Nernst plot

It is a good idea, when using such simple Nernst plots as an analytical method of determining an activity, to check that the intercept at jc = 0 is indeed the standard electrode potential. (There are many compendia listed in the Bibliography at the end of this book that cite large numbers of values, as does Appendix 3.)... 

Cornish-Bowden A, Koshland DE. 1975. Diagnostic uses of the Hill (logit and Nernst) plots. J Mol Biol 95 201. 

Fig. 22. UV-visible spectra of recombinant Rhodnius NPl-NO at pH 5.5 as a function of applied potential. In order of decreasing Soret band heights +200, —60, —70, —80, —90, —100, —110, and —400 mV vs Ag/AgCl, respectively (add 205 mV for potential vs NHE). Inset) Nernst plot of the spectroelectrochemicEil data. Reprinted with permission from Ref 49).

Using the spectral data of Fig. 22, and similar data obtained for the nitrophorins in the absence of NO and in the presence of histamine, imidazole, or 4-iodopyrazole, Nernst plots such as that shown in the insert of Fig. 22 were constructed, and the midpoint potentials of the nitrophorins and their NO and histamine complexes were calculated. The results are summarized in Table IV, where they are compared to those obtained earlier for NPl (49, 50, 55). All potentials are expressed vs NHE (+205 mV with respect to the Ag/AgCl electrode used in the spectroelectrochemical titrations and the Nernst plot shown in the insert of Fig. 22). It can be seen that the reduction potentials of all four nitrophorins in the absence of NO or histamine are within 20-40 mV of each other. The reduction potentials of their NO complexes, however, differ significantly from each other. For example, the reduction potential of NP4-NO is about 350 mV more positive than that of NP4 in the absence of NO, as compared to a 430 mV shift for NPl upon binding NO, and the positive shifts for NP2—NO and NP3—NO are somewhat smaller (318 and 336 mV, respectively, at pH 7.5) 49, 50). These differences relate to the ratios of the dissociation constants for the two oxidation states, as discussed later. 

Figure 3.16 (A) Spectra recorded during spectropotentiostatic experiment in optically transparent thin-layer electrode on 0.97 mM o-tolidine, 0.5 M acetic acid, 1.0 M HC104. Applied potentials A, 800 B, 660 C, 640 D, 620 E, 600 F, 580 G, 400 mV vs. SCE. (B) Nernst plot at 438 nm. [From Ref. 36.]...

Figure 17.12 Spectropotentiostatic experiment conducted with a 6.08 tnM solution of [Re3Cl,2]3 in the 49.0-51.0 mol% AlCl3-l-methyl-3-ethylimidazolium chloride melt at40°C using the cell shown in Figure 17.11. Applied potentials (V) (a) open circuit, (b) -0.266, (c) -0.303, (d) -0.325, (e) -0.340, (f) -0.352, (g) -0.364, (h) -0.383, (i) -0.405, (j) -0.550. Inset Nernst plot constructed from the spectra in this figure. [From S. K. D. Strubinger, I.-W. Sun, W. E. Cleland, and C. L. Hussey, Inorg. Chem. 29 993 (1990), with permission.]...

Methods. All solutions were prepared to be ImM Cytochrome c, 0.1mM DCIP, 0.10M alkali halide, and 0.10M phosphate buffer at pH 7.0 or pD 7.0. The DCIP served as a mediator-titrant for coupling the Cytochrome c with the electrode potential. E° values were measured using a previously described spectropotentiostatic technique using an optically transparent thin-layer electrode (OTTLE) (7,11,12). This method involved incrementally converting the cytochrome from its fully oxidized to fully reduced state by a series of applied potentials. For each potential a spectrum was recorded after equilibrium was attained. The formal redox potential was obtained from a Nernst plot. The n value... 

Oxidation by hydrogen peroxide ( and O) followed by reduction by ascorbate (A and X). The abscissa represents electron equivalents present per molecule of laccase. Insert Nernst plot of the type 3 copper against type 1 copper. Ox/Red represents the calculated ratio between concentration of the oxidized and reduced chromophores. For calculating OxSso the extinction valve for native oxidized laccase at 330 nm was used. 

Figure 8. Nernst plots of equilibrium constants (left) and oxidation rate (2e ) and nitroxide (le )9 respectively, as counterparts (data from Ref. 44.

Again, the value of Eq may be extracted from a Nernst plot of the logarithm of the concentration ratio [0]/[R] against E. In some cases, the measurements are made difficult by the presence of additional absorbances in the UV range due to the mediator couple. 

FIG. 19 Nernst plots of cathodic peak potentials vs. pH for oxidized carbon electrodes (a) CWZ—Ox (b) CWN2—Ox (c) RKD3—Ox. (Adapted from Refs. 27 and 194.)... 

Figure 5B, Double Nernst plot of the reduction by i.-ascorbate at 610 and 330 nm, phosphate buffer (vH 7.0), I = 0.20 M, J0.0 C, enzyme 28 fxM.

The Nernst plot slope in this case doesn t yield an n value that corresponds to the true number of electrons transferred and the observed apparent midpoint potential Eijf) is also shifted positive relative to the true Nernstian value. Consequently, the observed midpoint potential, associated with the... 

This same R T state equilibrium model can also be used to interpret the non-Nernstian behaviour of Fib redox monitored by spectroelectrochemistry. Reduction or oxidation of interacting electroactive centres in Fib gives rise to a nonlinear Nernst plot. The changing slope of the Nernst plot, n, can no longer be simply interpreted as the number of electrons transferred. Nonlinear Nernstian plots obtained from such spectroelectrochemical data require a special treatment. 

The n value and the constant change in slope (= (RTIF)n) along the curve cannot be interpreted as the number of electrons transferred during the oxi-dation/reduction process however, the n value at the midpoint potential, //2, is indicative of the level of cooperativity between different subunits. The 1/2 value allows the comparison of different Nernst plots as a function of the... 

Figure 2.3 Uncorrected (a) and corrected (b) Nernst plots for diferric (Fe2Tf), O C-terminal (FecTf) and T N-terminal (FeNTf) monoferric transferrin. Conditions [MV ] = 0.2-0.4mM [KCl] = 500 mM [MES] = 50mM at pH = 5.8 20 °C FcjTf (0.11-0.19mM in Fe) FccTf (0.18-0.22 mM in Fe) FeNTf (0.68 mM in Fe). Error bars represent standard deviations for the average of 2-3 independent experiments. Data obtained below -530 mV for Fe2Tf and FecTf in panel (a) were not used for the corrected Nernst plots in panel (b) due to the low absorbance changes measured at these low potentials. Figure adapted from ref. 6 and used with permission.

Figure 2.4 Plot illustrating various parameters that may be obtained from a Nernst plot of Hb that shows cooperativity. The lower line is a plot of eqn (2.3) for human hemoglobin A (Hb Aq). The upper line is a plot of the changing slope of the Nernst plot multiplied by 58.1 FjRT). These data serve to illustrate the parameters F1/2, 50 and Zmax- Figure adapted from ref.

The relationship between the concentration ratio of the oxidised to reduced form of the protein and the absorbance readings has been demonstrated in Section 2.3.1 and can be used to create a Nernst plot using eqn (2.3) (Figure 2.5). ° The result is a well-behaved Nernstian plot as evidenced by its slope ( / mV ) corresponding to a single electron-transfer process n= 1). The reduction is expected to facilitate the dissociation of the reduced Fe because of its increased lability and lower affinity for the protein. In the presence of a mediator, the equilibria involved in the OTTLF cell can be as follows,... 

Optical spectra of transferrin C-lobe docked with the transferrin receptor showed a characteristic broad absorption band centred at 465 nm, just as in the receptor-free /zo/o-protein (Figure 2.1 inset). The intensity of this absorbance band declined as more negative potentials were applied in a spectroelectrochemistry experiment, but did not qualitatively change in its overall features. An EPR spectrum of the Fec/TfR complex at pH 5.8, recovered from the OTTLE cell after completion of spectroelectrochemical studies allowed us to conclude that the first coordination shell of Fe " in transferrin is intact and unperturbed when C-lobe is complexed with TfR. Consequently, we assume that C-lobe and Fec/TfR complex have similar if not identical Fe " and Fe binding constants, and so we take for binding of Fe " in the protein-receptor complex to be 10 M as calculated for free Tf. This value was used to correct the observed Nernst plot data by accounting for the dissociation of Fe that occurs upon reduction. Nernst plots for the observed spectroelectrochemical data for FccTf/TfR, and data corrected for Fe dissociation, are presented in Figure 2.7. The corrected plot exhibits typical Nernstian behaviour for a one-electron transfer and a E1/2 value of —285 mV. 

Figure 2.8 compares corrected Nernst plots for C-lobe half-transferrin free in solution and bound to the transferrin receptor, at endosomal pH. These data clearly demonstrate that docking iron-loaded C-lobe transferrin at the transferrin receptor at pH 5.8 makes it energetically more favourable to reduce Fe " to Fe by 200 mV. Furthermore, receptor-docking places Fe reduction in a range accessible to NADH or NADPH cofactors, consistent with the hypothesis that reduction is the initial event in iron release from transferrin in the endosome. Fe " is bound by /zTf at least 14 orders of magnitude more weakly than Fe, so that reductive release of iron bound to HTi in the transferrin-transferrin receptor complex is then physiologically and thermodynamically feasible, and the barrier to transport across the endosomal membrane is lifted. The transferrin receptor, therefore, is more than a simple conveyor of... 

Representative Nernst plots corresponding to the reduction of Fe " in the Fec/TfR assembly. (A) Observed data ( ) Data corrected for Fe " dissociation from Fec/TfR. Conditions Au mesh electrode [FccTf/ TfR] = 0.19mM [MES] = 50mM at pH 5.8 [methyl viologen]= 1.4mM [KCl] = 500 mM. Figure adapted from ref. 19 and used with permission. 

The first coordination shell of Fe in Fib is identical to that of Mb, but there are other major differences that influence the spectroelectrochemical/Nernst plot profile of Hb and make it distinct from that of Mb. Hb is a tetrameric protein with four heme-containing subunits (a2p2), each of which is redox-active. Differences between Mb and Hb include amino-acid sequence, which results in different redox potentials for the a and (3 chains, plus subunit-subunit interactions that lead to allostery in Hb. This allosteric interaction leads to cooperative electron transfer that gives rise to a non-Nernstian redox profile that requires special consideration for data analysis and interpretation of results. ... 

A general trend is evident that ease of Hb oxidation (lower 1/2) generally correlates with increased O2 affinity (low F1/2) for various Mbs, Hbs and their mutants. This is illustrated in Figure 2.9 where the Nernst plot for horse Hb is displaced towards lower potentials relative to that of human Hb Aq. One of the structural differences between human and horse Hb is the substitution of a... 

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