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701—The interaction between bovine serum albumin and molybdate ions
Bioelectrochemisrry and Bioenergetics, 13 (1984) 329-342 A section of J. Electroanal. Chem., and constituting Vol. 174 (1984) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
329
701--THE INTERACTION BETWEEN BOVINE SERUM ALBUMIN AND MOLYBDATE
J.P.S. ARORA, Department (Revised
IONS
R.P. SINGH,
Smt. S. JAIN,
S.P. SINGH
of Chemistry, D.A. V. College, Muzaffarnagar
manuscript
received
October
and A. KUMAR (India)
18th 1984)
SUMMARY The interaction between molybdate ions and bovine serum albumin (BSA) has been studied employing transmittance measurements. In the acidic range, the stoichiometry of the combination between molybdate and protonated nitrogen groups of protein was found to be one-to-one. The pH-dependent binding result has been ascribed to the existence of anionic as well as cationic species of molybdenum(V1). The pH-metric titrations and ultraviolet absorption spectra of molybdenum(VI)-bovine serum albumin mixtures also supported the existence of polymerisation-depolymerisation of molybdenum as well as deprotonation and conformational changes in the protein molecule. The free energy, enthalpy and entropy of binding have been determined and the effect of molybdate ion on the heat of ionization and pKint, of BSA groups has’been discussed.
INTRODUCTION
The biological implications of polyanion-polycation interactions are well established. Several workers have investigated the stoichiometry of such combinations to explain the mechanism of some important biological processes [l-9]. Hummel and Witzel have used pH measurements to study the binding of nucleotides to pancreatic ribonuclease [lo], while other workers emphasized the valency states of metals in metalloproteins by this method [ll-141. .A few workers have also used turbidimetric titrations to determine the equivalence points in such complexations which involve polyions of opposite charges [15,16]. Owing to the heparin-like activity of phosphomolybdate [17,18] and the biological significance of molybdate ion [19,20], Malik and Arora [21-231 and Arora et al. [24,25] have extensively studied oxomolybdenum-protein interactions employing physico-chemical methods. Recently, the results on the binding of molybdenum(V1) with bovine serum albumin employing polarographic and equilibrium dialysis methods were reported [26]. However, this literature survey revealed that no such studies between oxomolybdenum(V1) and bovine serum albumin have been made so far using transmittance and pH measurements. In this paper the energetics of binding between oxomolybdenum ion and bovine serum albumin is reported employing transmittance and 0302-4598/84/$03.00
0 1984 Elsevier Sequoia
S.A.
330
pH measurements. cations for bovine
The thermodynamics of the interaction serum albumin are discussed.
and the structural
impli-
EXPERIMENTAL
Reagents A solution of bovine serum albumin (BSA) (Armor and Co. Product, 11z.w.69 000) was prepared in double distilled water. The strength of the protein solution was determined by drying its known volume in an air oven at 105110°C. Sodium molybdate (E. Merck) was dissolved in distilled water and its molybdenum content was determined gravimetrically. Buffers and other solutions were prepared from reagent grade chemicals. A potassium chloride (BDH) solution was used for the maintenance of ionic strength of the reaction mixtures.
Techniques Transmittance measurements The transmittance of BSA-Mo(V1) mixtures, which can be taken as an index of turbidity developed, was measured by means of the Bausch and Lomb Spectronic -20 instrument at 375 nm. For these measurements the following types of experiments were arranged: (i) 0.5 g/dm3 BSA was put in different boiling tubes and the total volume was kept at 10.0 cm3 by adding fixed amounts of different buffers and double distilled water; (ii) 0.5 g/dm3 BSA and 0.01 M Mo(V1) were put in different tubes and the total volume was made 10.0 cm3 as in (i); (iii) 2.0 g/dm3 BSA was put in different tubes and to each was added different amount of Mo(V1) and the total volume was made 10.0 cm3. Several different sets were prepared at different pH values (2.0 to 5.0). Similar sets were also prepared keeping the amount of molybdenum fixed and varying the amounts of protein. The effect of potassium chloride was studied at one specified pH to show the effect of ionic strength on the extent of complexation. The transmittance of all the mixtures was measured immediately after preparing the reaction mixtures.
pH-measurements These were made on a Systronic pH-meter using a wide range glass electrode. The instrument was standardized against 0.05 M potassium acid phthalate (pH 4.0) and 0.05 it4 borax (pH 9.2) for the acidic and basic ranges respectively. Three different sets of mixtures were prepared: (i) varying amounts of hydrochloric acid and potassium hydroxide (each 0.10 M) were put in different tubes and 1.0 cm3 of 3.0% isoionic BSA was added to each of them. The total volume was made 10.0 cm3 by addding water and enough 1.0 M KC1 to make the total ionic strength of the solution 0.15;
331
(ii) varying amounts of acid or base were mixed with 1.0 cm3 of 3.0% BSA and 1.0 cm3 of 0.01 M molybdenum(VI), keeping the total volume at 10.0 cm3 as in (i). These measurements were carried out at two temperatures, uiz. 23 and 33°C; (iii) 10.0 cm3 of 0.3% BSA solution of different pH values were titrated against 0.10 M sodium molybdate solution of the same pH as the protein. The pH values of the above mixtures were recorded immediately after mixing and after 24 hours. These measurements at pH 5.35, 9.85 and 11.50 were carried out at 25 and 40°C respectively. Ultraviolet spectroscopy
These measurements were made on a Carl Zeiss specord U.V. visible spectrophotometer. The spectra of a very diluted BSA solution (0.03 g/dm3) in the absence and presence of 1.0 X 10e5 M molybdenum(V1) were recorded at four different pH values (5.57, 7.5, 9.40 and 11.40). The shifts in the curve of BSA in the presence of molybdenum(V1) were taken as a qualitative evidence of interaction. RESULTS
AND DISCUSSION
Bovine serum albumin (BSA) formed insoluble complexes with molybdate ions below its isoelectric point (IEP). The different aspects of the insoluble complex formation such as pH, protein and molybdate concentration are critically discussed below. Effect of pH on transmittance
Figure 1 shows the percentage transmittance of BSA in the presence and absence of molybdenum at different pH values. A minimum in the vicinity of the isoelectric
1OOr
BSA(O.OS%) BSA(O.O5%) tO.OlOM Na,MOO/&
00s --O-=+7. z s 60 -.z $ e I40
?-?J,,, 20 3.2 2.4
PH 4.0
4.6
5.6
6.0
Fig. 1. Percentage transmittance plotted against pH for BSA in the presence molybdenum(V1). (0) 0.05% BSA alone; (0) 0.05% BSA-tO.010 M sodium molybdate. 0.15.
and absence Ionic strength
of =
332
point (pH 5.0) is obtained in the case of BSA. Similar types of maxima and minima were observed by Arora and Singh [27] in the case of denatured and S-ovalbumins, while Malik and Arora [21] were incapable of showing any such behaviour in the case of ovalbumin and gelatins (see also Ref. 22). It appears that all corpuscular proteins possess the property of salting out at their isoelectric points. It may be concluded that the presence of a large proportion of hydrophilic groups in gelatin imparts a greater stability over the entire pH range, while BSA and other related proteins having a smaller number of hydrophilic groups show a greater tendency towards precipitation in the vicinity of their isoelectric points. On addition of Mo(VI), the transmittance largely decreases in the BSA-Mo(V1) system. The minimum does not occur at the original isoelectric point, but at a much lower pH. The shift in the IEP of BSA from 5.0 to 4.6 on addition of Mo(V1) may be explained in terms of the reaction of the molybdate ions with the protonated basic groups of BSA. Owing to the binding of molybdate ions with the positively charged nitrogen groups, the total positive charge on the BSA molecule decreases. This explains the shift of IEP towards a lower pH value. Stoichiometty of Mo(VI)-BSA interaction The extent of binding of molybdate ions to BSA was computed by carrying out direct titrations (Fig. 2). The break in the transmittance uersus concentration curves indicates the point where the formation of the insoluble complex is complete. Furthermore, if this point does not indicate the completion of the formation of the insoluble complex, then with the addition of more molybdate there would be an even larger decrease in the transmittance. From the breaks in the curves, the number of moles of molybdate bound per mole of BSA ( VM) was found using the relationship, V, = C&P], where [P] is the molarity of protein used and C, the amount of
100
60
I
0
I
I
1
I
I
2
a
kol (IO-?rj) I’ I 4 4.5
i'i
I_
3
Fig. 2. Transmittance titration curves of fixed BSA (0.05%) against varying (ionic strength, 0.15) at different pH values (indicated at the curve).
concentrations
of molybdenum
333 TABLE
1
Binding of molybdenum 2). 30°C PH
to BSA (0.28
Bound molybdenum (G,) (1O-4 w
x
10m4 M) at different
Moles of molybdenum bound per mole of
pH values from transmittance
LogK
AG kcal/mol
7.00 7.03 7.07 7.15 7.21 7.29 7.58 8.10
- 9.597 -9.651 - 9.706 - 9.816 - 9.884 - 10.01 - 10.40 - 11.10 _
method
(Fig.
BSA(&I 1.0 1.5 2.0 2.6 3.0 3.5 4.0 4.6 5.0
35.0 32.5 29.4 25.0 22.0 18.0 10.0 2.8
125.0 116.0 105.0 90.0 82.0 65.0 35.0 10.0
molybdate (molarity) bound at the inflexion point. The values of V, at different pH values are given in Table 1. The equilibrium constant of the precipitation reaction is given by the following expression [28],
KS =[P]m;MO]z where KS = instability constant of the insoluble complex, which is the reciprocal of the solubility product, and m and z represent the number of moles of protein and molybdenum i.e. the concentration of protein and molybdenum in mol/dm3 at the equivalence point (breaks in titrations). In these studies at the inflexion point in the transmittance uersu.s concentration curves (Fig. 2) the molybdenum used (C,) is taken as the total concentration bound, [MO]‘, to the total protein, [Plm, taken at different pH values. The instability constants calculated in the case of the present irreversible systems with the help of above equation are presented in terms of log K in Table 1. These values of log K at 30°C were used to determine the free energy change at the respective pH and are given in Table 1. A perusal of the binding data i.e. V,, log K and AG, (Table 1) revealed that the amount of molybdenum bound at any pH is nearly the same as the total number of hydrogen ions bound to this protein, i.e. the total number of protonated cationic sites at any pH. It is also evident that V, increases with decreasing pH. The instability constants characterizing the irreversible precipitation reaction decrease as the pH decreases. This order of log K values is in agreement with log K for the molybdenum-OA system [27]. Approximately similar values of log K and free energy changes (Table 1) pointed to the significance of the involvement of the same cationic groups on the BSA molecule in the binding process. The enhanced binding in the low pH range can be explained in terms of the electrostatic attraction between
334
the cationic protein and the anions of the polyacid [21]. This suggests an interaction between the molybdate ion and protonated nitrogen atom as suggested by Steinhardt and Fugett [29] and Pankhurst and Smith [30] in the case of detergent anion-protein interaction. In these experiments the precipitate does not dissolve in excess of molybdate, but preliminary experiments revealed that excess of BSA dissolved the precipitate. The dissolution may be due to the unequal amount of charge on the protein which caused the shifting of the solubility product of molybdate-BSA complex. Furthermore, the BSA-molybdate complex exists as a colloidal precipitate which undergoes sedimentation on keeping, however, with the excess of protein a protective layer is formed keeping the complex in a stable colloidal state. There is evidence of the protection of many hydrophobic colloidal systems by the adsorption of gelatin on the surface of the sol particle through the undissociated carboxyl groups [31,32]. E;ffct of molybdenum on hydrogen ion equilibria of BSA Some evidence of binding of Mo(V1) to BSA has .been obtained from pH titrations. Figure 3 shows the titrations of BSA in the absence and presence of 0.001 M sodium molybdate solution. In the alkaline range the BSA curve lies above the corresponding curve of the BSA-Mo(V1) mixture, this indicates the removal of hydrogen ions from the cationic protein sites. However, in the acidic range there are two distinct regions; in one the Mo(VI)-BSA shows greater proton binding than the
2.5 2.0
1.0
0
1.0
2.0
2.5
Fig. 3. pH-titration curves of BSA (0.3%) in the absence and presence of 0.001 M sodium molybdate at ionic strength of 0.15; (0) BSA+ Mo(VI); (0) BSA alone.
protein, and in the other the complex binds less protons than the protein. The first region lies between pH 3.0 and 5.0, which is indicative of some change in the structure of the protein. Resnik and Klotz [33] observed similar results in the titrations of polyamines in the presence of sodium dodecylsulphate (SDS). In each titration with SDS an insoluble complex was formed ultimately. The formation of this complex produced upward drifts in pH, probably due to slow diffusion of protons in the insoluble complex. Continuous stirring resulted in reproducible results. In the pH range from 2.0 to 3.0, the anionic molybdate species undergoes changes into a cationic one, hence much more protons are free which results in a lowering of pH. The observed pH values (Fig. 3) obtained at constant ionic strength (0.15) were used to determine the number of hydrogen ions dissociated per mole of BSA (y) in the absence and presence of added molybdenum at two temperatures, uiz. 23 and 33°C using Tanford’s equation [34]. The hydrogen ions dissociated (y) determined at two temperatures were plotted against pH in view of predicting the effect of added molybdenum on the protein titrations. From the temperature dependence of y, the apparent heat of dissociation (AH ion), i.e., the difference of pH values at constant value of y and temperature, in the absence and presence of molybdenum was determined according to Wyman’s method [35], using the equation AHion= -2.303
RT
2 @pH)
dT
where R is the value of the gas constant (2 calorie), T is the absolute temperature, dT is the difference of two temperatures, and d pH is the difference of pH values at constant value of y. The apparent heat of ionization was plotted against the number of hydrogen ions dissociated per protein molecule ( y) and the values of the apparent heat of ionization for different dissociable groups of BSA in the absence and presence of molybdenum are given in Table 2. Owing to the involvement of guanidinium groups of arginyl residues in interaction with molybdenum the dissociable groups are removed from the titration and therefore it was not possible to determine their heat of ionization (A Hi,,). These values of heat of ionization show
TABLE 2 Apparent heat of ionization molybdenum Group
(kcal/mol)
Expected range for AHi,,
Carboxyl Imidazole Amino Guanidino
1.5-3.0 6.9-7.5 10.0-12.0 12.0-13.0
of dissociable
groups of BSA in the absence and presence of
AHi, in absence of molybdenum
AHion in presence of molybdenum
2.40 7.20 11.00
2.00 6.80 10.80
336
that the heat of ionization of carboxyl, imidazole and amino group is depressed to the extent of 17% in the presence of molybdate ions. Applying Tanford’s equation [34], a plot of pH/log (y/n - 1) uersus h gave straight lines (in this equation the quantity Z of the original equation was replaced by h, the number of protons bound per protein molecule). The value of W, the electrostatic interaction factor was determined from the slope of the straight line and was found to be 0.0138 and 0.0125 in the absence and presence of molybdate ions respectively. The values of intrinsic association constant for carboxyl groups was found to be 3.4 in the absence and 3.9 in the presence of molybdate ions. The increase in pK of carboxyls in the presence of molybdenum shows the formation of the BSA-molybdate complex and the value of extra dissociated hydrogen ions (r) suppresses the dissociation of carboxyl groups. Binding of molybdate ions to BSA by pH displacements
Arora and Singh [36] studied the effects of added sodium molybdate on the pH of the isoionic S-ovalbumin and observed that there was a decrease in pH with increasing concentration of molybdate. This decrease in pH was attributed to the preferential binding of one of the ions of the added electrolyte to the protein. In such cases a negative ApH indicates cation binding while a positive ApH is indicative of anion binding. In order to note whether molybdenum reacts as anion or cation or as a Lewis acid, its interactions with BSA at different fixed pH values were investigated from pH 1.0 to 12.0 and these titrations are shown in Fig. 4 in the form of ApH plotted against molar concentration (M) of molybdenum(V1) added. It is interesting to note that except at the pHs 3.0, 3.5 and 4.1, at all other pH values studied the ApH is found to be negative. The negative ApH values showed that
-0.6 0
I
I 0.2
Y O.lOH
I
a 0.4
Na, MO 04 cm3 I1
0.6
I
I 0.8
I
I 1.0
Fig. 4. pH titrations of BSA against sodium molybdate curve.
at different fixed pH values indicated on each
337
molybdenum binds in the form of a cation while the positive values indicated its interaction as anion. The nature of negative or positive ApH indicated the existence of anionic and cationic species of molybdenum. Similar types of pH displacements were observed by Hummel and Witzel in the interaction of nucleotids with pancreatic ribonuclease [lo]. The amount of ligand bound per mole of BSA (I’,) was calculated by the method of Scatchard and Black [37], from which the value of bound (C,) and free (C,) molar concentration of molybdenum was determined from the known molar concentration of BSA. The experimentally, determined binding data, uiz. V, and C, were analysed by means of Scatchard equation [38] in the form,
+Kn--KV, F
where V, and C, have their usual meanings, K is the average apparent association constant for binding at each protein site, and n is the average maximal number of ligand binding sites on a protein molecule with the same association constant K. If all the binding sites are equivalent and independent, a plot of V,/C, as a function of V, would give a straight line such that the intercept on the V,/C, axis is Kn, as V, approaches zero as a limit, and the intercept on the V, axis is n, as Q/C, approaches zero as a limit. Deviation from linearity occurs when binding takes place at more than one set of sites with different values of the association constant. The contribution of electrostatic factors may also produce deviations [23,24]. The Scatchard plots at pH values 5.35, 7.50 and 2.50 were found to be non-linear. The non-linear nature of the plots may be attributed either to the saturation of the same class of primary sites (nr) or to the involvement of second (n2) or subsequent classes of sites in the molybdenum interaction [39]. Arora el al. [26,40] have earlier reported that the non-linearity of the plots was due to the involvement of binding sites of different affinity in molybdenum-protein interactions. However, a linear relationship was found to exist between V,/C, uersus V, plots at pH values 9.85 and 11.50, which indicated the involvement of a single primary class of equivalent and independent sites in interaction; if a second class of sites existed it would have a much smaller association constant than the primary class of binding sites. The number of primary binding sites (nr) and their association constants (K,) determined from the intercept of the V, and V,/ C, axes are given in Table 3. To determine the number of sites in the second class ( n2) and their binding constant (K,), we plot V,,/C, uersuS V,,, where V,, = [V, - number of primary binding sites (nl)]. This procedure substracts out the contribution of the strung primary binding sites (nr) from the data. This is valid only when the two classes of sites are non-interacting, i.e., binding at one site does not affect binding at the other. Applying this procedure the number of secondary binding sites (n2) and association constant (K2) were found to be 6 and log K 2.48 at the two temperatures (25 and 40%) and at pH 5.35. The standard free energy change calculated from pK (at the given pH and temperature) was found to be - 3.40 kcal/mol. These results show that the secondary sites are weaker and destroyed at higher pH values.
338 TABLE 3 Binding constants of BSA-molybdate PH
n
25OC 5.34 7.50 9.50
11 9 8
9.85 11.50 40°C 5.34 7.50 9.50 9.85 11.50
log K
system from pH displacement method AGO kcal/mol
AS” Cal mol-’
3.95 3.78 3.61
- 5.415 -5.182 - 4.949
-
7 3
3.60 3.60
- 4.835 - 4.835
+ 14.0 + 8.6
9
3.81
- 5.486
+4.7 -
6 2
3.58 3.52
- 5.155 - 5.068
+ 14.6 + 6.0
K-’
AH’ kcal/mol
+5.0
-4.0 - 0.572 - 2.266
The small number of primary binding sites (n,) i.e. 11, 9, 8, 7 and 3 at pH values 5.35, 7.50, 9.50, 9.85 and 11.50, respectively, showed that not all the protonated nitrogen groups are equally available for interaction. The reason for the involvement of a smaller number of binding sites may be a conformational one. This protein possesses a significant structure brought about by hydrophobic interactions, hydrogen bonding and other intersegmental cohesive forces [41]. However, since the folded core of the globular protein is known to be inaccessible to water, a rough estimate of 40-608 of the total sites may be available to interact with the anions. Considering this constraint imposed by the chain folding of BSA, it appears that anion binding to this protein occurs in a restricted way [42]. The lesser value of binding sites (n) at pH 9.85 and 11.50 could also be explained on the basis of successive deprotonation of protein groups. The pK values for histidyl and lysyl amino acid residues are 6.90 and 9.85, while for guanidinium of arginyl residues it is greater than 12.0. Thus at pH 9.85 and 11.50 the sites involved in molybdenum interaction must be the guanidinium groups of the protein. This argument also goes support from the apparent heat of ionization of guanidinium groups in the presence of molybdate ions. However, the involvement of a smaller number of guanidinium sites could be due to a large amount of electrostatic repulsion between anionic protein and the molybdate ions, and also some of these groups may be irreversibly bonded to the carboxyl groups. Thermodynamic
parameters
The oxomolybdenum(V1) ion binding by BSA is observed to be modified by pH and temperature. The number of sites decreases with rising pH and temperature but log K remains nearly constant. The significance of constant values of log K (almost independent of pH and temperature is that only a single primary class of sites is reacting, and the appearance of their different number is therefore not responsible
339 0.4 -
-2 5 0.2 -g z O-
, 225
-0.1 Fig. 5. Absorption spectra of BSA (0.003%) in the absence and presence of molybdenum (1.0 X 10m5 M) at different pH vdues. (3) BSA + MO (pH 9.40); (1) BSA (pH 9.40); (4) BSA + MO (pH 7.50); (2) BSA (pH 7.50).
for decreased binding which may then be due to the decreased availability of the same class of sites owing to irreversible effects in the protein structure. The free energy (AGO), enthalpy (AH’) and entropy (AY) changes of the interaction were calculated using standard methods (Table 3). The positive entropy value probably indicates that water of hydration is released both from the protein and oxomolybdenum, and that the configuration of the polypeptide is changing to a random coil as the complex formation occurs. The higher values of entropy and enthalpy changes at pH 9.85 possibly indicate that all three factors mentioned above may be involved at this pH. ,Spectral studies
The changes of the absorption spectrum with pH, for BSA in the presence of molybdenum (Figs. 5 and 6), are assumed to be related to the changes in hydration and conformation discussed above. The molecular extinction coefficients of molybdenum-BSA complex and BSA were calculated at the wavelength of maximum absorption and are given in Table 4. At pH values 5.57, 7.50 and 9.40 both
0.6-
0,
I 225
235
‘.\,
\
245
255’%60
-O.lFig. 6. Absorption spectra of BSA (0.003%) in the absence and presence of molybdenum (1.0 X lo-’ (4) BSA+Mo (pH 11.4); (3) BSA (pH 11.4); (2) BSA+Mo (pH 5.57); (1) BSA (pH 5.57).
M);
340 TABLE 4 Molecular extinction coefficients (mol/dm)) for wavelength of maximum absorption denum-BSA complex and BSA alone at different pH values PH
X (nm)
Molybdenum + BSA x lo4
BSA alone X lo9
5.57 7.50 9.40 11.40
235 235 235 240
1.00 1.50 2.00 1.25
9.50 3.10 0.75 10.00
of molyb-
BSA and molybdenum-BSA complex have a maximum absorption at 235 nm, but at pH 11.40 it is shifted towards a higher wavelength, i.e. 240 nm. Moreover, the maxima at this pH are much broader than at other pH values. The shift of wavelength of maximum absorption from 235 to 240 nm at pH 11.50 may be ascribed to protein unfolding [43] which may cause a change in the configuration of some of the molybdenum binding sites on the protein molecule. The trend of the extinction coefficient (Table 4) of BSA-molybdenum complex is analogous to its enthalpy change while that of BSA is analogous to its entropy change. The values of the thermodynamic parameters and of the molecular extinction coefficients of molybdenum-BSA complex are in line with the fact that the nature of the binding sites is different in lower and higher pH. Nature of interaction The results discussed above showed that Mo(V1) reacts as anion or cation (Lewis acid) depending upon the pH of the system. In the acidic range, the interaction between polyacid anions and protonated groups of BSA can be formulated in analogy to the description of the ovalbumin-chloroaurate interaction [44]. Presumably, the anion is first attracted to the positive centres on the protein surface by coulombic forces, and when it is sufficiently near water molecules are removed, making the lone electron pair on nitrogen available for coordination with the molybdenum of the molybdate ion. The shift of the titration curve of Mo(VI)-BSA complex towards higher pH may perhaps be in line with the type of bonding reported in Mo(VI)-N-methyliminoacetic acid [45] and the enzyme nitrogenase [46] because hydrogen ion react with the molybdate ion to form Mo(OH)O, [47] which in turn is removed as water molecule on reacting with BSA. In the lower pH range, the complex molybdate ion may contain many hydroxyl groups [48], hence a polynuclear complex may be formed through coordination and hydrogen bonding with protonated nitrogen and undissociated carboxyl groups of BSA. A similar type of reaction was suggested by Malik and Arora [49] and Malik et al. [50] in the binding of silicic acid with proteins. However, in the higher pH range the decreased binding can be explained on the basis of the following points:
341
(i) The number of hydroxyl groups in the polyacid decreases and as a result the number of linkages also progressively decreases with decreasing metal oxide content; (ii) The increased negative charge on the protein molecule would contribute to a repulsive electrostatic effect on the polyanions; (iii) The progressive deprotonation of nitrogen atoms would result in a smaller number of positive loci, therefore resulting in a decreased binding. The ions bound are anions; however, these results do not allow us to determine which groups are involved in binding with molybdate ions. The deprotonation of cationic and carboxyl groups is responsible for the decreased binding above the IEP of BSA. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
AS. Michaels and R.G. Miekha, J. Phys. Chem., 65, (1961) 1705. A.S. Michaels, L. Mir and N.S. Schiender, J. Phys. Chem., 69, (1965) 1947. M.B. Mathews, B&hem. J., 96 (1965) 710. E. Goldsawor and F.W. Putnam, J. Phys. Colloid. Chem., 54 (1954) 79. J. Monod, J.P. Chargeux and F. Jacob, J. Mol. Biol., 6 (1963) 306. R.C.C. Huang, J. Booner and K. Murray, J. Mol. Biol., 8 (1964) 54. D.E. Olins, A.L. Olins and R.H. Von Hippel, J. Mol. Biol., 24 (1967) 157: A.M. Liquori, L. Constantino, V. Elia, and V. Vitagliano, J. Mol. Biol., 24 (1967) 113. M. Leong and G. Felsenfeld, Proc. Natl. Acad. Sci. USA, 56 (1966) 1325. J.P. Hummel and W. Witzel, J. Biol. Chem., 241 (1966) 1023. S. Lindskog, J. Biol. Chem., 238 (1963) 945; Biochemistry, 5 (1966) 2641. J.E. Coleman, J. Biol. Chem., 22 (1967) 5521. B.F. Van Gelder and F.S. Slater, Biochim. Biophys. Acta, 73 (1963) 663. B.F. Van Gelder and A.O. Muisfers, B&him. Biophys. Acta, 81 (1965) 405. R.M. Fuoss and H. Sadex, Science, 110 (1949) 552. H. Teerayama, J. Polym. Sci., 8 (1952) 243. J.H. Bradgon and R.J. Havel, Science, 120 (1954) 113. J.H. Bradgon and R.J. Havel, Circulation, 10 (1954) 591. B. Rosaff and H. Spencer, Nature (London), 202 (1964) 410. C.L. Comar, Nucleonics, 3 (1948) 34. W.U. Malik and J.P.S. Arora, J. Electroanal, Chem., 26 (1970) 414. W.U. Malik, S.M. Ashraf and J.P.S. Arora, J. Ind. Chem. Sot., 46 (1969) 609. W.U. Malik and J.P.S. Arora, J. Electroanal. Chem., 22 (1969) 539. J.P.S. Arora, R.P. Singh, D.S. Jadon and S.K. Mittal, J. Ind. Chem. Sot., 57 (1980) 180. J.P.S. Arora, R.P. Singh, S.P:Singh and V.K. Singhal, Stud. Biophys., 99 (1984) 97. J.P.S. Arora, R.P. Singh, D. Soam, S.P. Singh and R. Kumar, Bioelectrcchem. Bioenerg., 10 (198 3) 441. J.P.S. Arora and R.P. Singh, Kolloid. Z. (Comm.), 1983. J. Inczedy, in Analytical Application of Complex Equilibria, Wiley, New York, (1976) p. 182. J. Steinhardt and C.H. Fugett, J. Res. Natl. Bur. Standard, 29 (1942) 315. K.G.A. Pankhurst and R.C.M. Smith, Trans. Faraday Sot., 43 (1947) 506. A. Pactor and R. Matalon, Discuss. Trans. Faraday Sot., 18 (1954) 161. B. Jergensons, Macromolec. Chem., 6 (1951) 30. R.A. Resnik and I.M. Klotz, J. Am. Chem. Sot., 77 (1955) 5015. C. Tanford, J. Am. Chem. Sot., 72 (1950) 441. J. Wyman, J. Biol. Chem., 127 (1939) 1. J.P.S. Arora and R.P. Singh, Stud. Biophys., 83 (1981) 181.
342 37 38 39 40 41 42 43 44 45 46 47 48
G. Scatchard and E.S. Black, J. Phys. Chem., 53 (1949) 88. G. Scatchard, Ann. N.Y. Acad. Sci., 51 (1949) 660. N.D. Chasteen and J. Francavilla, J. Phys. Chem., 80 (1976) 867. J.P.S. Arora, R.P. Singh, S.P. Singh and S.P. Malik, Stud. Biophys. 29(2) (1984) 97. W. Kauzmann, Adv. Protein Chem., 14 (1959b) 1. S.F. Sun, N.O. Del-Rosario and L.A. Goldstein, Int. J. Peptide Protein Res., 5 (1973) 337. S.F. Sun, Arch. Biochem. Biophys., 129 (1969) 411. J.P. Craig (Jr.), A.G. Garrett and H.B. Williams, J. Am. Chem. Sot., 76 (1954) 1570. S.I. Chan, R.J. Kula and D.T. Sawyer, J. Am. Chem. Sot., 86 (1964) 377. R.C. Burns and R.W.F. Hardy, Biochem. Biophys. Res. Commun., 39 (1970) 90. P.F. Knowles and H. Deibler, Trans. Faraday Sot., 64, (1968) 977. H.J. Emleus and J.E. Anderson (Editors), Modern Aspects of Inorganic Chemistry, The English Language Book Society, London, (1961) p. 322. 49 W.U. Malik and J.P.S. Arora, J. Electroanal. Chem., 24 (1970) 459. 50 W.U. Malik, J.P.S. Arora, D.S. Jadon and R.P. Sin& Egypt. J. Chem., 23 (1980) 229.
329
701--THE INTERACTION BETWEEN BOVINE SERUM ALBUMIN AND MOLYBDATE
J.P.S. ARORA, Department (Revised
IONS
R.P. SINGH,
Smt. S. JAIN,
S.P. SINGH
of Chemistry, D.A. V. College, Muzaffarnagar
manuscript
received
October
and A. KUMAR (India)
18th 1984)
SUMMARY The interaction between molybdate ions and bovine serum albumin (BSA) has been studied employing transmittance measurements. In the acidic range, the stoichiometry of the combination between molybdate and protonated nitrogen groups of protein was found to be one-to-one. The pH-dependent binding result has been ascribed to the existence of anionic as well as cationic species of molybdenum(V1). The pH-metric titrations and ultraviolet absorption spectra of molybdenum(VI)-bovine serum albumin mixtures also supported the existence of polymerisation-depolymerisation of molybdenum as well as deprotonation and conformational changes in the protein molecule. The free energy, enthalpy and entropy of binding have been determined and the effect of molybdate ion on the heat of ionization and pKint, of BSA groups has’been discussed.
INTRODUCTION
The biological implications of polyanion-polycation interactions are well established. Several workers have investigated the stoichiometry of such combinations to explain the mechanism of some important biological processes [l-9]. Hummel and Witzel have used pH measurements to study the binding of nucleotides to pancreatic ribonuclease [lo], while other workers emphasized the valency states of metals in metalloproteins by this method [ll-141. .A few workers have also used turbidimetric titrations to determine the equivalence points in such complexations which involve polyions of opposite charges [15,16]. Owing to the heparin-like activity of phosphomolybdate [17,18] and the biological significance of molybdate ion [19,20], Malik and Arora [21-231 and Arora et al. [24,25] have extensively studied oxomolybdenum-protein interactions employing physico-chemical methods. Recently, the results on the binding of molybdenum(V1) with bovine serum albumin employing polarographic and equilibrium dialysis methods were reported [26]. However, this literature survey revealed that no such studies between oxomolybdenum(V1) and bovine serum albumin have been made so far using transmittance and pH measurements. In this paper the energetics of binding between oxomolybdenum ion and bovine serum albumin is reported employing transmittance and 0302-4598/84/$03.00
0 1984 Elsevier Sequoia
S.A.
330
pH measurements. cations for bovine
The thermodynamics of the interaction serum albumin are discussed.
and the structural
impli-
EXPERIMENTAL
Reagents A solution of bovine serum albumin (BSA) (Armor and Co. Product, 11z.w.69 000) was prepared in double distilled water. The strength of the protein solution was determined by drying its known volume in an air oven at 105110°C. Sodium molybdate (E. Merck) was dissolved in distilled water and its molybdenum content was determined gravimetrically. Buffers and other solutions were prepared from reagent grade chemicals. A potassium chloride (BDH) solution was used for the maintenance of ionic strength of the reaction mixtures.
Techniques Transmittance measurements The transmittance of BSA-Mo(V1) mixtures, which can be taken as an index of turbidity developed, was measured by means of the Bausch and Lomb Spectronic -20 instrument at 375 nm. For these measurements the following types of experiments were arranged: (i) 0.5 g/dm3 BSA was put in different boiling tubes and the total volume was kept at 10.0 cm3 by adding fixed amounts of different buffers and double distilled water; (ii) 0.5 g/dm3 BSA and 0.01 M Mo(V1) were put in different tubes and the total volume was made 10.0 cm3 as in (i); (iii) 2.0 g/dm3 BSA was put in different tubes and to each was added different amount of Mo(V1) and the total volume was made 10.0 cm3. Several different sets were prepared at different pH values (2.0 to 5.0). Similar sets were also prepared keeping the amount of molybdenum fixed and varying the amounts of protein. The effect of potassium chloride was studied at one specified pH to show the effect of ionic strength on the extent of complexation. The transmittance of all the mixtures was measured immediately after preparing the reaction mixtures.
pH-measurements These were made on a Systronic pH-meter using a wide range glass electrode. The instrument was standardized against 0.05 M potassium acid phthalate (pH 4.0) and 0.05 it4 borax (pH 9.2) for the acidic and basic ranges respectively. Three different sets of mixtures were prepared: (i) varying amounts of hydrochloric acid and potassium hydroxide (each 0.10 M) were put in different tubes and 1.0 cm3 of 3.0% isoionic BSA was added to each of them. The total volume was made 10.0 cm3 by addding water and enough 1.0 M KC1 to make the total ionic strength of the solution 0.15;
331
(ii) varying amounts of acid or base were mixed with 1.0 cm3 of 3.0% BSA and 1.0 cm3 of 0.01 M molybdenum(VI), keeping the total volume at 10.0 cm3 as in (i). These measurements were carried out at two temperatures, uiz. 23 and 33°C; (iii) 10.0 cm3 of 0.3% BSA solution of different pH values were titrated against 0.10 M sodium molybdate solution of the same pH as the protein. The pH values of the above mixtures were recorded immediately after mixing and after 24 hours. These measurements at pH 5.35, 9.85 and 11.50 were carried out at 25 and 40°C respectively. Ultraviolet spectroscopy
These measurements were made on a Carl Zeiss specord U.V. visible spectrophotometer. The spectra of a very diluted BSA solution (0.03 g/dm3) in the absence and presence of 1.0 X 10e5 M molybdenum(V1) were recorded at four different pH values (5.57, 7.5, 9.40 and 11.40). The shifts in the curve of BSA in the presence of molybdenum(V1) were taken as a qualitative evidence of interaction. RESULTS
AND DISCUSSION
Bovine serum albumin (BSA) formed insoluble complexes with molybdate ions below its isoelectric point (IEP). The different aspects of the insoluble complex formation such as pH, protein and molybdate concentration are critically discussed below. Effect of pH on transmittance
Figure 1 shows the percentage transmittance of BSA in the presence and absence of molybdenum at different pH values. A minimum in the vicinity of the isoelectric
1OOr
BSA(O.OS%) BSA(O.O5%) tO.OlOM Na,MOO/&
00s --O-=+7. z s 60 -.z $ e I40
?-?J,,, 20 3.2 2.4
PH 4.0
4.6
5.6
6.0
Fig. 1. Percentage transmittance plotted against pH for BSA in the presence molybdenum(V1). (0) 0.05% BSA alone; (0) 0.05% BSA-tO.010 M sodium molybdate. 0.15.
and absence Ionic strength
of =
332
point (pH 5.0) is obtained in the case of BSA. Similar types of maxima and minima were observed by Arora and Singh [27] in the case of denatured and S-ovalbumins, while Malik and Arora [21] were incapable of showing any such behaviour in the case of ovalbumin and gelatins (see also Ref. 22). It appears that all corpuscular proteins possess the property of salting out at their isoelectric points. It may be concluded that the presence of a large proportion of hydrophilic groups in gelatin imparts a greater stability over the entire pH range, while BSA and other related proteins having a smaller number of hydrophilic groups show a greater tendency towards precipitation in the vicinity of their isoelectric points. On addition of Mo(VI), the transmittance largely decreases in the BSA-Mo(V1) system. The minimum does not occur at the original isoelectric point, but at a much lower pH. The shift in the IEP of BSA from 5.0 to 4.6 on addition of Mo(V1) may be explained in terms of the reaction of the molybdate ions with the protonated basic groups of BSA. Owing to the binding of molybdate ions with the positively charged nitrogen groups, the total positive charge on the BSA molecule decreases. This explains the shift of IEP towards a lower pH value. Stoichiometty of Mo(VI)-BSA interaction The extent of binding of molybdate ions to BSA was computed by carrying out direct titrations (Fig. 2). The break in the transmittance uersus concentration curves indicates the point where the formation of the insoluble complex is complete. Furthermore, if this point does not indicate the completion of the formation of the insoluble complex, then with the addition of more molybdate there would be an even larger decrease in the transmittance. From the breaks in the curves, the number of moles of molybdate bound per mole of BSA ( VM) was found using the relationship, V, = C&P], where [P] is the molarity of protein used and C, the amount of
100
60
I
0
I
I
1
I
I
2
a
kol (IO-?rj) I’ I 4 4.5
i'i
I_
3
Fig. 2. Transmittance titration curves of fixed BSA (0.05%) against varying (ionic strength, 0.15) at different pH values (indicated at the curve).
concentrations
of molybdenum
333 TABLE
1
Binding of molybdenum 2). 30°C PH
to BSA (0.28
Bound molybdenum (G,) (1O-4 w
x
10m4 M) at different
Moles of molybdenum bound per mole of
pH values from transmittance
LogK
AG kcal/mol
7.00 7.03 7.07 7.15 7.21 7.29 7.58 8.10
- 9.597 -9.651 - 9.706 - 9.816 - 9.884 - 10.01 - 10.40 - 11.10 _
method
(Fig.
BSA(&I 1.0 1.5 2.0 2.6 3.0 3.5 4.0 4.6 5.0
35.0 32.5 29.4 25.0 22.0 18.0 10.0 2.8
125.0 116.0 105.0 90.0 82.0 65.0 35.0 10.0
molybdate (molarity) bound at the inflexion point. The values of V, at different pH values are given in Table 1. The equilibrium constant of the precipitation reaction is given by the following expression [28],
KS =[P]m;MO]z where KS = instability constant of the insoluble complex, which is the reciprocal of the solubility product, and m and z represent the number of moles of protein and molybdenum i.e. the concentration of protein and molybdenum in mol/dm3 at the equivalence point (breaks in titrations). In these studies at the inflexion point in the transmittance uersu.s concentration curves (Fig. 2) the molybdenum used (C,) is taken as the total concentration bound, [MO]‘, to the total protein, [Plm, taken at different pH values. The instability constants calculated in the case of the present irreversible systems with the help of above equation are presented in terms of log K in Table 1. These values of log K at 30°C were used to determine the free energy change at the respective pH and are given in Table 1. A perusal of the binding data i.e. V,, log K and AG, (Table 1) revealed that the amount of molybdenum bound at any pH is nearly the same as the total number of hydrogen ions bound to this protein, i.e. the total number of protonated cationic sites at any pH. It is also evident that V, increases with decreasing pH. The instability constants characterizing the irreversible precipitation reaction decrease as the pH decreases. This order of log K values is in agreement with log K for the molybdenum-OA system [27]. Approximately similar values of log K and free energy changes (Table 1) pointed to the significance of the involvement of the same cationic groups on the BSA molecule in the binding process. The enhanced binding in the low pH range can be explained in terms of the electrostatic attraction between
334
the cationic protein and the anions of the polyacid [21]. This suggests an interaction between the molybdate ion and protonated nitrogen atom as suggested by Steinhardt and Fugett [29] and Pankhurst and Smith [30] in the case of detergent anion-protein interaction. In these experiments the precipitate does not dissolve in excess of molybdate, but preliminary experiments revealed that excess of BSA dissolved the precipitate. The dissolution may be due to the unequal amount of charge on the protein which caused the shifting of the solubility product of molybdate-BSA complex. Furthermore, the BSA-molybdate complex exists as a colloidal precipitate which undergoes sedimentation on keeping, however, with the excess of protein a protective layer is formed keeping the complex in a stable colloidal state. There is evidence of the protection of many hydrophobic colloidal systems by the adsorption of gelatin on the surface of the sol particle through the undissociated carboxyl groups [31,32]. E;ffct of molybdenum on hydrogen ion equilibria of BSA Some evidence of binding of Mo(V1) to BSA has .been obtained from pH titrations. Figure 3 shows the titrations of BSA in the absence and presence of 0.001 M sodium molybdate solution. In the alkaline range the BSA curve lies above the corresponding curve of the BSA-Mo(V1) mixture, this indicates the removal of hydrogen ions from the cationic protein sites. However, in the acidic range there are two distinct regions; in one the Mo(VI)-BSA shows greater proton binding than the
2.5 2.0
1.0
0
1.0
2.0
2.5
Fig. 3. pH-titration curves of BSA (0.3%) in the absence and presence of 0.001 M sodium molybdate at ionic strength of 0.15; (0) BSA+ Mo(VI); (0) BSA alone.
protein, and in the other the complex binds less protons than the protein. The first region lies between pH 3.0 and 5.0, which is indicative of some change in the structure of the protein. Resnik and Klotz [33] observed similar results in the titrations of polyamines in the presence of sodium dodecylsulphate (SDS). In each titration with SDS an insoluble complex was formed ultimately. The formation of this complex produced upward drifts in pH, probably due to slow diffusion of protons in the insoluble complex. Continuous stirring resulted in reproducible results. In the pH range from 2.0 to 3.0, the anionic molybdate species undergoes changes into a cationic one, hence much more protons are free which results in a lowering of pH. The observed pH values (Fig. 3) obtained at constant ionic strength (0.15) were used to determine the number of hydrogen ions dissociated per mole of BSA (y) in the absence and presence of added molybdenum at two temperatures, uiz. 23 and 33°C using Tanford’s equation [34]. The hydrogen ions dissociated (y) determined at two temperatures were plotted against pH in view of predicting the effect of added molybdenum on the protein titrations. From the temperature dependence of y, the apparent heat of dissociation (AH ion), i.e., the difference of pH values at constant value of y and temperature, in the absence and presence of molybdenum was determined according to Wyman’s method [35], using the equation AHion= -2.303
RT
2 @pH)
dT
where R is the value of the gas constant (2 calorie), T is the absolute temperature, dT is the difference of two temperatures, and d pH is the difference of pH values at constant value of y. The apparent heat of ionization was plotted against the number of hydrogen ions dissociated per protein molecule ( y) and the values of the apparent heat of ionization for different dissociable groups of BSA in the absence and presence of molybdenum are given in Table 2. Owing to the involvement of guanidinium groups of arginyl residues in interaction with molybdenum the dissociable groups are removed from the titration and therefore it was not possible to determine their heat of ionization (A Hi,,). These values of heat of ionization show
TABLE 2 Apparent heat of ionization molybdenum Group
(kcal/mol)
Expected range for AHi,,
Carboxyl Imidazole Amino Guanidino
1.5-3.0 6.9-7.5 10.0-12.0 12.0-13.0
of dissociable
groups of BSA in the absence and presence of
AHi, in absence of molybdenum
AHion in presence of molybdenum
2.40 7.20 11.00
2.00 6.80 10.80
336
that the heat of ionization of carboxyl, imidazole and amino group is depressed to the extent of 17% in the presence of molybdate ions. Applying Tanford’s equation [34], a plot of pH/log (y/n - 1) uersus h gave straight lines (in this equation the quantity Z of the original equation was replaced by h, the number of protons bound per protein molecule). The value of W, the electrostatic interaction factor was determined from the slope of the straight line and was found to be 0.0138 and 0.0125 in the absence and presence of molybdate ions respectively. The values of intrinsic association constant for carboxyl groups was found to be 3.4 in the absence and 3.9 in the presence of molybdate ions. The increase in pK of carboxyls in the presence of molybdenum shows the formation of the BSA-molybdate complex and the value of extra dissociated hydrogen ions (r) suppresses the dissociation of carboxyl groups. Binding of molybdate ions to BSA by pH displacements
Arora and Singh [36] studied the effects of added sodium molybdate on the pH of the isoionic S-ovalbumin and observed that there was a decrease in pH with increasing concentration of molybdate. This decrease in pH was attributed to the preferential binding of one of the ions of the added electrolyte to the protein. In such cases a negative ApH indicates cation binding while a positive ApH is indicative of anion binding. In order to note whether molybdenum reacts as anion or cation or as a Lewis acid, its interactions with BSA at different fixed pH values were investigated from pH 1.0 to 12.0 and these titrations are shown in Fig. 4 in the form of ApH plotted against molar concentration (M) of molybdenum(V1) added. It is interesting to note that except at the pHs 3.0, 3.5 and 4.1, at all other pH values studied the ApH is found to be negative. The negative ApH values showed that
-0.6 0
I
I 0.2
Y O.lOH
I
a 0.4
Na, MO 04 cm3 I1
0.6
I
I 0.8
I
I 1.0
Fig. 4. pH titrations of BSA against sodium molybdate curve.
at different fixed pH values indicated on each
337
molybdenum binds in the form of a cation while the positive values indicated its interaction as anion. The nature of negative or positive ApH indicated the existence of anionic and cationic species of molybdenum. Similar types of pH displacements were observed by Hummel and Witzel in the interaction of nucleotids with pancreatic ribonuclease [lo]. The amount of ligand bound per mole of BSA (I’,) was calculated by the method of Scatchard and Black [37], from which the value of bound (C,) and free (C,) molar concentration of molybdenum was determined from the known molar concentration of BSA. The experimentally, determined binding data, uiz. V, and C, were analysed by means of Scatchard equation [38] in the form,
+Kn--KV, F
where V, and C, have their usual meanings, K is the average apparent association constant for binding at each protein site, and n is the average maximal number of ligand binding sites on a protein molecule with the same association constant K. If all the binding sites are equivalent and independent, a plot of V,/C, as a function of V, would give a straight line such that the intercept on the V,/C, axis is Kn, as V, approaches zero as a limit, and the intercept on the V, axis is n, as Q/C, approaches zero as a limit. Deviation from linearity occurs when binding takes place at more than one set of sites with different values of the association constant. The contribution of electrostatic factors may also produce deviations [23,24]. The Scatchard plots at pH values 5.35, 7.50 and 2.50 were found to be non-linear. The non-linear nature of the plots may be attributed either to the saturation of the same class of primary sites (nr) or to the involvement of second (n2) or subsequent classes of sites in the molybdenum interaction [39]. Arora el al. [26,40] have earlier reported that the non-linearity of the plots was due to the involvement of binding sites of different affinity in molybdenum-protein interactions. However, a linear relationship was found to exist between V,/C, uersus V, plots at pH values 9.85 and 11.50, which indicated the involvement of a single primary class of equivalent and independent sites in interaction; if a second class of sites existed it would have a much smaller association constant than the primary class of binding sites. The number of primary binding sites (nr) and their association constants (K,) determined from the intercept of the V, and V,/ C, axes are given in Table 3. To determine the number of sites in the second class ( n2) and their binding constant (K,), we plot V,,/C, uersuS V,,, where V,, = [V, - number of primary binding sites (nl)]. This procedure substracts out the contribution of the strung primary binding sites (nr) from the data. This is valid only when the two classes of sites are non-interacting, i.e., binding at one site does not affect binding at the other. Applying this procedure the number of secondary binding sites (n2) and association constant (K2) were found to be 6 and log K 2.48 at the two temperatures (25 and 40%) and at pH 5.35. The standard free energy change calculated from pK (at the given pH and temperature) was found to be - 3.40 kcal/mol. These results show that the secondary sites are weaker and destroyed at higher pH values.
338 TABLE 3 Binding constants of BSA-molybdate PH
n
25OC 5.34 7.50 9.50
11 9 8
9.85 11.50 40°C 5.34 7.50 9.50 9.85 11.50
log K
system from pH displacement method AGO kcal/mol
AS” Cal mol-’
3.95 3.78 3.61
- 5.415 -5.182 - 4.949
-
7 3
3.60 3.60
- 4.835 - 4.835
+ 14.0 + 8.6
9
3.81
- 5.486
+4.7 -
6 2
3.58 3.52
- 5.155 - 5.068
+ 14.6 + 6.0
K-’
AH’ kcal/mol
+5.0
-4.0 - 0.572 - 2.266
The small number of primary binding sites (n,) i.e. 11, 9, 8, 7 and 3 at pH values 5.35, 7.50, 9.50, 9.85 and 11.50, respectively, showed that not all the protonated nitrogen groups are equally available for interaction. The reason for the involvement of a smaller number of binding sites may be a conformational one. This protein possesses a significant structure brought about by hydrophobic interactions, hydrogen bonding and other intersegmental cohesive forces [41]. However, since the folded core of the globular protein is known to be inaccessible to water, a rough estimate of 40-608 of the total sites may be available to interact with the anions. Considering this constraint imposed by the chain folding of BSA, it appears that anion binding to this protein occurs in a restricted way [42]. The lesser value of binding sites (n) at pH 9.85 and 11.50 could also be explained on the basis of successive deprotonation of protein groups. The pK values for histidyl and lysyl amino acid residues are 6.90 and 9.85, while for guanidinium of arginyl residues it is greater than 12.0. Thus at pH 9.85 and 11.50 the sites involved in molybdenum interaction must be the guanidinium groups of the protein. This argument also goes support from the apparent heat of ionization of guanidinium groups in the presence of molybdate ions. However, the involvement of a smaller number of guanidinium sites could be due to a large amount of electrostatic repulsion between anionic protein and the molybdate ions, and also some of these groups may be irreversibly bonded to the carboxyl groups. Thermodynamic
parameters
The oxomolybdenum(V1) ion binding by BSA is observed to be modified by pH and temperature. The number of sites decreases with rising pH and temperature but log K remains nearly constant. The significance of constant values of log K (almost independent of pH and temperature is that only a single primary class of sites is reacting, and the appearance of their different number is therefore not responsible
339 0.4 -
-2 5 0.2 -g z O-
, 225
-0.1 Fig. 5. Absorption spectra of BSA (0.003%) in the absence and presence of molybdenum (1.0 X 10m5 M) at different pH vdues. (3) BSA + MO (pH 9.40); (1) BSA (pH 9.40); (4) BSA + MO (pH 7.50); (2) BSA (pH 7.50).
for decreased binding which may then be due to the decreased availability of the same class of sites owing to irreversible effects in the protein structure. The free energy (AGO), enthalpy (AH’) and entropy (AY) changes of the interaction were calculated using standard methods (Table 3). The positive entropy value probably indicates that water of hydration is released both from the protein and oxomolybdenum, and that the configuration of the polypeptide is changing to a random coil as the complex formation occurs. The higher values of entropy and enthalpy changes at pH 9.85 possibly indicate that all three factors mentioned above may be involved at this pH. ,Spectral studies
The changes of the absorption spectrum with pH, for BSA in the presence of molybdenum (Figs. 5 and 6), are assumed to be related to the changes in hydration and conformation discussed above. The molecular extinction coefficients of molybdenum-BSA complex and BSA were calculated at the wavelength of maximum absorption and are given in Table 4. At pH values 5.57, 7.50 and 9.40 both
0.6-
0,
I 225
235
‘.\,
\
245
255’%60
-O.lFig. 6. Absorption spectra of BSA (0.003%) in the absence and presence of molybdenum (1.0 X lo-’ (4) BSA+Mo (pH 11.4); (3) BSA (pH 11.4); (2) BSA+Mo (pH 5.57); (1) BSA (pH 5.57).
M);
340 TABLE 4 Molecular extinction coefficients (mol/dm)) for wavelength of maximum absorption denum-BSA complex and BSA alone at different pH values PH
X (nm)
Molybdenum + BSA x lo4
BSA alone X lo9
5.57 7.50 9.40 11.40
235 235 235 240
1.00 1.50 2.00 1.25
9.50 3.10 0.75 10.00
of molyb-
BSA and molybdenum-BSA complex have a maximum absorption at 235 nm, but at pH 11.40 it is shifted towards a higher wavelength, i.e. 240 nm. Moreover, the maxima at this pH are much broader than at other pH values. The shift of wavelength of maximum absorption from 235 to 240 nm at pH 11.50 may be ascribed to protein unfolding [43] which may cause a change in the configuration of some of the molybdenum binding sites on the protein molecule. The trend of the extinction coefficient (Table 4) of BSA-molybdenum complex is analogous to its enthalpy change while that of BSA is analogous to its entropy change. The values of the thermodynamic parameters and of the molecular extinction coefficients of molybdenum-BSA complex are in line with the fact that the nature of the binding sites is different in lower and higher pH. Nature of interaction The results discussed above showed that Mo(V1) reacts as anion or cation (Lewis acid) depending upon the pH of the system. In the acidic range, the interaction between polyacid anions and protonated groups of BSA can be formulated in analogy to the description of the ovalbumin-chloroaurate interaction [44]. Presumably, the anion is first attracted to the positive centres on the protein surface by coulombic forces, and when it is sufficiently near water molecules are removed, making the lone electron pair on nitrogen available for coordination with the molybdenum of the molybdate ion. The shift of the titration curve of Mo(VI)-BSA complex towards higher pH may perhaps be in line with the type of bonding reported in Mo(VI)-N-methyliminoacetic acid [45] and the enzyme nitrogenase [46] because hydrogen ion react with the molybdate ion to form Mo(OH)O, [47] which in turn is removed as water molecule on reacting with BSA. In the lower pH range, the complex molybdate ion may contain many hydroxyl groups [48], hence a polynuclear complex may be formed through coordination and hydrogen bonding with protonated nitrogen and undissociated carboxyl groups of BSA. A similar type of reaction was suggested by Malik and Arora [49] and Malik et al. [50] in the binding of silicic acid with proteins. However, in the higher pH range the decreased binding can be explained on the basis of the following points:
341
(i) The number of hydroxyl groups in the polyacid decreases and as a result the number of linkages also progressively decreases with decreasing metal oxide content; (ii) The increased negative charge on the protein molecule would contribute to a repulsive electrostatic effect on the polyanions; (iii) The progressive deprotonation of nitrogen atoms would result in a smaller number of positive loci, therefore resulting in a decreased binding. The ions bound are anions; however, these results do not allow us to determine which groups are involved in binding with molybdate ions. The deprotonation of cationic and carboxyl groups is responsible for the decreased binding above the IEP of BSA. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
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