Studying the influence of metal ions on the structural stability of carbonic anhydrase with the Zetasizer Nano ZSP: An orthogonal approach

Summary

Proteins may be stabilized both by their local buffer solvent environment and by internal bonds. Metal ion specific and non-specific binding is a key influence on the structural integrity of many proteins. The interactions of a protein with certain metal ions may result in pronounced structural changes, with such conformational alterations hypothesized to promote the pathogenicity and virulence of certain infectious agents, and regulate cytotoxicity in prion protein[1].

The primary investigation detailed in this application note compares the structural stability of carbonic anhydrase (CBA) in solution, with and without excess metal ions within the solvent, across a temperature gradient and at different pH values. The z-average size (radius in nm) and electrophoretic mobility (µmcm/Vs) were automatically measured across a temperature range of 20ºC to 70ºC for each sample, and the data sets analyzed.

All measurements were acquired using the new Zetasizer Nano ZSP. The diffusion barrier method, described in application note MRK1651-02, was used during acquisition of zeta potential measurements in order to prevent instrument-induced degradation of the samples.

This note highlights the benefits of an orthogonal approach to measurements of protein structural stability.

Figure 1: Carbonic anhydrase II, bovine, 195 Å resolution [8] IV9E Source RCSB PDB

Method

All samples were prepared at a concentration of 10 mg/mL in 10 mM HEPES buffer (Sigma-Aldrich, Poole, UK), which was diluted from 10x stock with Fresenius sterile water (Fresenius Kabi, UK). The buffer was then equally split into two sterile 500 mL bottles and zinc acetate (Sigma Aldrich, Poole, UK) was added to a final concentration of 5 mM to one of the bottles of buffer. All buffers were pH-adjusted with 1 M sodium hydroxide or 1 M hydrochloric acid and vacuum sterile filtered to 0.02 µm. Protein samples were prepared in the appropriate buffer and again sterile-syringe filtered to 0.02 µm (Fisher Scientific, 50-822-149). The diffusion barrier method was used for the zeta potential measurements of the proteins unless otherwise specified, to prevent protein degradation and denaturation. Duplicate data sets were collected and the average values plotted, with the plot inflection point taken as the point of aggregation/denaturation. The instrument was set up with default parameters, unless otherwise specified in the Appendix: Tables 1 & 2.

Results & Discussion

Carbonic anhydrase

Bovine carbonic anhydrase (CBA) catalyzes the reaction of carbon dioxide and water to produce carbonic acid and hydrogen.

[(-hys)3Zn(H2O)]2+ + CO2 → [(-hys)3Zn]2+ + HCO3- + H+

Hydration is inhibited by imidazole and dehydration is inhibited by aromatic or heterocyclic sulphonamides. Zinc is required for carbonic anhydrase to adopt an active conformation, a zinc ion being bound to three histidyl residues (His 94, His 96 and His 119) in the active enzyme (see figure 1). The potent inhibitor acetazolamide (2-acetylamino-1,3,4-thiadiazole-5-sulphonamide) is unable to bind to the catalytic site in the zinc-free isoform[2].

Thermal trend monitoring of the average size of CBA (figure 2, blue) shows the point of heat denaturation or aggregation onset at 58ºC which corresponds with the published melting point (Tm) value (carbonic anhydrase II - 25 mM PIPES, 100 mM NaCl, 0.5 mM EDTA at pH 7.0)[3]. CBA with excess addition of 5mM ZnAc (figure 2, red) shows a significant reduction in thermal protein structural stability with aggregation onset at 40ºC.

Figure 2: The above chart shows the change in size of CBA as an effect of temperature: CBA in 10mM HEPES buffer pH 7 (blue); CBA in 10 mM HEPES buffer pH 7 and 5 mM zinc acetate (red). The point of aggregation for CBA without metal is estimated at 58ºC with the zinc-associated enzyme aggregating at 40ºC

Figure 3: The above chart shows the change in electrophoretic mobility (µmcm/Vs) of CBA as an effect of temperature: CBA in 10mM HEPES buffer pH 7 (blue); CBA in 10 mM HEPES buffer pH 7 and 5 mM zinc acetate (red). The point of aggregation onset or denaturation is 43ºC for CBA in the presence, or 60ºC in the absence of excess metal in the buffer

The effect of temperature on the electrophoretic mobility of CBA in HEPES buffer in the presence and absence of 5mM zinc acetate was investigated. The collected data (figure 3) demonstrates that CBA in the absence of zinc has an electrophoretic mobility of -1.5 µmcm/Vs (figure 3, blue) that is reduced in magnitude at temperatures greater than or equal to 60ºC, suggesting the onset of protein denaturation/aggregation. This supports the comparative size/temperature trend (figure 2, blue) which illustrates aggregation onset at 58ºC. Addition of 5 mM zinc acetate to the buffer reduces the electrophoretic mobility of CBA, with a point of positive inflection at approximately 48ºC, indicating that protein denaturation/aggregation begins at this temperature. This data supports the comparative size/temperature trend (figure 2, red).

The reduced stability of CBA-Zn and lack of global charge over the pH range 5 to 7 suggests that zinc is involved in, and disrupts, intermolecular interactions.

Effect of pH on CBA Electrophoretic Mobility

Figure 4: The electrophoretic mobility of CBA in 10mM HEPES with (red) and without (blue) 5 mM zinc acetate between pH 3 and 10 were compared. The data shows the effect of the presence of zinc on the protein's electrophoretic mobility.

The kinetic reactivity of CBA is dependent on a solvent pH of between 5 and 8, with the maximum observed rate of reaction at pH 8. The mid-point for activity is pH 7, suggesting that the catalytically active group has a pKa of 7 and that the high pH deprotonated isoform is more catalytically active. This would support the hypothesis: that it is a zinc-bound hydroxide ion that drives this simple mechanism for hydration of carbon dioxide[4].

CBA is only catalytically active at pH 5 to 8 with a bound zinc ion at its active site. The data collected shows how the protein's electrophoretic mobility varies at different pH values, with and without excess free zinc in the buffer. The CBA without zinc (figure 4, blue) has no overall charge at pH 4.5. CBA in the presence of excess zinc (figure 4, red) remains near neutral between pH 5 and 8, the pH range within which the enzyme is known to be in its active conformation and capable of substrate catalysis. In the previous experiments (figures 2 & 3) it was demonstrated that CBA in the presence of excess free zinc has a significantly lower (greater than or equal to 48ºC) melting temperature than CBA in the absence of zinc (greater than or equal to 60ºC). Therefore by comparing its electrophoretic mobility at different pH values, we can elucidate how a particular formulation may influence the overall charge of certain proteins and further understand the solution state of drug candidates or proteins of interest. In conclusion, the Zetasizer Nano ZSP provides an invaluable tool for assessing protein structural stability with two orthogonal approaches within one system.

References:

1. 'Effect of metal ions on de novo aggregation of full-length prion protein'. Biochemical and Biophysical Research Communications, Vol. 320, Issue 4 (2004), 1240-124.; A.Giese, J. Levin, U. Bertsch, H. Kretzschmar.
2. 'Metal ion dependent binding of Sulphonilamide to Carbonic Anhydrase'. Letters to Nature vol 214, 193 - 194 (April 1967). J. E. Coleman
3. 'Thermodynamic stability of carbonic anhydrase: measurements of binding. affinity and stoichiometry using ThermoFluor'. Biochemistry Vol 44 (2005), 5258-66). D. Matulis, J. K. Kranz, F. Salemme and M. J. Todd
4. 'Biochemistry, 5th Edition' Freeman Publishing, New York (2002). J. Berg, J. Tymoczko and L. Stryer

Appendix

Instrument Settings