the transfer free-energy values A/z 2. This parameter is independent of both the interaction mechanisms between the protein and the solvent component and the protein state whether natural or denaturalized. Fitting (Ol~2/Om3) to the polynomial function,

(OtZz/Om 3) = 350.18 - 2.39m 3 + 4.35m32 - 2.39m~ ~ A/~e can be determined by integration of the last function: A/z 2 = f°(350.18 - 2.39m 3 + 4.35m32 - 2.39m33) drn 3 where m 3 is the salt concentration in millimolars. Figure 4 shows A/~ 2 as a function of AIC13 concentration. The transfer free energy is positive at salt concentrations lower than approximately 4 mM, so the

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FIGURE 4. Variation of transfer free energy from water to salt solution for lysozyme A/z2 as a function of A1CI 3 concentration. The initial lysozyme concentration was 1% w/v, and five dilutions were made.

protein solutions in the salt medium is thermodynamically unfavorable compared to the solution in water, with the highest parameter values at salt concentrations of 2 mM. At concentrations greater than 4 raM, from a thermodynamical point of view the protein solution in the solvent mixture is better than that in water. According to the results, the ALE13, salt can be a salting-in or salting-out agent, depending on the salt concentration. At lower concentrations the salt appears to be a salting-out agent, whereas at higher concentrations it behaves as a salting-in agent. In order to confirm this behavior of A I C I 3 for lysozyme, its solubility in the solvent mixtures at a constant chemical potential was determined according to Arakawa and Timasheff [13]. In all cases, no precipitate was obtained after dialysis, indicating that AIC13 is a salting-in agent for lysozyme at all the salt concentrations studied. However, at lower salt concentrations the solvent mixture was less thermodynamically favorable. In order to determine the influence of this salting-in agent on the protein conformation, both viscometric and refractometric measures were carried out.

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301

From Figure 5, we can observe that the intrinsic viscosity of lysozyme as a function of AICI 3 concentration is not linear, indicating conformational transition of the protein [38]. The intrinsic viscosity decreases suddenly at 1 mM of salt, reflecting a more compact lysozyme conformation, which could be due to the preferential adsorption of A l f l 3. As indicated in the preferential adsorption parameter (Figure 1), when the preferential adsorption salt decreases, the lysozyme spreads out, thus causing in turn an increase in the intrinsic viscosity, although the lysozyme conformation is not the same as for water. The conformation transition of lysozyme at 1-mM AIC13 is confirmed by the change in the specific refractive index increment with the protein concentration, (dn/dc), as a function of salt concentration (Fig. 6), which also gives a minimum at the l-raM salt concentration. The influence of these structural changes on lysozyme function was studied, evaluating its lytic activity in the solvent mixtures (Fig. 7). The lysozyme activity decreases almost linearly as a function of AlE13 concentration. At 10 mM of salt, the activity is 75% its initial value, implying that the conformational change observed in the intrinsic viscosity and refractive index increment (dn/dc) with the protein concentration do not involve the active site of the protein. Taniguchi et al. [39] have carried out kinetic studies of binding of Cu 2+, Tb ~+, and Fe 3+ to chicken ovotransferrin. They show that the binding of Cu 2+ and Tb 3+ is very similar and there is only a difference in the relative binding affinity of two protein sizes; this signifies that the different ion charges do not seem to

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be a decisive factor in the binding. A number of interaction studies between lysozyme and cations such as zinc and mercury have been carried out [40, 41]. Both cations can interact with the four disulfide bridges of the lysozyme. Mercury causes an almost absolute loss of lysozyme activity at low concentrations [40] due to its binding at the Asp-52, which is on the active site, whereas the zinc interaction does not yield a complete loss of lysozyme activity because this cation does not directly join on the active site of the protein. The activity data show that A1C1a behavior is similar to Z n A c 2. Both yield a linear loss of activity as a function of salt concentration although this is smaller for A1CI3; at 15 mM of salt, lysozyme activity is approximately 65% for A1C13 and 35% for ZnAc 2. This difference could be due to either the distinct cation size or the charge. Basing our studies on Taniguchi et al. [39] and Olmo et al. [40], the cation size seems to have a greater influence than its charge in the interaction with proteins. The smaller size of the aluminum cation compared to the zinc leads to less distortion of the active site environment, with thus less influence on lysozyme activity. On the other hand, in the case of zinc, A13+ does not interact directly with the catalytic residues of the active site because a total loss of lytic activity is not observed. From the data obtained, we can therefore deduce that behaves as a salting-in agent to lysozyme. At low salt concentrations, a conformational transition is observed that brings about greater preferential binding of the aluminum salt and its smaller solubility power, in accordance with the larger

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preferential hydration and interaction. Despite all this, a drastic change in enzyme activity does not occur, because the protein is in a good solvent medium and aluminum cation binding does not take place on the active site residues.

REFERENCES 1. W. Y. Boadi, J. Urbach, J. M. Brandes, and S. Yannai, Environ. Res. 57, 96 (1992). 2. P. Cammer and A. Johansson, Environ. Health Perspect. 97, 185 (1992). 3. J. M. Nelson, P. G. Duane, K. L. Rice, and D. E. Niewoehner, Am. J. Respir. Cell. Mol. Biol. 5, 328, (1991). 4. U. V. Leontiev, V. N. Uversky, E. A. Permyakov, and A. G. Murzing, Biochim. Biophys. Acta 1162, 84, (1993). 5. M. Buck, S. E. Randoif, and C. M. Dobson, Biochemistry 32, 669, (1993). 6. M. J. Sutcliffe and C. M. Dobson, Proteins 10, 117 (1991). 7. N. Ohno and D. C. Morrison, Eur. J. Biochem. 186, 621 (1989). 8. N. Ohno and D. C. Morrison, Eur. J. Biochem. 186, 629 (1989). 9. J. C. Lee, K. Gekko, and S. N. Timasheff, Methods Enzymol. 61, 26 (1979). 10. E. F. Cassasa, and H. Eisenberg, Adv. Protein Chem. 19, 287 (1964). 11. G. Cohen and H. Eisenberg, Biopolymers 6, 1077 (1968). 12. E. Reisler, Y. Haik, and H. Eisenberg, Biochemistry 16 197 (1977). 13. T. Arakawa and S. N. Timasheff, Biochemistry 29, 1914 (1990). 14. P. H. Von Hippel and K. Y. Wong, J. Biol. Chem. 240, 3908 (1965).

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15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.

P. Tuengler and H. Durchschlag, Anal. Biochem. 98, 481 (1979). T. Arakawa and S. N. Timasheff, Biochemistry 21, 6545 (1982). T. Arakawa and S. N. Timasheff, Biochemistry 23, 5912 (1984). K. D. Collins and M. W. Washabaugh, Q. Rev. Biophys. 18, 323 (1985). N. Imay, H. Akimoto, M. Oda, M. Okazaki, S. Ishibashi, and M. Kurokawa, Mol. Cell. Biochem. 81, 37 (1988). J. M. Teij6n, M. D. Blanco, A. Rodrlguez, J. A. Onrubia, and I. Katime, Rev. Esp. Fisiol. 43, 81 (1987). P. O. Ganrot, Environ. Health Perspect. 86, 363 (1986). I. Katime, J. R. Ochoa, and J. M. Teij6n, J. Chem. Soc. Faraday Trans. 81, 783 (1985). R. M. Fuoss and U. H. Strauss, Po!ym. Sci. 3, 602 (1948). E. Reisler and H. Eisenberg, Biopolymers 6, 1077 (1969). I. Katime and C. Strazielle, Makromol. Chem., 178, 2295 (1946). P. Kratochvil, J. Poychly, and B. Sedlacek, J. Polym. Sci. 10, 2057 (1972). G. J. Scatchard, J. Am. Chem. Soc. 68, 2315 (1946). W. H. Stockmayer, J. Chem. Phys. 18, 58 (1950). S. N. Timasheff and H. J. Kronman, Arch. Biochem. Biophys. 83, 60 (1959). E. F. Cassasa and H. Eisenberg, J. Phys. Chem. 65, 427 (1961). E. F. Cassasa and H. Eisenberg, Adv. Protein Chem. 19, 287 (1964). V. Vlachy and S. Lapanje, Biopolymers 17, 2041 (1978). T. Arakawa and S. N. Timasheff, Methods Enzyimol. 114, 49 (1985). O. Shugar, Biochim. Biophys. Acta. 3, 303 (1952). W. Melander and C. Horvath, Arch. Biochem. Biophys. 183, 200 (1977). D. Kuntz, J. Am. Chem. Soc. 93, 514 (1971). I. D. Kuntz and W. Kauzmann, Adv. Protein Chem. 28, 239 (1974). T. Koseki, N. K. Tabatake, and E. Doi, J. Biochem. 103, 425 (1988). T. Taniguchi, K. Ichimura, S. Kawashirna, T. Yamamura, Y. Tachi'iri, K. Satake, and H. Kihara, Eur. Biophys. 18, 1 (1990). R. Olmo, P. Huerta, M. D. Blanco, and J. M. Teij6n, J. lnorg. Biochem. 47, 89 (1992). J. J. Pesek and J. F. Scheider, J. lnorg. Biochem. 32, 233 (1988).

Received March 15, 1994; accepted July 15, 1994