Comparison of binding of vanadium(V) with bovine serum albumin and bovine pancreatic trypsin

Bioelectrochemistry and Bioenergetics, 10 (1983) 57-67 A section of J. Electroanal. Chem., and constituting Vol. 155 (1983) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

57

JoI)-COMPARISON OF BINDING OF VANADIUM(V) WITH BOVINE SERUM ALBUMIN AND BOVINE PANCREATIC TRVPSIN

J.P.S. ARORA, R.P. SINGH, D. SOAM and R. SHARMA Department of Chemistry, D.A. V. (P.G.) College, Muzaffamagar 251001, U.P. (India) (Manuscript received January 29th 1982)

SUMMARY The binding of vanadium(V) ion has been studied with bovine serum albumin and bovine pancreatic trypsin at different pH values and temperatures by the polarographic technique. The binding data were found to be pH and temperature dependent. The intrinsic association constants (K) and the number of binding sites (n) were calculated from Scatchard plots and found to be at the maximum at lower pH and at lower temperatures. The values of enthalpy change (AH’) and entropy change (AS’) at pH 7.50 have been found to be - 2.090 kcal mole- ’ and + 6.372 cal mole- ’ degree- ’ respectively, for the vanadium (V)-bovine serum albumin system. The free energy change (AGO) of the combining sites were least at the higher pH and highest at the low pH; therefore, a lower temperature and lower pH offered more sites in the protein molecule for interaction with vanadium(V) ions. Statistical effects seem to be more significant at lower vanadium(V) ion concentrations, while at higher concentrations electrostatic effects and heterogeneity of sites are more significant.

INTRODUCTION

Although polarography is of little use in the study of anion-protein interactions, yet it may be employed particularly with those systems where other electrometric methods fail to give vital information. The binding of methyl orange anions with serum albumin has been studied by Strick and Kolthoff [l]. They even determined the molecular weight of serum albumin with the help of the diffusion current of the methyl orange-serum albumin complex. Saroff and Mark [2] studied the dye anion-protein interaction polarographically. Malik and co-workers [3,4] made systematic polarographic studies on the binding of metal-oxoanion and anionic dyes with proteins. On the basis of a diffusion-controlled wave of vanadium(V) [5], Ashraf [6] successfully employed the polarographic method in the study of vanadium(V)-protein interaction. Owing to numerous significant roles of vanadium on biological systems [7-91 and macromolecules [lo- 131,it was thought, worth while carrying out polarographically binding studies. In this paper the results of the polarographic study on the binding of oxovanadium(V) with bovine serum albumin and bovine pancreatic trypsin are reported. 03024598/83/$03.00

Q 1983 Elsevier Sequoia S.A.

58 EXPERIMENTAL

Proteins

Crystalline bovine serum albumin (BSA, m.w. = 69000) was obtained from V.P. Chest Institute, New Delhi. For the preparation of stock solution, the water content of the powdered protein was first determined by drying a small weighed sample for approximately 1 h at 1OO’C. The stock solution was then made up to the desired weight of protein per unit volume of solution by mixing the calculated weight of deionized twice-distilled water and protein, assuming a specific volume of 0.75 cm3/g for the protein [14]. Trypsin (bovine pancreatic, m.w. 24000, Sigma Chemical Co.) was dissolved in deionized twice-distilled water. The concentrations of the protein solution were determined by evaporating a known aliquot in an oven at lOO”C, as well as by the biuret technique. Reagents

Sodium-metavanadate (E. Merck) was dissolved in twice-distilled water and estimated gravimetrically [ 151. Sodium acetate and acetic acid (BDH) were used for the preparation of acetate buffers, while ammonia and ammonium chloride were used for the preparation of ammonia buffers from reagent grade chemicals. Potassium chloride (BDH) solution was used for the adjustment of ionic strength (0.15 M). pH-measurements

These measurements were carried out by a Systronic pH-meter using a wide-range glass electrode. The instrument was standardized against 0.05 M potassium acid phthalate (pH 4.00) and 0.05 M sodium borate (pH 9.20) for acidic and basic ranges respectively. Diffusion current measurements

These measurements were carried out on a Toshniwal polarograph Model Cl 02A in conjunction with an Osaw galvanometer in the external circuit. An H-shaped polarographic cell as recommended by Tanford was used [ 161.Nitrogen, purified by passing through a solution of chromous chloride and alkaline pyrogallol, was passed through the solution for producing an inert atmosphere. Triply distilled mercury was used for the d.m.e.. The cell was immersed in water thermostat maintained at 25 or 40 f 0.1%. The capillary used had a flow rate of about 2.2 mg/s with a drop time of 3.5 s. The rate of flow of mercury drops was determined by Lingane’s method [17]. Since the capillary characteristics (m, t) have a marked effect on diffusion current (which is directly proportional to m2i3, t’j6), these factors were therefore controlled carefully throughout these experiments.

59

Procedure

The following sets of solution were prepared for diffusion current measurements: (1) Varying concentrations of BSA (0.25% to 3%) were taken in Pyrex tubes and 1.0 cm3 of 0.01 it4 sodium metavanadate solution was added in each tube. The total volume was made up to 10 cm3 by adding the fixed amount of buffers of different pH-values (5.57; 7.50 and 9.50), 1.0 M potassium chloride solution (to make the ionic strength 0.15 M) and the requisite amount of twice-distilled water. (2) Here, 1.0 cm3 of BSA (5%) and different concentrations of sodium metavanadate (2.5 x 10v4 M to 50 X 10d4 M) were added. The total volume was made up to 10 cn? by adding the fixed amount of buffers (pH 5.57, 7.50 and 9.50), 1.0 M potassium chloride solution (for ionic strength 0.15 M) and the requisite amount of twice-distilled water. (3) Similar sets as in (2) were prepared in the absence of protein. Similar sets of solution were also prepared for trypsin. The above experiments were carried out at 25 IIIO.l’C at all pH values 5.57, 7.50 and 9.50 in both cases, while in case of BSA the measurements were also made at 40 f O.l”C at pH 7.50. RESULTS AND DISCUSSION

When a reducible substance is bound to a protein there is reduction in the diffusion current, and this decrease in the diffusion current may be used to calculate 0.95-

BSA c% 0.55, 0

I 0.5

I 1.0

I 1:s

I 2.0

I 2.5

Fig. 1. Effect of BSA concentration on Z,/(Z,,), (25°C).

I 3.0

1 3.5

at fiied (1 X 10m3M) vanadium(V) ion concentration

60

o.so-

O.L5 -

Trypsin 040

0

I 0.5

I 1.0

(c%) I 1.5

Fig. 2. Effect of trypsin concentration (25T).

I 2.0

I 2.5

on I,,/(&),

I 3.0

at fixed (1

I 3.5 x

10m3 M) vanadium(V)

ion concentration

the binding constants [ 161. Denoting Id and (Id),, the diffusion current of vanadium(V) ion in the presence and absence of protein respectively, and co, cb and ct the concentrations of total, bound and free vanadium(V) ion respectively,

where k (the fractional coefficient) is the limiting value of the diffusion current ratio (Zd/ld)o when so much protein has been added that all the vanadium(V) is protein

61

bound. Its value is obtained by an extrapolation method

&=k limit c, + 0 and found to be 0.58, 0.60 and 0.63 for BSA (Fig. 1) and 0.43, 0.47 and 0.49 for trypsin (Fig. 2) at pH-values 5.57, 7.50 and 9.50 respectively (25’C). The value of k was found to be 0.54 at pH 7.50 (40°C) for BSA. The mole .of vanadium(V) bound per mole of protein (V,) is then given by the relation, V, = c,/P, where P is the molarity of the protein. The pH-dependent values of the fractional coefficient in the case of two proteins could be explained on the basis of the following two facts. Firstly, at lower pH, the higher polyvanadates exist [18] which, being capillary active [ 191, cause a much larger reduction in the diffusion current, while towards the higher pH-value the smaller species of vanadium [20], being capillary inactive, do not affect the diffusion current to any great extent. Secondly, towards lower pH, there is a larger electrostatic attraction between vanadate anion and the cationic protein, while at higher pH there is a tendency for electrostatic repulsion. Effect of protein concentration on V,

Figures 1 and 2 show the effect of protein concentration on diffusion current ratio, while Table 1 shows VM at different protein concentrations. Although trypsin causes a much larger depression in the diffusion current than BSA, yet the average moles of anion bound per mole of protein are different. This is due to the fact that for BSA calculations were made from a molecular weight of 69,000 while in trypsin from a molecular weight of 24000. If the protein were to cause a lowering of

TABLE

1

Binding constants of the vanadium(V)-BSA Protein

Binding constant

and vanadium(V)-trypsin

pH 5.51 T= 2Y’C

systems

pH 7.50

pH 9.50 T= 25°C

T = 25°C

T=40°C 676.1 34 - 4.079 - 2.090 + 6.354

Bovine serum albumin

K ” AC0 kcal mole-’ A Ho kcal mole-’ AS0 cal mole-’ deg-’

859.0 50 - 4.027 -

799.8 40 - 3.989 - 2.090 + 6.372

Trypsin

K :GO

219.8 20

207.0 14

-

200.0 10

kcal mole-’

-3.211

- 3.170

-

-3.156

379.3 29 -3.541 -

62

diffusion current by some other process in addition to complexation, then the apparent value of V, at higher protein concentration would be higher than expected. It is observed that the values of V, are relatively higher at very low concentrations of protein. This may be either due to some loosening of protein structure favoured by a large net charge on the protein resulting in greater binding [21], or to a saturation effect on the binding sites owing to relatively higher concentrations of anion in comparison to protein. Perlmann [22] has reported as many as 80 phosphate ions bound to serum albumin, and has accounted for this on the basis of the saturation of the binding sites. Calculation of binding parameters

The binding data, viz. V, and c, were analysed by means of Scatchard’s equation [23-251

IV

Fig. 3. Plots of V,/c,

=

7.50 AT

40%

versus V, for BSA-vanadium(V)

ion systems.

63

Fig. 4. Plots of V,/c,

versus V, for trypsin-vanadium(V)

ion systems.

where K is the average apparent association constant for the binding at each site and n the average maximal number of binding site on protein with the same association constant K. The value of V, and cr (free anion concentration) were computed from polarographic measurements for a given concentration of small molecules. If all the sites are equivalent and independent, a plot of &/c, as a function of I$, would give a linear relation such that the intercept on the VM/cf axis is Kn, as V, approaches zero-as a limit, and the intercept on the V, axis is n, as VM/c, approaches zero as a limit. Deviation from linearity occurs when binding takes place at more than one set

TABLE 2 Effect of protein concentration on V, at pH 5.57 (T= 25’C, vanadium(V) concentration fixed = 1.0 x 10-3M).

Protein c (W

BSA

Trypsin

0.25 0.50 0.75 1.00 1.50 2.00 2.25 2.50 2.75 3.00

14.86 8.26 5.73 5.55 4.12 3.30 3.07 2.77 2.52 -

3.37 2.36

1.68 1.40 1.22 1.06 1.01 0.95 -

64

of sites with different values of the association constant. The contribution of electrostatic factors may also produce curvature [23-261. The results computed from polarographic measurements are plotted according to the above function in Figs. 3 and 4 for the two proteins. At pH 9.50 for serum albumin and pH 5.57 to 9.50 for trypsin, a linear relation exists between VM/cr and V,. It can be seen that the linear behaviour is due to the involvement of one class of sites which are equivalent and independent. Figures 3 and 4 (pH 5.57 and 7.50) for serum albumin show curvature, perhaps due to the involvement of more than one set of sites in the interaction. The values of the average apparent association constant and the maximal number of binding sites calculated from Scatchard plots are given in Table 2. It is clear from Table 2 that the small value found for n in the case of BSA shows that all the positive groups of. this protein are not available for interaction with oxovanadium(V) anion. In fact, out of the total 100 cationic groups [27] present in BSA only 50, 40 and 29, are involved in the interaction at pH 5.57, 7.50 and 9.50 respectively. Thus, the lesser number of binding sites does not allow us to determine correctly the groups (arginine, lysine or imidazole) which are involved in binding with vanadium(V). Effect of pH and temperature on binding parameters

The vanadium(V) ion binding by serum albumin is observed to be modified by pH and temperature. The binding regularly decreases from pH 5.57 to 9.50 at 25°C while at pH 7.50 it decreases with a rise in temperature, viz. from 25 to 40°C. The value of binding sites decreases with rise in pH and temperature in both the cases, but the value of K showed this behaviour only in the case of BSA not in that of trypsin. In the latter case, although the value of the binding sites are different, the K values are constant. At pH 9.50, the lysyl ~-amino groups (pK = 9.8) lose a major portion of the positive charge while the guanidinium groups @K = 12) are predominantly in the positively charged form, although the reduction in extent of binding at pH 9.50 appears to be mainly attributable to the loss in positive charge on catibnic binding sites in serum albumin molecule. Other factors may also contribute to the reduced binding, one of these could arise from an increase in the net negative charge on albumin leading to electrostatic repulsive forces between the protein and the anions. The effect of pH on the vanadate anion binding is in accordance with the theory of Scatchard et al. [28] who believed that binding of anion by the protein increased with the binding of protons. In the case of serum albumin the uniformity of K at pH-values 5.57 and 7.50 is in agreement with the involvement of similar groups, while a lower value of K at pH 9.50 with linear plot favours equivalent and independent one-class sites. In the case of trypsin tbe uniformity of K supports the fact that all the binding sites are equivalent and independent in the pH range 5.57 to 9.50. The significance of the uniformity of K at all pH values is that a single class of sites is reacting with

65

vanadate anion. The appearance of different sites (Table 2) is therefore not responsible for the increased binding which may then be due to the increased availability of the same class of sites. The increasing value of n as the pH decreases is an indication of this. The overall binding results are in agreement with the fact that the available sites are pH dependent, yet they have the same association constants. It is seen that the number of binding sites in the case of trypsin is nearly the same as the total number of cationic groups present [29], while only 50% of the total cationic groups in BSA molecule are involved in interaction at any particular pH. The large difference in the number of binding sites for the two proteins may be due to several reasons: (1) The larger fraction of the anionic groups in BSA [27] inhibits the electrostatic attraction between vanadate anion and the protonated nitrogen groups. (2) In BSA the geometry of all the cationic groups is not in a suitable juxtaposition, while in trypsin the geometry of these groups is in much favour for anion binding. (3) Trypsin is devoid of any configurational adaptability while serum albumin is a subject of this type of adaptability [30]. (4) In the acid range, trypsin is not salted out by vanadium ion while serum albumin does so readily. This observation points towards a much greater hydrophobic character of serum albumin than trypsin. (5) It is reasonable to assume that guanidinium groups of arginine residues are principal sites for binding vanadate anion. There is evidence in support of this fact because protamine, having 18 to 19 a&nine residues, is capable of forming a precipitate with NaVO, at pH 9.50 and below [31]. All the guanidinium and other such sites are the potential binding sites for anions. In the case of BSA, involvement of only 50% sites may also be explained by assuming duplet or triplet clustering of the cationic sites together. In fact, a model having two cationic groups together could satisfactorily explain the binding of one vanadate anion. However, this suggestion fails to explain the binding behaviour of trypsin. Intrinsic association constant and thermodynamic parameters The binding experiments with serum albumin (pH 7.50) were carried out at 25 and 40°C with a view to predicting the nature of the anion-protein bond, The values of the thermodynamic parameter, as calculated from standard thermodynamic procedures of the two systems, are given in Table 1. A comparison of the free energy change shows that BSA involved a greater free energy change in the interaction compared to trypsin. The enthalpy and entropy changes at pH 7.50 for vanadium(V)-serum albumin could give an idea about the nature of the anion-protein bond. According to Sharma et al. [32], in the case of divalent metal-amino acid complexes, the negative enthalpy change establishes the covalent bond formation between the metal atom and the amino groups of the amino acid. In the vanadium(V)-serum albumin system the magnitude of negative enthalpy change indicates a covalent linking between the vanadium atom and the nitrogen-containing

66

side-chain groups in the protein molecule. On the other hand, the positive entropy values indicate the replacement of water molecules from the hydrated charged sites and the interacting vanadate ions. In all, the negative enthalpy value and the additional positive entropy values provide evidence for a reaction similar to that suggested by Craig et al. [33] in the chloroaurate-ovalbumin reaction. Rlotz [34] suggested that the positive entropy changes observed for the association of various anions with serum albumin may arise from the release of water molecules as a result of neutralization of charged hydrate sites on the protein molecule and the interacting small molecule. Although the results presented for the binding of two proteins at all pH-values follow the order of reactivity: BSA > trypsin, yet at all pH-values trypsin showed identical sites, whereas BSA showed two classes of sites up to pH 7.50 and a single class of sites of pH 9.50. The binding behaviour of this anion is analogous to the well-known binding behaviour of other anions by proteins [4,35-381. In all these anion-protein interactions, the charged nitrogen loci of amino acids are assumed to be the points of attachment on the protein molecule. This view would further find support in that, unlike other metal cations, the binding of vanadium(V) ion decrease with increases in pH. It also seems that owing to the deprotonation of cationic sites, the proteins offer a smaller number of positive sites for interaction. ACKNOWLEDGEMENTS

The authors are indebted to Professor V.P. Agrawal, Principal, D.A.V. College, Muzaffarnagar for providing the necessary facilities. Thanks are also due to the Council of Scientific and Industrial Research, New Delhi, for providing a Junior Research Fellowship for one of us (R.S.). REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

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