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).

Analysis of Cytochrome C.

In order to begin experimenting on Cytochrome C itself, it is first necessary to establish a baseline of comparison. Therefore a series of experiments was conducted to analyse the electrochemical characterisitcs of Cytochrome C. The following blog entries will focus on describing these experiments.

The series of experiments was conducted in 3 stages. The first involved the preperation of an 0.5mM solution containing horse heart Cytochrome C. UV spectrophotometry was then performed on this solution whilst a reducing agent (ascorbic acid) was added in aliquots. A second reducing agent (thiosulphate) was then used to promote further reduction of the protein.

The Second stage of this experimental series involves performing cyclic voltammetry on a solution of horse heart CytC. The third and final stage involved performing a similar analysis in a fully anaerobic environment. This blog entry will detail the first stage of this experiment.

Stage 1: Using UV spectrophotometry to determine the electrochemical characteristics of horse heart CytC.

The process of UV spectrophotometry can be summarised simply below. Using a device called a spectrophotometer a beam of electromagnetic radiation (light) of a single wavelength can be fired through a sample. The sample will then absorb a certain portion of the radiation, causing a discrepency between the intensity of the light fired into the sample and that leaving the sample. This difference can be measured and recorded as the absorbance. The wavelength can then be varied over time. At any given specific wavelength of light a specific molecule will absorb a certain amount of light, defined by the following equation.

A=ECl

Where A=absorbance of light in arbitrary units.

E=The Extinction Coefficient of the given molecule at the specific wavelength used.

C=Concentration of the sample gievn in M.

l= The pathlength of the sample chamber in cm.

This is known as the Beer-Lambert law. From this we can see that as the concentration of a sample changes the absorbance will change proportionally. As the absorbance is dependent on the structure of the target molecule, if the structure is altered the absorbance will be changed as well.

When the horse heart CytC was purchased it was noted that other studies had identified 2 distinct reduction potentials for the molecule. The 2nd potential () however is not present in every sample of CytC. As this value falls below the potential for the reduction of oxygen it is important to determine wether our sample purchased contains cytc with this potential. It was determined that using a weak reducing agent, ascorbic acid, this could be determined. Ascorbic acid is capable of reducing at the higher reduction potential of CytC () but is not a powerful enough agent to reduce at the lower potential. To reduce CytC at this potential a more powerful reducing agent, dithionite, would be used.

By adding aliquots of ascorbic acid to the CytC solution, and meauring the resultant change in absorbance the complete reduction of the CytC sample containing the higher, by which I mean less negative, redution potential could be established. This would be the point when no further significant changes to the absorbance were detected after adding an aliquot containing ascorbic acid. At this point aliquots containing the dithionite solution could be added to see if further reduction would occur.

Methods: It was calculated that (assuming a MW of 12385 for CytC) 0.006193g of CytC added to 1 mL of buffer solution would generate an 0.5mM CytC solution. A mass of 0.0063g was added to a measured volume of 1mL of Buffer solution to generate a calculated concentration of 0.508mM (3s.f). Using the beer-lambert law with a known E value for CytC at 400nM of 105,000 /M/cm this would yield an absorbance value of 52.5. Such a value is too high to measure using a spectrophotometer and so a 500:1 dilution was prepared by adding 6 uL of CytC solution to 3mL of buffer. This solution has a calculated concentration of 1.02uM (3s.f).

A spectra was then run on the background air with a range of wavelengths of 350nm to 700nm. This established the baseline variation in the machine. Secondly a scan containing just the buffer solution was run. In each case the variation detected was only 0.004, i.e. quite small. After this a scan of the solution (of 1 ml volume) containing CytC (1.02uM) was run. At 409 nm a peak of 0.10085 was detected. When the value for the buffer solution at that peak (0.01899) was subtracted a concentration of 0.946uM (3s.f) was calculated.

A solution of ascorbic acid was then prepared. 0.0210g of acid was measured out and dissolved in 10ml buffer solution. a 100:1 dilution was achieved of this by adding 0.01ml of this acid solution to 0.99ml of buffer solution. A cincentration of 119uM was achieved. this solution was flushed with argon gas and sealed to prevent premature oxidation of the acid. A series of aliquots (5ul) of this acid solution were added to the CytC solution until the absorbance meaured remained consistent over 2 measurements. At this point full reduction of the higher reduction potential was determined to have occured.

A mass of 0.0184g of dithionite solution was weighed out and dissolved in 3mL of buffer solution. 25uL aliquots of this solution were then added to the CytC solution until 2 consistent absorbances were measured and full reduction of the CytC determined to have taken place. The experiment was then repeated using 20 uL and 10 uL aliquots of the ascorbic acid solution and 25 uL aliquots of the dithionite solution.

Results: Both sets of results will be discussed in this section. One potential problem of note exists however. During the first experiment a massive drop in absorbance was measured. This included a fall in the value of absorbance for the pure buffer solution as well. However the absorbance for the cytochrome C solution, measured as a difference from the buffer solution, was unaffected. This drop did not occur in the repeat experiment, indicating a greater reliability in the results. Some possible reasons for this will be discussed briefly at the end of this blog entry.

The attatched Spectra demonstrate the shape of the buffer and completed oxidised cytochrome C solutions. The major peak on the CytC spectra is the peak at 409nm, with a second broad peak seen clearly at roughly 530nm. After the addition of the ascorbic acid the spectra changed in shape. The major peak was shifted towards a longer wavelength proportional to the volume of ascorbic acid added. Also the peak height was increased. Secondly the broad peak at 530nm began to seperate into 2 distinct peaks. In addition the baseline itself was seen to move slightly over the course of the experiment. The movement of these peaks in such dramatic ways led me to choose a more stable region of the spectra for analysis. A section of the baseline was found at 600nm that exhibited no peak activity. A final minor peak was found at 550nm. Unlike the 409nm peak the position did not shift as ascorbic acid was added. Thus to track the reduction of CytC in the solution the difference between the 550nm peak and the baseline at 600nm was recorded.

The results from the scans are summarised in the table attatched. The Table clearly shows that upon addition of ascorbic acid the chemical composition of the solution changes reflected in the change in the UV spectra (i.e the CytC is reduced). The graph in the attatched shows that the reductive process occurs steadily at first and then stops as the increase in ascorbic acid produces no discernable effect. Then the addition of the dithionite solution again triggers a large change corresponding to further reduction of the cytochrome C.

Discussion: The graph clearly demonstrates that beyond a specific point the addition of ascorbic acid was no longer able to produce any clear reduction of the CytC solution. However the fact that dithionite was able to further reduce the solution suggests that both of the reduction potentials are present in the CytC posessed by the lab.

The most perplexing element of the experiment is the massive drop in absorbance recorded. As stated however the profile of the spectra remained constant when this occured, and when the buffer and CytC solutions were scanned again they remained at the new values. In essence the entire spectra was shifted down the y-axis by a massive amount. The consistency of the results suggests that the problem was not related to the chemicals involved in the experiment. This being so the only likely explaination is that there was a change in the machine that led to the drop, either in the software or in the hardware. As stated though the results obatined were consistent, and I believe them to be reliable.