Anaerobic Analysis of Cytochrome C

The purpose of this blog entry is to discuss the preperation of an anaerobic environment and solutions for the electrochemical analysis of Cytochrome C. The results of these experiments will be discussed in the next blog entry.

Introduction: As the previous entries have established there are a number of severe limitations to performing Voltammetry on CytC on the benchtop. The greatest issue in this process is the fact that once a potential difference (Voltage, V) of -0.2V is reached the reduction of the O2 in the air begins. Whilst attempts to remove O2 from the air had some impact and greatly affected the results the desired analysis of CytC could not be achieved. To succeed it became necessary to perform the analysis within a completely anaerobic environment. Also as far as possible it was determined to remove any O2 from the solutions being used i.e. the buffer and CytC solutions. Also of note in this experiment is the difference between the results obtained when the electrodes being used was altered. During the previous experiments the working electrodes (WE) the carbon point of the electrode was confidured such that it lay "end-on" to the solution. Graphite posesses several key structural features. The most important of these is it's layered effect whereby the C atoms form a large structure of connected 6 member rings, each C atom bonded directly to 3 others (see image below). The non bonding electrons then delocalise over the enitre structure. In an end-on electrode the grphite is arranged so that the planar surface of the graphite sheets is in contact with the solution.

By contrast with an edge-on carbon electrode the graphite arrangement is alterred so that it orientates to the edge of each planar sheet. When polished this provides a far greater surface area for the analytes to associate with and for the electrons to move through increasing the sensitivity of the electrode.

Method: Using a method as described in previous entries a fresh buffer solution was prepared. This solution contained 0.5834g of NaCl and 0.4771g HEPEs in 100mL of DI water for an overall 0.0998M/0.0200M solution (NaCl/HEPEs). When the pH was measured it was determined to  be 6.99. The solution was then placed in a sealed container and argon gas bubbled through the solution for 25 mins to remove any O2 present. This was then immideately transferred into an N2 chamber. Voltammograms were then taken of the previously prepared CytC solutions (100uM and 250uM solutions) using a standard "end-on" carbon electrode. The results were then analysed and further steps taken to remove residual traces of O2 found.

A set of 5 new working electrodes were prepared with a "side-on" arrangement. These electrodes were ground, polished and used in a series of Voltammagrams to determine their effacy. Of these, 3 electrodes labelled 2, 4 and 5 were selected and repeatedly cleaned and polished using fine sandpaper and aluminium oxide until a clean baseline was detected when using them to scan the deoxygenated buffer solution. This process took repeated attempts. Electrode 5 was determined to produce the best signal and was then subjected to repeated polishings until a clean baseline scan using the buffer solution was detected.

A fresh solution of CytC was prepared. 0.0126g of CytC was measured out and transferred into the N2 chamber. Once placed insisde the chamber this was dissolved in 2mL of deoxygented buffer solution to produce a stock solution of 508.65uM (referred to as Stock 1). 2:1, 5:1 and 20:1 dilutions of this stock were prepared to a volume of 1mL each. Once the buffer siganl was determined to be sufficiently clean a set of cyclic voltammagrams were taken of each of these solutions. The scan conditions, unless otherwise noted, were as follows for all scans conducted during this process. Scan rate 50mV/s. Step potential 5mV. Range chosen -0.6V-0.35V vs standard carbon electrode (SCE).