Electrostatic Interaction and Complex Formation between Gum Arabic

Biomacromolecules 2010, 11, 3367–3374

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Electrostatic Interaction and Complex Formation between Gum Arabic and Bovine Serum Albumin T. Vinayahan, P. A. Williams,* and G. O. Phillips Centre for Water-Soluble Polymers, Glyndwr University, Plas Coch, Mold Road, Wrexham, LL11 2AW, United Kingdom Received July 27, 2010; Revised Manuscript Received October 28, 2010

The interaction of gum arabic (GA) and bovine serum albumin (BSA) has been investigated through turbidity and light scattering intensity measurements and by the use of dynamic light scattering, laser Doppler velocimetry, and isothermal titration calorimetry. It has been shown that GA and BSA can form soluble and insoluble complexes depending on the solution pH and the mixing ratio and is a function of the net charge on the complex. Soluble complexes were obtained when the electrophoretic mobility was greater than (1. 5 µm s-1 V-1 cm-1. Changes in the value of the isoelectric point of the complexes with mixing ratio and isothermal titration calorimetric data indicated that complexes formed at pHs 3 and 4 consisted of ∼60 BSA molecules for every GA molecule, while at pH 5 there were ∼10 BSA molecules per GA molecule. Calorimetric studies also indicated that the interaction occurred in two stages at both pH 3 and pH 4, but that the nature of the interaction at these two pH values was significantly different. This was attributed to differences in the relative magnitude of the positive and negative charges on the BSA and GA, respectively, and possibly due to changes in the BSA conformation. The fact that there is an interaction at pH 5, which is above the isoelectric point of the BSA, is due to the interaction of the carboxylate groups on the GA with positive patches on the BSA or to the charge regulation of the protein-polysaccharide system brought about by changes in dissociation equilibria. Complexation is reduced as the ionic strength of the solvent increases and is prevented at a NaCl concentration of 120 mM.

Introduction There has been considerable interest in polysaccharide-protein complexes in recent years, not least because of their potential applications in a range of areas including protein entrapment and release, enzyme immobilization and recovery, biosensors, protein separation processes, emulsification, foam stabilization, and encapsulation.1-9 Bungenberg de Jong and Kruyt undertook the first systematic study of electrostatic interactions between polysaccharides and proteins using gum Arabic (GA) and gelatin as long ago as the 1920s and they referred to the complexes formed as complex coacervates as they were liquid-like rather than as solid precipitates.10 Overbeek and Voorn developed a theoretical treatment to explain the results,11-16 and a number of other theoretical models have since been proposed.17-19 Veis and Arayani considered coacervation as a two-step process rather than a spontaneous process, as assumed by Voorn and Overbeek. The first step is formation of soluble complexes which then aggregate to form a coacervate phase.17,19 GA - gelatin coacervates are still used today to encapsulate flavor oils.20 Despite their use in encapsulation processes and the obvious relevance of polysaccharide-protein interactions in the biological field, surprisingly little further work has been reported in the scientific literature until relatively recently. It has been shown that polysaccharide-protein electrostatic interaction is influenced by a number of factors, particularly the polysaccharide to protein ratio, the pH, and the ionic strength.21-30 Interaction will occur under pH conditions where the polysaccharide and protein have opposite charges and the complexes formed may be in the form of coacervates/precipitates or indeed may be soluble depending on the polysaccharide-protein ratio. The * To whom correspondence should be addressed. E-mail: williamspa@ glyndwr.ac.uk.

driving force for the interaction is the enthalpic contribution associated with the interaction of oppositely charged groups and the increase in entropy due to the release of their counterions. It has also been demonstrated by a number of workers that for certain systems electrostatic interaction can occur above the isoelectric point of the protein where both biopolymers carry a net negative charge.9,22 The interaction in this case has been attributed to the association between positively charged patches on the protein and the anionic groups on the polymer, although some workers have argued that it is due to a shift in the dissociation equilibria induced by complexation.31 Schmitt et al. studied the interaction between β-lactoglobulin and GA at varying mixing ratios, pHs, and total biopolymer concentrations and reported that both soluble and insoluble complexes were formed.30 They also investigated the influence of β-lactoglobulin aggregates on complex formation.32 Weinbreck et al.23,24,26,33-36 have undertaken a series of studies on whey protein-GA complexes. They monitored the turbidity and scattering intensity of 2:1 whey protein/GA systems as the pH was gradually reduced in situ as a result of the slow hydrolysis of glucono-δ-lactone (GDL). The turbidity remained constant as the system pH was reduced from 5.7 to 4.75 and then it increased dramatically due to complex coacervate formation. Interestingly, the scattering intensity showed an initial increase when the pH was reduced to 5.3 and then a further increase at 4.7 when the system became turbid. The increase in scattering intensity at pH 5.3 without a simultaneous rise in turbidity provided evidence for the formation of soluble complexes. Using this approach, Weinbreck et al. were able to construct a phase diagram for interaction at varying pH. They also demonstrated that the pH regions for complex formation are a function of the electrolyte concentration and that above ∼50 mM NaCl complexation is prevented.

10.1021/bm100486p  2010 American Chemical Society Published on Web 11/11/2010

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Biomacromolecules, Vol. 11, No. 12, 2010

Vinayahan et al.

Figure 1. Electrophoretic mobility of GA and BSA (1 mg/mL) as a function of pH.

This paper sets out to gain further insight into polysaccharide-protein complexation by investigating the phase behavior of GA and bovine serum albumin (BSA) mixtures as a function of pH, mixing ratio, and electrolyte.

Experimental Section Materials. Bovine serum albumin used in this study was obtained from Sigma Aldrich, Gillingham, U.K. According to the supplier, it was bovine serum albumin fraction V g98% and was in the form of a lyophilized powder. The GA sample was from Acacia senegal trees and was kindly supplied by Agrisales Ltd. (London). Details of its chemical and physicochemical characteristics of this sample have been previously described.37 In summary, it was found to consist of 40% arabinose, 34% galactose, and 12% rhamnose and has a protein content of 2.15%. The molecular mass of the arabinogalactan, arabinogalactanprotein, and glycoprotein components of the gum Arabic sample were found to be 4 × 105, 2 × 106, and 2 × 105, respectively, and the average molecular mass was 8.6 × 105. Methods. Phase BehaVior. GA (1% w/w) and BSA (1% w/w) stock solutions were prepared on a dry weight basis in deionized water [Purite Select water purification system] at room temperature under general stirring for 5 h and stored at 4 °C overnight to fully hydrate. They were then filtered through a 3 µm filter. GA and BSA solutions were adjusted to the required pH using HCl and were added together to obtain mixtures with a BSA/GA w/w ratio, R, of 0.1-50 at varying pH. Phase separation was evidenced by an increase in turbidity that was monitored immediately after preparing the mixtures by measuring the absorbance at a wavelength of 600 nm. A second set of stock solutions was prepared at natural pH (for 1% w/w GA, pH ) 4.89; for 1% w/w BSA, pH ) 6.79). Mixtures were produced at different ratios, and the pH was adjusted in situ using the approach of Weinbreck et al.23,24,26,33-36 by addition of GDL powder, which slowly hydrolyzed, giving rise to a gradual decrease in the pH. Samples were taken as required to monitor turbidity by measuring the absorbance at 600 nm using a UV spectrophotometer and also the scattering intensity by dynamic light scattering using the Malvern Zeta Nano ZS photon correlation spectrophotometer. An increase in the scattering intensity without a simultaneous rise in turbidity was taken to indicate the formation of soluble electrostatic complexes while the rise in turbidity was attributed to the formation of insoluble complexes. A phase diagram was produced by plotting the pH at which soluble or insoluble complexes were formed as a function of the mixing ratio. The influence of an electrolyte on the phase behavior of GA/BSA complexes was determined by monitoring the scattering intensity at varying pH and varying NaCl concentrations using the Malvern Zeta Nano ZS at 25 °C. Complexes were prepared at different w/w ratios

of GA/BSA at a total concentration of 0.25% (w/w) using 10% (w/w) stock solutions. The concentration of NaCl was varied from 0 to 0.14 M by adding 1 M NaCl stock solution, keeping the total volume of the complexes at 50 mL. The complex pH was varied by titrating with 0.5 M HCl using an autotitrator and the scattering intensities were measured in triplicate at each pH. Dynamic Light Scattering. The hydrodynamic diameter of GA/BSA complexes were determined as a function of pH by dynamic light scattering using the Malvern Zetasizer 1000HSA, which is equipped with a He-Ne laser with a wavelength of 633 nm. Data analysis was undertaken using the CONTIN program. A total of 15 mL of a solution containing GA and BSA (R ) 2; 1 mg/mL) was introduced into a foursided clear cell and the hydrodynamic diameter was measured at various pH levels. Measurements were performed in triplicate. Electrophoretic Mobility. The electrophoretic mobilities of the BSA, GA and their complexes were determined by Laser Doppler Velocimetry using the Malvern Zeta Nano ZS. A total of 15 mL of BSA and GA solutions (1 mg/mL) were introduced into a capillary cell connected to an autotitrator and titrated against 0.5 M HCl and 0.5 M NaOH. The electrophoretic mobilities were recorded at each pH and the experiment performed in triplicate. GA/BSA complexes were prepared in deionized water at different w/w ratios according to the procedure described above. The gum arabic concentration was kept at 0.5% (w/w), and the BSA was added at different volumes to obtain different ratios. The concentration of BSA changed from 0.5 to 7.5% (w/w). The electrophoretic mobility of the systems was measured at 25 °C and each experiment was repeated three times at each pH. Isothermal Titration Calorimetry (ITC). ITC experiments were performed using the Calorimetry Sciences Corporation Isothermal Titration Calorimeter. Aliquots of 20 µL of BSA (13%) were injected into a 1.3 mL titration cell initially containing either 5 mM acetate buffer solution or 0.03 mM GA dissolved in 5 mM acetate buffer. Each injection lasted 100 s, and there was an interval of 500 s between successive injections. The temperature in the titration cell was 25 ( 0.2 °C, and the solution was stirred at 299 rpm throughout the experiment. Measurements were carried out in duplicate. Enthalpy changes were calculated by subtracting the enthalpy change when BSA was titrated into buffer solution from that obtained when BSA was titrated into GA solution.

Results and Discussion Electrophoretic Mobility of GA and BSA. The electrophoretic mobilities of GA and BSA are given as a function of pH in Figure 1. It is noted that for GA the mobility changes from ∼0 µm s-1 V-1 cm-1 to -1.5 µm s-1 V-1 cm-1 on

Gum Arabic and Bovine Serum Albumin


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Figure 2. Absorbance of GA solutions (0.5% w/w) at varying mixing ratios on decreasing the pH using glucono-δ-lactone.

Figure 3. Scattering intensity (count rate/second) for GA solutions (0.5% w/w) at varying mixing ratios on decreasing the pH using glucono-δlactone.

increasing the pH from 2-4 and then remains constant up to pH 8. The change in electrophoretic mobility is attributed to the increased dissociation of the glucuronic acid groups present in the GA as the pH increases. Schmitt et al. carried out measurements over the pH range 3.6 to 5.0 and reported similar values.30 For BSA, the electrophoretic mobility changes from +1.20 5 µm s-1 V-1 cm-1 to -0.90 µm s-1 V-1 cm-1 on increasing the pH from 2 to 8 due to changes in the degree of ionization of the amine and carboxylate groups. The isoelectric point was found to be at pH 4.9, which is in agreement with literature values. Effect of pH and Mixing Ratio on Complex Formation. The absorbance values on decreasing the pH of solutions of GA in the presence of varying concentrations of BSA are given in Figure 2. At a 1:1 ratio, the absorbance is seen to increase when the pH is lowered to 4.75, and this is attributed to the formation of insoluble electrostatic complexes due to the interaction between cationic amine groups on the BSA and anionic carboxylate groups on the GA. The solubility of the complexes will depend on the GA/BSA mixing ratio, the degree of ionization of the GA carboxyl groups, and the degree of ionization of the BSA carboxyl and amine groups. At higher BSA additions (R ) 2-5), the pH at which turbidity was first observed increased to pH 5.1, reflecting the delicate interplay of the charge on the GA and BSA molecules and the mixing ratio. The pH at which turbidity was observed remained constant

at higher R, indicating that maximum binding had been achieved. Figure 3 shows the light scattering intensity of the same solutions and it is noted that the scattering intensity increases for all systems at pH 5.5. The fact that there is an increase in scattering intensity without a corresponding increase in absorbance indicates the formation of soluble complexes as discussed by Weinbreck et al.23,24,26,33-36 A phase diagram was constructed from absorbance and light scattering intensity measurements of GA/BSA mixtures at various mixing ratios as a function of pH and this is shown in Figure 4. At R values e1 the complexes formed are soluble over a broad pH range. It is seen that at pH values above ∼pH 5.4 there is no interaction at any mixing ratio between the GA and BSA because both carry an overall negative charge (see Figure 1). However, soluble complexes are formed between pH values of 4.8 and 5.4, which are above the isoelectric point of the BSA. This may be due to the presence of positive patches on the BSA, which are able to interact with the carboxylate groups on the GA molecules, as suggested by Park et al., who studied the phase behavior of a number of protein-polyelectrolyte systems22 or due to charge regulation as noted above.31 At pH values between ∼4.8 and ∼3.0 (depending on the mixing ratio), the GA and BSA carry opposite charges and insoluble complexes are formed. At pH values below ∼3, the GA carries only a very small negative charge, and the BSA carries a significant positive charge, hence, there may be soluble complexes formed or indeed no complexation at all.

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Figure 4. Phase diagram for GA/BSA mixtures as a function of pH and mixing ratio obtained from the turbidity and scattering intensity data presented in Figures 2 and 3.

Further insight into the interaction between GA and BSA has been obtained by dynamic light scattering. The scattering intensity profiles of GA, BSA, and their complexes (R ) 2) at different pHs are shown in Figure 5. It should be noted at this point that the magnitude of the light scattering intensities is highly sensitive to molecular size and that the peak areas are not a true representation of the concentration of the molecular species. In addition the measurements were made at a single concentration and hence the hydrodynamic diameters quoted are apparent values only. It is noted that the BSA alone has a peak maximum at ∼5 nm and that for GA alone there are three peaks with peak maximum values of ∼15, 60, and 300 nm. The fact there are three peaks observed for GA is due to the fact that it contains a number of molecular fractions that differ in molecular mass and protein composition and also because it readily self-associates in solution.38-40 For BSA/GA mixtures at pH 6 there are four separate peaks. The peak at 5 nm is attributed to BSA and the other three peaks at 15, 120, and about 500 nm are attributed to GA. The fact that there are four peaks confirms that there is no interaction between the BSA and GA, which is as expected because GA and BSA are both negatively charged at this pH. At pH 5, the BSA peak is no longer present and there are two peaks observed, one at about 20 nm and the other at 100 nm. These are attributed to the formation of complexes, which are soluble as discussed above. At pH 4.7 there is only one peak at around 450 nm, and it is concluded that the BSA and GA interact to form complexes that, as noted in the phase behavior studies, are insoluble. The particle diameter of the complexes is much larger compared to the soluble complexes formed at pH 5. At pH 3.5, again there is only one peak that appears around 170 nm. The smaller particle diameter at pH 3.5 compared to the complexes at pH 4.7 may be due to the fact that the interaction between GA and BSA is reduced due to the lower charge of the GA molecules. At pH 3.1, which is close to the phase boundary (Figure 4), the expected profile for GA is evident, but not for BSA. It is likely that both soluble and insoluble complexes are formed. At pH 2.7, in addition to the expected peaks for GA there is also a peak at ∼8 nm, which may be attributed to the BSA. The value is slightly higher than at pH 6 because the BSA molecules unfold and adopt a linear rather than globular conformation at low pH.41,42 It is evident at this low pH that there is no interaction between the GA and BSA molecules. The phase diagram was also determined in the presence of an electrolyte from absorbance and scattering intensity measurements of GA/BSA mixtures (R ) 1), and this is shown in Figure 6. It is noted that the pH range over which insoluble complexes are formed is reduced with increasing electrolyte concentration and that no complexation occurs above an electrolyte concentra-

Figure 5. Apparent particle diameters for BSA/GA mixtures at R ) 2 at varying pH in deionized water.


Figure 6. Phase diagram of BSA/GA mixtures (R ) 1) as a function of pH in the presence of electrolyte at a total polymer concentration is 0.25%.

tion of 120 mM NaCl. This is significantly higher than the concentration of ∼55 mM NaCl reported by Weinbreck et al.35 in their studies involving the interaction of gum arabic with β-lactoglobulin. Seyrek at al. undertook a detailed investigation of the effect of electrolyte on the phase behavior of a number of systems including BSA and a hydrophobically modified poly(acrylic acid).43 They concluded that the distribution of charges around the surface of the protein plays a major role in the ionic strength dependence of its binding to polyelectrolytes and, hence, may explain the higher value we observed for the GA/BSA system. Electrophoretic Mobility of Gum Arabic/BSA Complexes. The electrophoretic mobilities of GA/BSA mixtures at varying mixing ratios are given as a function of pH in Figure 7. The electrophoretic mobility of the complexes was found to vary as a function of pH, and the isoelectric points obtained for each are shown in Figure 8. It is noted that the isoelectric point increased with increasing BSA content up to a pH of 4.4, corresponding to R ) 5 and that, at higher ratios, the isoelectric point remained constant. Schmitt et al. also reported that the isoelectric point of β-lactoglobulin/GA complexes changed with the mixing ratio.30 The fact that the isoelectric point becomes constant at R ) 5 indicates that binding is at a maximum. This value is consistent with the earlier assumptions made from the turbidimetric studies. The weight average molecular mass of the GA is ∼860000 Da, while that for BSA is 67000 Da. This means that there are approximately 60 BSA molecules interacting with a single GA molecule.


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Figure 8. Isoelectric points for BSA/GA complexes at different ratios obtained from Figure 7.

The electrophoretic mobility of GA/BSA complexes at varying pH and mixing ratios are given in Figure 9. It was found that the complexes with an electrophoretic mobility greater than (1. 5 µm s-1 V-1 cm-1 were soluble and that those with lower values were insoluble. As the charge of the complex increases above a critical value aggregation is prevented. Isothermal Titration Calorimetry. The enthalpy of the interaction of BSA and GA was determined using ITC at varying pH and the results obtained are presented in Figure 10a-d. The points represent the experimental data and the lines have been fitted using the instrument BindWorks software assuming two sets of multiple binding sites. The corresponding turbidity data are also presented in order to assist in the interpretation of the results. The figures show that the enthalpy change on titration with BSA is very much dependent on the pH. At pH 3, the ITC data indicates that there is a strong exothermic interaction between GA and BSA. The enthalpy decreases in magnitude on addition of BSA above R ∼ 4 and reaches a minimum value at R ) 5. This ratio corresponds to maximum binding between the GA and BSA, as noted above. The interaction is initially accompanied by a large increase in absorbance (turbidity) on addition of the BSA, which is consistent with the formation of insoluble complexes. The absorbance decreases at higher BSA additions due to the formation of soluble complexes or to no complex formation at all, consistent with the studies on phase behavior and hydrodynamic diameter of the molecules reported above. At pH 4.0, the ITC data indicates that there is a strong exothermic interaction between the BSA and GA, which decreases with increasing BSA, reaching a minimum at R ) 5.

Figure 7. Electrophoretic mobility of BSA/GA complexes at varying mixing ratio as a function of pH in deionized water.

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Figure 9. Electrophoretic mobility of GA/BSA complexes as a function of mixing ratio and pH.

There is a corresponding increase in the absorbance, confirming the formation of insoluble GA/BSA complexes. At pH 5.0 there is a small endothermic interaction observed on titration of BSA into the ITC cell. The interaction is not accompanied by an increase in turbidity, and hence, it is suggested that the complex formed is soluble, in agreement with the dynamic light scattering data presented in Figure 5. At this pH, individual BSA molecules have very little net charge because it is close to the isoelectric point. As noted above, it is assumed that interaction occurs between the negatively charged carboxylate groups on the GA and positively charged patches on the surfaces of the BSA molecules or possibly to a shift in the dissociation equilibria induced by complexation. Harnsilawat et al. studied the interaction between β-lactoglobulin and sodium alginate. On titrating the alginate into the reaction cell containing β-lactoglobulin, they showed that the interaction was highly exothermic at pH 3 and 4 but was endothermic at pH 5,27 which is consistent with our observations for GA and BSA. The enthalpy of binding, ∆H, the binding constant, K, and the number of binding sites, N, determined from the ITC experiments using the instrument software, assuming two sets of multiple binding sites, are presented in Table 1. Although we need to be careful in the interpretation of this data using such mathematical models, it is interesting to note that at pH 3 and 4 the total number of BSA molecules interacting with a single GA molecule (i.e., N1 + N2) is ∼68 and 62, respectively, which is in close agreement with findings reported above based on turbidity and isoelectric point measurements. The number of molecules interacting at pH 5 is significantly less, as might be expected since both the BSA and gum arabic are negatively charged. Schmitt and Sanchez have reviewed recent work in the literature undertaken using ITC to study the interaction of polyelectrolytes and proteins.1 In the case of strong polyelectrolytes, complexation is generally characterized by endothermic peaks, which have been attributed to the entropy contribution due to the release of counterions. For weak polyelectrolytes, exothermic peaks are commonly observed and it has been argued that complexation is mainly driven by the enthalpic contribution of the electrostatic interaction. Interestingly, Schmitt et al.44 observed an exothermic followed by an endothermic signal for a single titration of GA into β-lactoglobulin at pH 4.2. Aberkane et al.45 also studied the interaction of GA with β-lactoglobulin

using ITC and concluded that the “two sets of sites” binding model provided the best fit to their data. They argued, however, that the notion of two different and independent binding sites is not realistic when considering the interaction between macromolecules. Instead, it was proposed that the model indicated the presence of two distinct structuring stages during the interaction. In the first stage it was noted that, at pH 4.2, ∼90 β-lactoglobulin dimers interacted with one GA molecule and in the second stage ∼80 β-lactoglobulin dimers were involved. The two stage structuring model was previously used by Girard et al.,46 investigating the interaction between β-lactoglobulin and pectin. When this approach is used for our data presented in Table 1, it is noted that the number of BSA molecules bound to GA in the first stage, N1, and the binding affinity, K1, are significantly greater at pH 3 than at pH 4, although the enthalpy of interaction (∆H1) is much more exothermic at pH 4. It is evident that the BSA will have significantly more positive sites at pH 3, while the GA will have significantly less ionized carboxylate groups present, which may account for the lower enthalpy change at pH 3. The increase in the number and strength of binding at pH 3 may be due to the fact that the BSA molecules unfold and adopt extended structures at this lower pH, facilitating hydrogen and hydrophobic bonding in addition to electrostatic interaction. The hydrophobic bonding could involve the GA rhamnose residues or methyl ester glucuronic acid groups, which have been reported to be at the periphery of the molecules. In the second stage of the interaction, the number of BSA molecules bound (N2) is greatest at pH 4, although the binding affinity (K2) is much lower than at pH 3. The enthalpy change (∆H2) is approximately the same at the two pH values. In this second stage of the interaction, at pH 4 the BSA molecules can interact with vacant sites on the GA molecules, while at pH 3 most of the sites are already occupied. The total number of BSA molecules bound is similar at the two pH values.

Conclusions This study has shown that GA and BSA can form soluble and insoluble electrostatic complexes depending on the solution pH and the mixing ratio. Solubility is a function of the net charge on the complex and it occurs when the electrophoretic mobility



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is greater than (1.5 µm s-1 V-1 cm-1. The evidence obtained by turbidity, electrophoresis, and ITC measurements indicates that the complexes at pH 3 and 4 consist of ∼60 BSA molecules for every GA molecule. At pH 5, the ITC experiments indicated that there are ∼10 BSA molecules per GA molecule. The ITC data has been analyzed in terms of two stages of interaction at pH 3 and pH 4. At pH 3 most of the binding occurs at stage one, while at pH 4 most occurs at the second stage. The difference in the interaction behavior has been attributed to changes in the degree of ionization of the ionizable groups on the protein and GA molecules. The fact that there is an interaction at and above the isoelectric point of the BSA suggests that the carboxylate groups on the GA interact with positive patches on the BSA molecules or that there is charge regulation due to shifts in the dissociation equilibria brought about by complex formation. Complexation is reduced as the ionic strength of the solvent increases and is prevented at NaCl concentrations >120 mM. This relatively high value may be due to the fact that there are charge patches present on the BSA molecules.

References and Notes

Figure 10. (a) Enthalpy of interaction on titrating 13% BSA into 0.03% GA at pH 3.0 and the corresponding absorbance measured at 600 nm. (b) Enthalpy of interaction on titrating 13% BSA into 0.03% GA at pH 4.0 and the corresponding absorbance measured at 600 nm. (c) Enthalpy of interaction on titrating 13% BSA into 0.03% GA at pH 5.0 and the corresponding absorbance measured at 600 nm. (d) Shows the comparison of ITC data only at pH 3, 4, and 5. Table 1. Parameters Calculated from ITC pH parameters

5.0

10.9 ( 3.7 N1 K1 (1.5 ( 0.194) × 103 ∆H1 kJ/mol 170.8 ( 48.2 N2 K2 ∆H2 kJ/mol

4.0

3.0

12.5 ( 2.1 (4.8 ( 0.87) × 103 -1082.4 ( 827.7 56.02 ( 10.62 (1.145 ( 0.564) × 103 -614.2 ( 192.9

57.83 ( 0.92 (1.38 ( 0.4) × 106 -50.1 ( 0.37 4.1 ( 2.1 (1.4 ( 0.166) × 104 -780.8 ( 456.7

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