2.E.72.E.7
2.E.7Alternative protein/precipitant systems for future workAlternative protein/precipitant systems for future work Alternative protein/precipitant systems for future workAlternative protein/precipitant systems for future work
Phase diagrams of the type compiled by Iwai et al.22 (for lysozyme), appear to be nonexistent or at least not easily available for other protein/precipitant systems. There does however appear to be fragmented pieces of data within the literature which we have collated (vide infra) to give one an idea of how another
protein/precipitant system may fare under the methodology utilised and presented herein61. Basic requirements that need to be met by such an alternative system would be the following
1) pH dependence on protein solubility.
2) A precipitant that is able to undergo either oxidation or reduction on the electrode surface at reasonable potential.
3) A reasonably sized metastable zone width.
4) Comprise protein which is easily available and in high quantity. 5) Protein that does not degrade under electrolysis
These basic requirements appear to be mostly met by the well known and well studied glucose isomerase/ ammonium sulphate system.
Chapter 2 Page 134
Figure 2.31 The solubility of crystalline glucose isomerase in the presence of ammonium sulphate taken at approximately pH 5.5 from work by Chayen61. Panel (b) same as (a) except that the solubility
has been plotted on a log scale. Figures taken from ref. 61
Ammonium sulphate dissociates into NH+4 and 2- 4
SO ions in solution and some sample reduction reactions and associated potentials (vs. NHE)62 for both the cation and anion are given below
2- + - 4 2 2 SO +4H +2e ←→ SO +2H O (+0.17 V) (2.9) 2- + - 2- 4 2 6 2 2SO +4H +2e ←→ S O +H O (-0.22 V) (2.10) 2- + - 2- 4 3 2 SO +4H +2e ←→ SO +H O (-0.93 V) (2.11)
(
)
- + - 2 4 2 4 N H +4H O+2e ←→ NH +4OH +0.11V (2.12)By application of relatively small positive and negative potentials, one may be able to decrease the concentration of either cation or anion in the vicinity of the electrode. Although some sample reactions62, particularly for the sulphate ion indicate the situation appears more advanced than the relatively more simple electrochemical processes evident with a NaCl precipitant. In any case, the major advantage with the glucose isomerase/(NH ) SO4 2 4 system is fact the lower potentials may be possibly used reduce the precipitant at the electrode surface, thus enabling the separation of water oxidation/reduction from precipitant oxidation/reduction.
Chapter 2 Page 136 The glucose isomerase/ammonium sulphate system appears to be a good
system for future work. Although electrochemically, the reactions taking place at the electrode will be far from simple. One should also note that the isoelectric point of glucose isomerase63 is around pH 5 and glucose isomerase readily forms crystals between pH 6 – pH 8, but is known to rapidly degraded below pH 5 and thus only cathodic potentials may possibly be used. The protein is also known to show solubility decreases with increasing temperature64, enabling further degrees of control over protein solubility via control of temperature.
Having identified another possible model system with which one could further investigate our proposed conceptual framework, it may be useful to consider protein/precipitant systems which would be considered unsuitable for this method.
We have conducted some preliminary work on ferritin (Figure 2.34), which we have found to be unsuitable at this present time. This ‘unsuitably’ was thought to be mostly due to the lack of control caused by a very short metastable zone65. The short width of the metastable zone is particular to the ferritin/CdSO4 system (Figure 2.33). It is thought that due to the short metastable zone width one is able to move from being supersaturated to undersaturated (via pH changes and cadmium
Additionally, we considered our level of control over Cd2+reduction
insufficient as large cadmium formations (islands and whiskers) were found to form on the platinum electrode surface (Figure 2.34). A neater, more controllable system could be formed by using stripping voltammetry (with a mercury droplet), which would enable fine control over the Cd2+
concentration at the droplet surface. This lack of control overCd2+
concentration was further evidenced by what appeared to be ‘drifting’ between mass nucleation, dendritic skeletal crystal morphology and no crystals at all using the same experimental conditions.
Figure 2.33 A phase diagram for the ferritin/ CdSO4 system in 0.2 M sodium acetate buffer (pH 5). Note the very short width of the metastable zone. The solubility curve is the solid line and the dashed
lines indicate the beginning of the nucleation zone. Figure taken from ref. 65.
Ferritin ‘electrocrystallization’ was previously attempted by Moreno5 et al. who found that triangular wave pulses (incorporating both cathodic and anodic potentials) resulted in changes to crystal morphology (a change from dendritic to cubic). We found that although ferritin crystals appeared to grow preferentially under cathodic regimes, the crystal morphology became skeletal and dendritic via what appears to be a mechanism based on polyhedral instability66.
Chapter 2 Page 138 With regards our own findings, Cd2+ became reduced on the electrode surface at cathodic potentials which produced large islands and sometimes ‘whiskers’ of cadmium on the electrode surface. Although deemed unsuitable at this point in time, the ferritin/CdSO4system does represent a good challenge and a yardstick against
which one may measure the degree of ones control over the concentration gradients at the electrode surface via electrolysis.
Figure 2.34 The crystallization of ferritin on a 2mm diameter Pt disc electrode under a 2 A/m2 cathodic
regime after 2 hours. Black areas are cadmium islands. Crystal morphology is showing signs of becoming dendritic.