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J. Phys. Chem. B 2005, 109, 14752-14754
Reply to “Comments on ‘Hofmeister Series: Hydrolytic Activity of Aspergillus niger Lipase Depends on Specific Anion Effects’” M. Cristina Pinna,† Andrea Salis,† Maura Monduzzi,*,† and Barry W. Ninham‡ Dipartimento di Scienze Chimiche, UniVersita` di Cagliari - CSGI, S.S. 554 BiVio Sestu 09042, Monserrato (CA), Italy, and Department of Applied Mathematics, A.N.U. Canberra, Australia ReceiVed: May 12, 2005 We would like to thank A. F. Dexter for her comment on our recent letter1 that allows us to explain some presumably unclear points by further confirming the occurrence of Hofmeister effects. The first objection of the Comment concerns pH measurements. It is true that if pH measurements through a glass electrode in the presence of high concentrations of background electrolytes are used to estimate the real proton activity in the aqueous solution, specific ion effects are to be expected and should be accounted for properly. How to do that is still a matter of debate, as demonstrated by a wide variety of recent literature where different models are proposed to rationalize intermolecular interactions. Widespread criticism is a natural consequence. The two articles2,3 mentioned in the comment are just an example. Activity coefficients change as a function of ionic strength. Moreover, at the water/glass interface, some ionic adsorption due to non-electrostatic electrodynamic forces can yield an apparent pH (surface pH) that can be different from the bulk pH. However, our goal was to show that, independently of the possible effects of ion adsorption at the electrode surface on pH measurements, ion-induced pH variations are not the main cause of the observed ion-specific increases in the enzymatic activity. This effect is different for different anions and follows the Hofmeister series. Concerning the comment that raises doubts on possible artifacts in enzyme activity measurements, we remark that in the original letter1 we did not mention all the experimental details. Nor did we present a detailed discussion. The pH data reported in Table 21 may need additional comments. If the extended Debye-Huckel (D-H) approximate equations are used to predict the activity coefficients of the phosphate species, pH variations can be estimated.4 Such estimates require several unknown parameters that represent ion ‘sizes’. A pH of 6.34 can be predicted as a result of 1 M 1:1 electrolyte addition such as NaBr. This value agrees with those experimentally measured in the case of NaCl and NaBr but not for NaClO4. A pH of 6.24 can be calculated for a 2 M concentration of added salt, but in this case, the concentration is largely outside the range of validity of the extended D-H equations. The Debye length is less than 3 Å so that electrostatics is irrelevant. Similar results are obtained for phosphate buffer at initial pH of 6. The calculated pHs are 5.34 and 5.26, respectively, for 1 M and 2 * Prof. Maura Monduzzi. Tel +39 070 6754385. Fax +39 070 6754388. E-mail:
[email protected]. † Universita ` di Cagliari. ‡ A.N.U. Canberra. Visiting Professor at Universities of Cagliari and Florence.
TABLE 1: Observed Molar Extinction Coefficients (Eobs) of p-nitrophenol (p-NP-OH)/p-nitrophenloate (p-NP-O-) Determined from Calibration Lines Performed at Different Sodium Salt Concentrations salt concentration (M)
obs (NaCl)
obs (NaNO3)
obs (NaBr)
obs (NaClO4)
0 0.1 0.3 0.5 1 2
8990 6220 4770 4200 2910 2050
8990 6260 4740 3980 2830 1760
8990 6070 4690 4000 2750 1400
8990 6160 4620 3590 2380 1210
M concentrations of added electrolyte. Clearly pH measurements are affected by ion-specific effects. These may be related either to surface or to bulk phenomena, or both. But the real value of pH is not relevant to our point. We know that pH decreases as a result of added salts. Consider now enzyme activity measurements that are based on the p-nitrophenyl acetate (p-NPA) hydrolysis. Spectrophotometric measurements were carried out after a determination of calibration lines of p-nitrophenol absorbance/concentration, at all pH values, and for all salt types and concentrations. Each calibration line gave a different angular coefficient that, for the Lambert-Beer law, is proportional to the molar extinction coefficient () at a determined wavelength (λ). The experimental values are reported in Table 1. Each calibration was performed by reading the absorbance at 400 nm of several p-nitrophenol (p-NP-OH) standards dissolved in the phosphate buffer solution at the given sodium salt concentration. Correlation coefficients in the range 0.996-0.999 were determined. This confirms the validity of the Lambert-Beer law in our systems. The obtained values can be defined as “observed” molar extinction coefficients, since they are determined by the equilibrium
pKa ) 7.15 (thermodynamic value) The obs is the result of the of the two absorbing species, p-nitrophenol and p-nitrophenolate (p-NP-O-) at λ ) 400 nm (pNPO- . pNPOH). It should be noted that, in these calibration buffers, added salts and the main absorbing enzymatic hydrolysis species are considered. These obs values reflect the state of p-NP-OH dissociation as a result of salt addition. As demonstrated by obs value decreases, dissociation decreases with increasing salt concentration. It is remarkable that this effect is not only due to ionic strength increasing but, as shown in Table 1, follows the Hofmeister series (Cl- > NO3- > Br- > ClO4at salt concentration g 0.3 M). When enzymatic hydrolysis of p-NPA occurs, three simultaneous equilibria are present, namely eqs 1, 2, and 3.
H2PO4- + H2O a HPO42- + H3O+
(2)
CH3COOH + H2O a CH3COO- + H3O+
(3)
with pKa (thermodynamic value) of 7.21 for eq 2 and 4.84 for eq 3.
10.1021/jp052491l CCC: $30.25 © 2005 American Chemical Society Published on Web 07/08/2005
Comments
Figure 1. Cation effect on enzymatic activity of lipase A (Aspergillus niger) in phosphate buffer (5 mM, pH 7) solution with increasing concentration of bromide salts.
At very low reaction times (initial rate determination), when reagent conversion is still low, equilibrium 2 is the most important one and has the power of shifting eqs 1 and 3 toward the left. Hence, no overestimation of enzymatic activities is likely to occur. Eventually, the small contribution of CH3COOH produced in the enzymatic reaction (not considered in the calibration) should lead to an underestimate of the enzymatic activity. Moreover, at pH e 6 (without added salt), where the buffer acts weakly, the eventual decrease of pH due to equilibrium 3 should give, in the worst case, a further decrease of the measured enzymatic activity (see Figure 1 in Pinna et al.1). Thus, no overestimates of activity are possible in any case. As to the trends of enzymatic activity with increasing salt concentration, no model or mechanism was suggested. We agree that a plateau of activity with increasing salt concentration may be expected, but we did not observed it in the investigated concentration range. The salt-induced superactivity of enzymes is not a novelty. Many examples are reported in the literature.5-7 Sometimes biochemists refer to the superactivity phenomena as salt-induced ‘osmotic stress’. Some anions promote the stress, some others counteract this stress. When different anions and also cations are compared, very often a direct or reverse Hofmeister series can be identified. The detailed mechanisms behind these phenomenological effects are still unclear. But they exist, and it has been demonstrated on many occasions that they cannot be accounted for by simply invoking pH changes or ionic strength effects. In our system, salt-induced superactivity is assessed by the calibrations performed for each activity measurement; thus, any possible artifact can be ruled out. Specific ion effects start to occur at salt concentration greater than 0.1 M, and increase with increasing electrolyte concentration. It is reasonable that non-electrostatic dispersion forces can play a significant role at high concentrations. They include ion type properties such as ion volume, ion charge, ion hydration, and ion polarizability. Just as an example we may cite here some previous reports describing similar effects.6,8-13 To provide further support to the previous findings and related conclusions,1 we present here additional data that demonstrate specific ion effects for monovalent cations also. Enzymatic activity measurements were performed in phosphate buffer 5 mM at initial pH of 7 containing bromide salts (Figure 1) with the same experimental procedures used for anions (calibrations for each activity measurement).
J. Phys. Chem. B, Vol. 109, No. 30, 2005 14753
Figure 2. Enzymatic activity (filled symbols, left y axis) and pH (empty symbols, right y axis) of lipase A (Aspergillus niger) in phosphate buffer (5 mM, pH 6) solution with increasing concentration of sodium salts NaCl (b), NaBr (9), NaSCN ([), and NaClO4 (2).
Again, a Hofmeister series is obtained for cations in the order Li+ > Na+ > K+ > Cs+ > TMA+ (tetramethylammonium) at 1 M salt concentration. A more significant effect is obtained at 2 M (LiBr was not soluble at this concentration). This allows us to indicate NaBr as the most effective specific ion pair that, when added at 2 M concentration, promotes a 3.7-fold increase of the hydrolytic activity of Aspergillus niger lipase in phosphate buffer 5 mM at initial pH of 7. Besides the anion-specific effect shown in the previous letter,1 Hofmeister effects can also be seen if the enzymatic specific activity is measured using a phosphate buffer at initial pH of 6. In this case, we replaced the anion SCN- (the highest chaotropic ion in the Hofmeister series) for the more neutral NO3-. Activity and pH data are shown in Figure 2 for 1 and 2 M concentrations of added salts. Data were also corrected for spontaneous hydrolysis of p-NPA induced by the salt and/or the pH. While pH values decrease with increasing salt concentrations as expected, different trends of the enzymatic activity are observed for the different anions. Independently of the salt-induced pH decrease, it is remarkable that a high concentration of the most chaotropic anions, namely SCN- and ClO4-, causes a decrease of activity below the value measured in the buffer at initial pH of 6. On the contrary, NaCl and NaBr induce similar superactivities. These trends should be compared to those observed in the phosphate buffer at initial pH of 7 where all salts increased enzyme activity in the order Br- > Cl- ≈ NO3- > ClO4-.1 Finally, enzymes as Aspergillus niger lipase are mainly used for biotechnological applications in ester hydrolysis, esterification, and transesterification (the last two processes occur in nonconventional media).14,15 These applications do not generally require a highly pure enzyme, both because of high purification costs and also because some “impurities” (usually carbohydrates) are stabilizers of the preparation or inert diluents added by the manufacturer to standardize activity of enzymes obtained from different production batches.16 Acknowledgment. MIUR-PRIN 40% (Italy) and Consorzio Sistemi Grande Interfase (CSGI-Firenze) are acknowledged for financial support. References and Notes (1) Pinna, M. C.; Salis, A.; Monduzzi, M.; Ninham, B. W. J. Phys. Chem. B 2005, 109, 5406.
14754 J. Phys. Chem. B, Vol. 109, No. 30, 2005 (2) Bostro¨m, M.; Craig, V. S. J.; Albion, R.; Williams, D. R. M.; Ninham, B. W. J. Phys. Chem. B 2003, 107, 2875. (3) Spitzer, J. J. J. Phys. Chem. B 2003, 107, 10319. (4) Butler, J. N.; Cogley, D. R. Activity coefficients and pH. In Ionic Equlibrium: Solubility and pH Calculations; John Wiley & Sons: New York, 1998; p 41. (5) Amaya, I.; Botella, M. A.; de la Calle, M.; Medina, M. I.; Heredia, A.; Bressan, R. A.; Hasegawa, P. M.; Quesada, M. A.; Valpuesta, V. FEBS Lett. 1999, 457, 80. (6) Zolda`k, G.; Sprinzl, M.; Sedla`k, E. Eur. J. Biochem. 2004, 271, 48. (7) Yancey, P. H.; Clark, M. E.; Hand, S. C.; Bowlus, R. D.; Somero, G. N. Science 1982, 217, 1214. (8) Warren, J. C.; Stowring, L.; Morales, M. F. J. Biol. Chem. 1966, 241, 309.
Comments (9) Park, C.; Raines, R. T. J. Am. Chem. Soc. 2001, 123, 11472. (10) Wondrak, E. M.; Louis, J. M.; Oroszlan, S. FEBS Lett. 1991, 280, 344. (11) Kim, H.-K.; Tuite, E.; Norde´n, B.; Ninham, B. W. Eur. Phys. J. E 2001, 4, 411. (12) Wright, D. B.; Banks, D. D.; Lohman, J. R.; Hilsenbeck, J. L.; Gloss, L. M. J. Mol. Biol. 2002, 323, 327. (13) Bismuto, E.; Nucci, R.; Febbraio, F.; Tanfani, F.; Gentile, F.; Briante, R.; Scire`, A.; Bertoli, E.; Amodeo, P. Eur. Biophys. J. 2004, 33, 38. (14) Jaeger, K. E.; Reetz, M. T. TIBTECH 1998, 16, 396. (15) Salis, A.; Pinna, M. C.; Murgia, S.; Monduzzi, M. J. Mol. Catal., B: Enzymatic 2004, 27, 139. (16) Bjurlin, M. A.; Bloomer, S.; Hass, M. J. J. Am. Oil Chem. Soc. 2001, 78, 153.
