Behaviour of bovine serum albumin in aqueous solutions of ...nopr.niscair.res.in/bitstream/123456789/15795/1/IJCA 38A(7) 651-65… · salts and DT in 0.02 g cm-3 aqueous bovin serum

1

Indian Journal of Chemistry Vol. 38A, July 1999, pp.651-655

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 Gil1* & Vazid Ali

Department of Chemistry, Panjab University, Chandigarh 160014, Indi a

Received 15 October 1998; revised 22 Febmary 1999

Ultrasoni c velocity (£I), viscosity (11 ) and density (p) of aqueous solutions of disod ium succ inate (DSS), trisodium citrate (TSC), disodium ethylenediaminetetraacetate (DSEDTA), tetraethyl ammoni um bromide (TEAB) and dex trose (DT) have been measured in the concentrati on range 0.01 to 0.40 mol dm·3 at 308.15 K. Such measurement s have also been made at different concentrati ons of these salts and DT in 0.02 g cm-3 aqueous bovin seru m albumin (BSA) and also at varying concentrations of BSA in the range 0 .003 to 0.040 g cm·3 in 0.1480 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= Ilu2p. The isentropic compressibility contribution (~Ks) 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 resu lts show that BSA interacts strong ly with all these salts and DT. TEAB, TSC and DT stabilize the protein by interaction at all BSA concentnit ions. At low concentrations of BSA, 0. 1480 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 water2

, metal ions3.4, organic com­pounds5.(, including some organic solvents7 and sodium dodecyl sulphate6 and other surfactantsH has been inves­tigated. The denaturation ofBSA with guanidine hydro­chlorides has also been studied. Many physicochemical methods such as viscosity>, densit/, NMR6.lo.11 dielec­tric relaxation2.12.13 and ultrasonic absorption 14 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 .15 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 10-7 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%, tri so­dium 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 l­ometer 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 104. 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 1999

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2

1 /~ 14eO 0 +----+--_--

D.DOO 0.010 0-D20 Q.O;JO Q.04O O.oeo 0.000 0.0100.02 _ _ .:J_!, 0 .040 0.0:50

1~ 1~~-----_-----__ ~ 1.100

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Gg.cm·J

Fig. I - Plot of ultrasonic velocity (u) and viscosity (ll) versus concentratioh '(C) of BSA in water at 308. 15K.

" Il. N

'I ~ '-" '"

500

475

450

42~

• 400

375

350 0.000 0 .1 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 308.15 K .

. DSS; 0, DSEDTA;., DT; .... . TEAB ; 6 ; TSC.

ed ly made to check reproducibil ity of results. The over­all accuracy of viscosity measurements was ± 0 .1 %. Densities of all solutions were measured with reproduc­ibility of ±1 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 308.15 ± 0.0 I K.

Results and Discussion Ultrasonic velocity (u). viscosity (11), density (p) and

electrical conductivity (K) of BSA solutions in water in the concentration range 0.003 to 0.040 g cm3 have been measured at 308. I 5K and the results are presented in Fig. I ~ Such studies have also been made at different salt

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425.0

400.0

375.0

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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 308.15 K [Symbols as in Fig. 2).

8r---------------------------.

'--2 <I

-16

-2g0:l:0--0---0.-+~-1 0----0-.0+-12-0 ----0.-+~3-0--0-.0+-14-0· ---10

.050

CI g. em-3

Fig. 4 -Plot of difference of isentropic compressibi lity (6Ks) between 0.1480 mol dm3 salt or dextrose in aqueous BSA solutions & 0.1480 mol dm·J salt in water versus con­centration (C) of BSA at 308. 15 K [ • • BSA and all other symbols as in Fig. 2).

GILL el aL.: BEHAVIOUR OF BOVINE SERUM ALBUMIN IN AQUEOUS SALT SOLUTIONS 653

1.0-.----'----------,

0 .9

0..

~ <J.B s::-

0 .7

O. 6 ~--+--_+_-_+_-_+_-___i 0 .00 0.10 0.20 0.30 0.40 0.50

M/ mol dm-3

Fig. 5 - Plot of viscosity (TJ) versus molarity (M) for some salts and dextrose (DT) in water at 308.15 K

1.1~---~--~------------------,

•

n. 0 .9

~

0.7+----+----+----1----+----1 0.0 0.1 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 308.15 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 0.1480 mol dmJ 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 = IIu2 p

The plots of Ks versus salt concentration (M) (with­out BSA) are shown in Fig. 2. The Ks values for the

, \

1.1

1.0

n. 0.9 0

"-"'. 0.8

O.7+-~-+----+----I---_+_---'--1 . 0.00 0.01 0.02 0 .03 0.04 0 .05

C/ !I.cm-J

Fig. 7 - Plot of viscosity (T]) of BSA in 0.1480 mol dm') solu ­tions of different salts & dextrose versus concentration of BSA( C) at 308.15K [Symbols as in Fig. 2].

0.240

0.210

0 .1 80

0.150 Q. u

0.120 "-. c:-<I 0.090

.. 0.0110

0.0.30

0.000 ' 0.000 0.010 0.020 0.0.30 0 .040 0.050

C/ g.cm-J

Fig. 8 - Plot of difference of viscosity (t1T]) between 0.1480 mol dm') salt or dextrose in aqueous BSA solutions & 0.1480 mol dm') salt in water versus concentration (C) of BSA at 308.15 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 versus M for salts or DT in 0.02 g cm') BSA solution s 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 0.1480 mol dm) salt solution in water, the Ks values for 0.1480 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. 0.1480 mol dm3 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 1999

Il. lJ

"-'" ~ . .,.

<l

20.0

15.0

5.0

0.0 0.0 0.1 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 308.15 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 15 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 15. 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 0.015 g cm3 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 11 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 11 of BSA as a function of protein concentration (C) in 0.1480 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 (Ll11) 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 .015 g cm" ) Ll11 values for BSA in the presence of DSEDTA and DSS fall on higher side of the reference plot. High Ll11 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 Ll11 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 15 g cm') BSA denaturate the prote in 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 differ­

ent 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

4N+

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 0.1480 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 compari son 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 (1412) / 96 - EMR II.

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