Protein film voltammetry

[citation needed] Provided that it makes suitable contact with the electrode surface (electron transfer between the electrode and the protein is direct) and provided that it is not denatured, the protein can be fruitfully interrogated by monitoring current as a function of electrode potential and other experimental parameters.

[1] Special electrode designs are required to address membrane-bound proteins.

[2] Small redox proteins such as cytochromes and ferredoxins can be investigated on condition that their electroactive coverage (the amount of protein undergoing direct electron transfer) is large enough (in practice, greater than a fraction of pmol/cm2).

Electrochemical data obtained with small proteins can be used to measure the redox potentials of the protein's redox sites,[3] the rate of electron transfer between the protein and the electrode,[4] or the rates of chemical reactions (such as protonations) that are coupled to electron transfer.

[5] In a cyclic voltammetry experiment carried out with an adsorbed redox protein, the oxidation and reduction of each redox site shows as a pair of positive and negative peaks.

[3] The same is true for experiments performed with non-biological redox molecules adsorbed onto electrodes.

[7] Since both this faradaic current (which results from the oxidation/reduction of the adsorbed molecule) and the capacitive current (which results from electrode charging) increase in proportion to scan rate, the peaks should remain visible when the scan rate is increased.

In contrast, when the redox analyte is in solution and diffuses to/from the electrode, the peak current is proportional to the square root of the scan rate (see: Randles–Sevcik equation).

Irrespective of scan rate, the area under the peak (in units of AV) is equal to

At slow scan rates there should be no separation between the oxidative and reductive peaks.

is the exchange electron transfer rate constant in Butler Volmer theory.

Laviron equation[4],[8],[9] predicts that at fast scan rates, the peaks separate in proportion to

The binding of a small molecule (other than the proton) may also be coupled to a redox reaction.

Two cases must be considered depending on whether the coupled reaction is slow or fast (meaning that the time scale of the coupled reaction is larger or smaller than the voltammetric time scale[10]

The electroactive coverage of large redox enzymes (such as laccase, hydrogenase etc.)

is often too low to detect any signal in the absence of substrate, but the electrochemical signal is amplified by catalysis: indeed, the catalytic current is proportional to turnover rate times electroactive coverage.

The effect of varying the electrode potential, the pH or the concentration of substrates and inhibitors etc.

is the electroactive coverage, and TOF is the turnover frequency (or "turnover number"), that is, the number of substrate molecules transformed per second and per molecule of adsorbed enzyme).The latter can be deduced from the absolute value of the current only on condition that

The factors that may influence the TOF are (i) the mass transport of substrate towards the electrode where the enzyme is immobilised (diffusion and convection), (ii) the rate of electron transfer between the electrode and the enzyme (interfacial electron transfer), and (iii) the "intrinsic" activity of the enzyme, all of which may depend on electrode potential.

In that case, mass transport of substrate towards the electrode where the enzyme is adsorbed may not be influential.

Under very oxidising or very reducing conditions, the steady-state catalytic current sometimes tends to a limiting value (a plateau) which (still provided there is no mass transport limitation) relates to the activity of the fully oxidised or fully reduced enzyme, respectively.

If interfacial electron transfer is slow and if there is a distribution of electron transfer rates (resulting from a distribution of orientations of the enzymes molecules on the electrode), the current keeps increasing linearly with potential instead of reaching a plateau; in that case the limiting slope is proportional to the turnover rate of the fully oxidised or fully reduced enzyme.

[8] The change in steady-state current against potential is often complex (e.g. not merely sigmoidal).

[12] Another level of complexity comes from the existence of slow redox-driven reactions that may change the activity of the enzyme and make the response depart from steady-state.

If a RDE is used, these slow (in)activations are detected by a hysteresis in the catalytic voltammogram that is not due to mass-transport.

The hysteresis may disappear at very fast scan rates (if the inactivation has no time to proceed) or at very slow scan rates (if the (in)activation reaction reaches a steady-state).

[14] Conventional voltammetry offers a limited picture of the enzyme-electrode interface and on the structure of the species involved in the reaction.

Complementing standard electrochemistry with other methods can provide a more complete picture of catalysis.

Fig 1. Voltammetric signals for a one-electron (A) or two-electron (B) redox species adsorbed onto an electrode in the limit of slow scan rate. The current plotted here is normalized by , implying that the measured current increases in proportion to scan rate ( ).
Fig 2. Effect of scan rate on the voltammetry of a one-electron redox species adsorbed onto an electrode (the current shown here is normalized by , implying that the measured current increases in proportion to scan rate ( )) calculated for , K. The scan rate increases from blue to red. A: voltammograms. B: peak potentials