Indian Journal of Chemistry Vol. 38A, July 999, pp.65-655 Behaviour of bovine serum albumin in aqueous solutions of some sodium salts of organic acids, tetraethylammonium bromide and dextrose investigated by ultrasonic velocity, viscosity and density measurements Dip Singh Gil* & Vazid Ali Department of Chemistry, Panjab University, Chandigarh 6004, India Received 5 October 998; revised 22 Febmary 999 Ultrasonic velocity ( I), viscosity ( ) and density (p) of aqueous solutions of disodium succinate (DSS), trisodium citrate (TSC), disodium ethylenediaminetetraacetate (DSEDTA), tetraethylammonium bromide (TEAB) and dextrose (DT) have been measured in the concentration range 0.0 to 0.40 mol dm 3 at 308.5 K. Such measurements have also been made at different concentrati ons of these salts and DT in 0.02 g cm- 3 aqueous bovin serum albumin (BSA) and also at varying concentrations of BSA in the range 0.003 to 0.040 g cm 3 in 0.480 mol dm 3 solution of these salts or DT in water. The isentropic compressibilities (Ks) of all the solutions have been calculated from the relation: Ks= Ilu 2 p. The isentropic compressibility contribution (K s) and viscosity contribution (ll) due to BSA in various salt solutions as well as in pure water have been calculated at different concentrations of the protein and plotted against BSA concentration (C). The results show that BSA interacts strongly with all these salts and DT. TEAB, TSC and DT stabilize the protein by interaction at all BSA concentnitions. At low concentrations of BSA, 0. 480 mol dm ) DSS, TSC and DSEDTA denaturate or dissociate (destabilize) the protein by interaction. At high concentrations of protein only DSEDTA denaturates or dissociates the protein while all other salts and DT have stabili zing effect. Investigations of bovine semm albumin (BSA), which is appreciably soluble in water and in many salt solutions, have been made in water undet different conditions of ph, temperature, ionic strength and in the presence of varying concentrations of denaturants l. The interaction ofbsa with water 2, metal ions 3.4, organic compounds5.(, including some organic solvents 7 and sodium dodecyl sulphate 6 and other surfactants H has been investigated. The denaturation ofbsa with guanidine hydrochlorides has also been studied. Many physicochemical methods such as viscosity>, densit/, NMR6.lo. dielectric relaxation2.2.3 and ultrasonic absorption 4 have been used to study the interaction ofbsa with the above molecules. Ultrasonic velocity and isentropic compressibility measurements of BSA in many solvent systems and salt solutions are still lacking. In the present paper, ultrasonie velocity, viscosity and density measurements of BSA have been made in some interesting salt solutions at 308.5 K to get infonnation on the interactions of thi s protein with some sodium salts of organic acids and with tetraethylammonium bromide (TEAB) and dextrose (DT). Materials and Methods Doubly distilled water with conductivity 2-4 x 0-7 S cm- I was used for all measurements. Disodium succinate hexahydrate (DSS) 99% and tetraethylammonium bromide (TEAB) >98% (both from Sisco Research laboratories, Bombay), disodium ethylenediaminetetraacetate dihydrate (DSEDTA) 98%, dextrose (DT) >98%, trisodium citrate dihydrate (TSC) > 99% (all from $.D. Fine Chemicals, Boisar) and BSA (Fraction V, Fluka) were used as received. Desired concentrations of BSA were prepared by weighing the protein and dissolving it in the appropriate volume of water or in the desired salt or dextrose solutions. Vigorous stirring was avoided to prevent foam formation during preparation of protein solutions in all cases. Ultrasonic velocities we,:e measured at 2 MHz frequency using an ultrasonic time in terva l ometer model UTI-lO I from Innovative Instmments, Hyderabad by a pulse echo overlap technique. The absolute accuracy of sound velocity measurements was 2 parts in 0 4. Vi scosities of the salt solutions as well as of the protein solutions were measured using an Ubbelohde suspended bulb viscometer. Measurements were repeat-
652 INDIAN J CHEM. SEC A. JULY 999 j'!.a-------------------- 500 i en Ii.2.. '/ / 4eO D.DOO 0.00 0-D20 Q.O;JO Q.04O O.oeo 0.000 0.000.02.:J_!, 0.040 0.0:50 4 3 2 0 +----+--_-- -----_-----.t...00 D.IIOO '? Ii :::.000 ij.axj o.aoo. +-------.-- o.ooa 0.00 D.02a 0.030 0.040 O.oao. 0.000 0.000.020 0.039 O.D-«I D.OSO Gg.cm J Fig. I - Plot of ultrasonic velocity (u) and viscosity (ll) versus concentratioh'(c) of BSA in water at 308. 5K. 450.0 Il. N 'I '- ' 500 475 450 42 400 375 350 0.000 0. 00 0.200. 0.300 0.400. 0.500 M/mol dm- 3 Fig. 2 - Plot of isentropic compressibility (Ks) versus mol arity (M) for some salts & dextrose (DT) in water at 308.5 K.. DSS; 0, DSEDTA;., DT;... TEAB ; 6 ; TSC. edly made to check reproducibility of results. The overall accuracy of viscosity measurements was ± 0. %. Densities of all solutions were measured with reproducibility of ± x IO g c m 3 by Anton Paar digital densitimeter model 60 and a calibrated cell type 602. All physicochemical measurements were made in a water thermostat bath maintained at 308.5 ± 0.0 I K. Results and Discussion Ultrasonic velocity (u). viscosity (), density (p) and electrical conductivity (K) of BSA solutions in water in the concentration range 0.003 to 0.040 g cm 3 have been measured at 308. I 5K and the results are presented in Fig. I Such studies have also been made at different salt. N I 0.:::. '.... o '- -2 <I 425.0 400.0 375.0 350.0+----+----,,---+- ------ +------ 0.00 0.0 0.20 0.30 0.40 0.50 M/moldm-3 Fig. 3 - Plot of isentropic compressibility (Ks) versus molarity (M) for some salts and dextrose (DT) in aqueous O.02g cm 3 BSA solutions at 308.5 K [Symbol s as in Fig. 2). -6 8r---------------------------. -2g0:l:0--0---0.-+-0----0-.0+- 2-0 ----0.-+3-0--0-. 0+- 4-0 ---. 0 050 CI g. em- 3 Fig. 4 -Plot of difference of isentropic compressibi lity (6Ks) between 0.480 mol dm 3 salt or dextrose in aqueous BSA solutions & 0.480 mol dm J salt in water versus concentration (C) of BSA at 308. 5 K [ BSA and all other symbols as in Fig. 2).
GILL el al.: BEHAVIOUR OF BOVINE SERUM ALBUMIN IN AQUEOUS SALT SOLUTIONS 653.0-.----'----------, 0.9 0.. <J.B s::- 0.7 O. 6 --+--_+_-_+_-_+_- i 0.00 0.0 0.20 0.30 0.40 0.50 M/ mol dm-3..0 n. 0.9 0 - '. 0.8 O.7+--+----+----I---_+_---'--. 0.00 0.0 0.02 0.03 0.04 0.05 C/!I.cm-J Fig. 7 - Plot of viscosity (T]) of BSA in 0.480 mol dm') solutions of different salts & dextrose versus concentration of BSA( C) at 308.5K [Symbols as in Fig. 2]. Fig. 5 - Plot of viscosity (TJ) versus molarity (M) for some salts and dextrose (DT) in water at 308.5 K.-----------------------, Q. u.- c:- <I 0.240 0.20 0. 80 0.50 0.20 0.090 n. 0.9 0.7+----+----+--------+---- 0.0 0. 0.2 0.3 0.4 0.5 Mlmol dm-.3 Fig. 6 - Plot of viscosity.(t]) versus molarity (M) for some salts & dextrose (DT) in aqueous 0.02 gem') BSA solutions at 308.5 K [Symbols as in Fig. 2]. concentrations of DSS, TSC, DSEDTA, TEAB and DT in water and in 0.02 g cm) BSA solutions in water and also in 0.480 mol dm J salt solutions in water with varying BSA concentrations. The salt or DT concentration employed in the present study was between 0.0 I and 0040 dm') and the BSA concentrations between 0.003 and 0.04 g cm'). By using ultrasonic velocity (u) and density (p) data for various solutions, the isentropic compressibility (Ks) for various solutions was calculated by using the equation Ks = IIu 2 p The plots of Ks versus salt concentration (M) (without BSA) are shown in Fig. 2. The Ks values for the, \.. 0.00 0.0.30 0.000' 0.000 0.00 0.020 0.0.30 0.040 0.050 C/ g.cm- J Fig. 8 - Plot of difference of viscosity (tt]) between 0.480 mol dm') salt or dextrose in aqueous BSA solutions & 0.480 mol dm') salt in water versus concentration (C) of BSA at 308.5 K. [, BSA and all other symbols as in Fig. 2]. salts (solutes) investigated decrease in the order: TEAB>DT>DSS>DSEDTA>TSC (Fig.2). The plots of K s versus M for salts or DT in 0.02 g cm') BSA solution in water are shown in Fig.3. In Fig.3, the Ks values at each salt concentration is lower in 0.02 g cm) BSA as compared to the corresponding values for the salts or DT in pure water in Fig. 2. From the Ks values for BSA in 0.480 mol dm) salt solution in water, the Ks values for 0.480 mol dm') salt solution in water in each case obtained from Fig. 2 ere substrated to get isentropic compressibility contribution (tiks) due to BSA at dif ferent BSA concentrations. 0.480 mol dm 3 salt concentration was only a selected value and is close to midway concentration for most of the salt concentrations used in the present study. These tiks values are plotted as a function of BSA concentration (C) to examine the effect of different salts on BSA. The plots of tiks versus protein concentration(c) are shown in Fig. 4. The Ks
654 INDIAN J CHEM. SEC A, JULY 999 Il. lj -. '.,. <l 20.0 5.0 5.0 0.0 0.0 0. 0.2 0.3 0.4 0.5 M/mol dm-3 Fig.9 - Plot of difference of viscosity (TJ) between 0.02 gem') BSA in salts or dextrose solution & 0.02 gem') BSA in water versus concentration of salt (M) at 308.5 K [., BSA and all other symbols as in Fig. 2]. values for BSA without salt at different concentrations of protein were obtained by subtracting the isentropic compressibility of pure water from that of BSA solutions in water at the respective BSA concentrations. These LlKs values without any salt are also plotted against BSA concentrations in Fig. 4 as a reference plot for comparison with other plots for various salts. It is seen that a very interesting effect is obtained from Fig. 4. LlKs values for DSEDTA and DSS fall on the more negative value side of the reference plot upto about 0.0 5 g cm ofbsa, while they fall on the more positive value side or less negative value side for all salts in the higher protein concentration region. More negative LlKs values are due to more structural effects 5. When BSA denaturates or dissociates it occupies more volume of space and due to increased volume more structure is produced. On the other hand when BSA interacts with ions and the protein structure becomes more compact only in certain regions of solutions and the LlKs value will become more positive or less negative. This result indicates that all the salts and dextrose interact with BSA and the protein shows denaturation or dissociation only with DSEDTA and DSS upto 0.05 g cm 3 of the protein. In the higher protein concentration i'ange, all salts show stabilization effect on BSA with the result protein shows no denaturation effect. Similar result is also obtained from viscosity measurements. In Fig. 5 is shown the variation of viscosity (rt) of various salts and DT with salts concentration (M) without BSA. Here the order of change of viscosity is: TSC>DSEDTA>DSS>DT>TEAB.In Fig. 6 is shown the plot of variation of with salt concentration (M) in the presence of 0.02 g cm BSA. The viscosity of the salt solutions in the presence of 0.02 g cm BSA significantly increases from the corresponding values of the salts without BSA. The order of viscosity in Fig. 6 becomes: DSEDTA>TSC>DSS>DT>TEAB which shows that DSEDTA affects the viscosity of BSA much more than any other salt. In Fig. 7, the plot of variation of of BSA as a function of protein concentration (C) in 0.480 mol dm' salt solution of each salt and DT is shown. Like in Fig. 4 where LlKs was plotted as a function of BSA concentration, the contribution of viscosity due to BSA (Ll) for each salt have been calculated and plotted against protein concentrations (C). Viscosity contribution due to BSA in water at different concentrations of protein is also plotted in Fig. 8 as a reference plot. At low BSA concentrations (upto 0.05 g cm ) Ll values for BSA in the presence of DSEDTA and DSS fall on higher side of the reference plot. High Ll values indicate more strong structure which can arise due to denaturation or di ssociation of BSA. In the viscosity effect plots (Fig. 8), DSEDTA throughout the protein concentration range gives Ll value which is higher than the corresponding value of protein in water (reference plot). Viscosity results show that DSEDTA over the whole protein concentration range studied and DSS upto 0.0 5 g cm') BSA denaturate the protein TEAB and DT both show protein stabilising effect at all the concentrations ofbsa. The viscosity results are also in agreement with the isentropic compressibility results., DSS and TSC are the salts of acids of slightly different chain length and differing in some functional group, but they produce almost similar effect on BSA. The effect of DSEDTA on BSA is different due to its long chain and different nature of an ion in volved. Na+ is strongly solvated in water. It is structure promotor in water. If only Na+ ions are involved to produce the effect on BSA, then the effect of DSS and DSEDTA should have been similar because of the same concentration of Na+ ions produced from a specific concentration of salt in both cases. Since DSEDTA strongly produces denaturation ofbsa over the whole protein concentration range, while DSS and TSC produce only in a limited range, therefore, the ethylenediaminetetraacetate ion also plays major role than Na+ ion towards denaturation of BSA. In electrolyte solutions, the behaviour of proteins depend upon the neighbouring environment of the protein molecules. The water structure enhancement by the salts in the neighbourhood of protein will produce the denaturation or stabilization of the protein I. The breaking of hydrogen bonding cf protein by interaction with anion
GILL el al.: BEHAVIOUR OF BOVINE SERUM ALBUMIN IN AQUEOUS SALT SOLUTIONS 655 of the added salt or the breaking of hydrogen bonding of the neighbouring water molecules due to presence of anion will be the reason for protein denaturation in the case of DSEDTA. Two 'ionisable hydrogens which are present in DSEDTA may also be responsible in producing strong denaturation of BSA due to lowering of ph of the solution. Such ph effect, however, is not present in DSS or TSC. Similarly in the case of TEAB, Et 4 N+ ion plays important role for interaction with BSA. This ion has hydrophobic interaction with protein and thus has strong effect on BSA due to hydrogen bonding enhancement. Dextrose interacts with BSA predominantly by hydrogen bonding and thus enhances hydrogen bonding in BSA either by direct interaction or interaction through neighbouring water molecules with the result the protein remains very stable in this solution. The ultrasonic velocity, viscosity and density data for salt solutions, at varying concentrations in 0.02 g cm BSA have also been utilized to obtain information on the interactions of protein with these salts and DT. The results obtained from these measurements are' also in agreement with the results explained above from ultrasonic velocity and viscosity measurements in water and in 0.480 mol dm' salt solutions. When the isentropic compressibility and viscosity of the salt solutions were subtracted from the corresponding values of the salts in 0.02 g cm BSA, the contribution of 0.02 g cm protein was obtained. For illustration, such viscosity contribution effect of 0.02 gem BSA in different salt solutions is plotted in Fig. 9. The effect of 0.02 g cm BSA in water is also plotted as a reference plot for comparison with other plots. In the case of DSEDTA, the tlll values for BSA are higher than the reference plot for BSA in pure water. These results also confirm that DSEDTA destabilizes BSA and produces higher viscosity of protein in salt solution as compared to that in pure water. Acknowledgement DSG thanks the CSIR, New Delhi, for a research grant under the scheme I (42) / 96 - EMR II. References Arakawa T, Bhat R & Timasheff S N, Biochem, 29( 990) 924. 2 Bone S, Gascoyne P R C & Pethig R, J chern Soc Faraday Tran s, 73(977) 605. 3 Sun S F, Chang T S & Del Rosario N 0, Inl J Pept Prot Res, 6 ( 974) 87. 4 l anado M, Yano Y, Nishida H & Nishida T, J soln Chelll, 5 (986) 839. 5 Lee C & Timasheff S N, Biochem, 3 ( 973) 257. 6 Oakes, J chem Soc Faraday Trails, 70 ( 974) 2200. 7 Gill D S, Sharma R, Ali V, Khajuria R & Singh, fndian J Chel, 35A(996) 4. 8 Nozaki Y, Reynolds A & Tanford C, J bioi Chel, 249 ( 974) 4452. 9 Hunter M, J phys Chem, 70 (966) 3285. 0 Nome J E, Lilja H, Lindman B, Einarsson R & Zeppezauer M, EurJ Biochem, 59( 975) 463. Oakes J, Eur J Biochel, 36 ( 973) 553. 2 Grant E H, Keefe S E & Takaschima S, J phys Chem, 72 ( 968) 4373. 3 Grant E H, J motec Biol, 9 (966) 33. 4 Barnes C, Evans A & Lewis T J, J aco4sl Soc Am, 80 ( 986) 29. 5 Gill D S, Kaur T, Kaur H, Joshi I M & Singh J, J chern Soc Faraday Trans, 89, (993) 737.