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.

Science Project: Voltammetry Practise, Method.

This Blog entry is a write up of my first experiments involving Voltammetry. the aims and methods are described here, whilst the results, graphs and discussion shall be uploaded in the next entry.

Introduction: As a great many of the experiments likely to occur during the course of this project may include Voltammetry, specifically cyclic voltammetry, a simple experiment was designed to familiarise myself with the method and process. The experiment itself concerns a voltammteric analysis of Potassium Ferricyanide (K3Fe(CN)6). The experiment itself was conducted in 2 stages on seperate days. On the first day a HEPES/NaCl buffer solution was prepared and scanned using voltammetry. On the second day this buffer was used in the preparation of the K3Fe(CN)6 solution. 2 seperate dilutions of this solution were scanned and the data collected for analysis. Due to the nature of the exerirment, and the well known charcateristics of Ferricyanide the following predictions were made.

1) An average redox potential of 220mV against a standard Ag/AgCl reference electrode.

2) Increased seperation fo the oxidation/reduction peaks on the voltammogram as the scan rate is increased.

3) The current detected at a specific scan rate to be proportional to the concentration of the Ferricyanide solution i.e. a higher current at the higher concentration.

Method: Stage one involved the preperation of 100mL of 0.02M HEPES/0.1M NaCl buffer solution.

As a general note, each piece of equipment was thoroughly washed with deionised water and dried before use.

A small quantity od=f Deionised water (DI water) was placed in a beaker for easy access and use. A mass of 0.4758g (4d.p) of HEPES was weighed out. This mass was placed in a seperate beaker and dissolved in roughly 10mL of DI water. A mass of 0.5835g (4d.p) of NaCl was then weighed out and again dissolved in roughly 10mL of DI water. The salt solution was then stirred using a magnetic stirrer and DI water addede until the salts had completely dissolved (~40mL) and a clear colourless solution formed.

Using a pH meter, calibrated using pH 4 and 7 stock solutions, the pH of the salt solution was measured as 5.37. Small volumes of 0.1M NaOH solution were added until a pH of 6.99 was recorded. The buffer solution was then transferred into a 100mL volumetric flask and made up to measure using DI water. The pH was again recorded aa 6.94. The final concentrations of the buffer solution are calculated as 0.1000M NaCl/0.0199M HEPES (4d.p). A series of Voltammograms were run for this buffer at 3 seperate scan rates. Graphs 1-3 of the next blog entry show the results of the scans. The buffer was then labeled and stored in a chilled room overnight.

On the second day of the experiment a mass of 0.0162g (4d.p) of K3Fe(CN)6 was weighed out. This was dissolved in 2mL of Buffer solution and a clear yellow solution formed. This solution was then transferred into a 10mL volumetric flask andmade up to volume using buffer solution. The final concentration of ferricyanide was calculated at 4.9201mM (4d.p).

An initial voltammogram of the buffer solution alone was run to calibrate and test the equipment. Following this the working electrode was cleaned and prepared. A 9:1 diltution of the ferricyanide was prepared with a volume of 1 mL, thus containing 0.1mL ferricyanide solution and 0.9 mL of buffer solution, and placed in the voltammogram. This yields a concentration of 0.4920mM (4d.p). A series of scans with the following conditions and perameters were conducted.

Scan Rate. Step Potential. E(initial) E(max) E(min)

1)20mV/s 2mV/s 0.4V 0.5V -0.1V

2)20mV/s 2mV/s 0.4V 0.6V -0.2V
3)10mV/s 1mV/s 0.4V 0.6V -0.2V
4)50mV/s 5mV/s 0.4V 0.6V -0.2V

Multiple scans (10) were run for each set of conditions, and the results collected. An overlay graph showing each set of conditions is shown in the next entry in graph 4.

The voltammogram was emptied and cleaned and the working electrode washed. Finally an 0.00492mM solution of ferricyanide was prepared and a set of scans corresponding to condition 2-4 were conducted. See graph 5 for a similar overlay showing the results.

Notes:

1) HEPES=4-(2 hydroxyethyl)-1-piperazineethanesulfonic acid.

Structure shown on the right.

2) The voltammograms done use a 3 electrode set up, with a working electrode, reference electrode and counter electrode. The working electrode was submerged in the buffer/ferricyanide solutions and the reference electrode in the buffer solution. These are seperated and connected via a ceramic plug. The entire apparatus was placed in a Faraday cage which was connected to an earth wire, to reduce noise in the signal.

Cytochrome C, Cardiolipin and Apoptosis

The purpose of this blog is to explain in brief the nature of the protein Cytochrome C and also to discuss it's place in the process of cell Apoptosis, or programmed cell death. I also hope to mention briefly a few pertinent details regarding the molecule Cardiolipin, it's structure and the interactions it forms with Cytochrome C during Apoptosis. In this entry the information is becoming more specialised. As of writing a general area of study has been decided upon for the research project. This area is to analyse the electochemistry of the compounds Cytochrome C, Cardiolipin and a specific cardiolipin/cytochrome C complex which forms during apoptosis. The exact queries have not been decided upon however. Rather it is hoped that through a review of available studies and data and preliminary voltammetric analysis of these compounds specific questions will arise which suggest a direction of study. Again the information is taken from a variety of sources. Primarily these are, "Molecular Biology of the Cell" 5th Edition, "Biochemistry" Garrett and Grisham and the scientific review "Free Radical Biology and Medicine", Volume 46, Issue 11, 1 June 2009 Pages 1439-1453. A typical undergraduate knowledge of Biochemistry is assumed.

To begin with a basic description of Cytochrome C, it's function and biochemistry is needed. Cytochrome C is a member of a general class of electron transport proteins. These proteins all contain Heme groups (the Fe containing group found also in Hemeoglobin). In humans cytochromes are restricted in location to the mitochondrial inner membrane where they form part of the electron transport chain of mitochondrial respiration. As such they are an extremely redox active series of proteins. As mentioned several classes of Cytochrome exist, including Cytochromes a, b, c, and e. The classification of the cytochrome is primarily dependent on the specific heme group attatched to the protein. Thus cytochrome C (CytC henceforth) posesses an attached c heme group. As mentioned cytochromes are primarily electron transport proteins. CytC specifically acts by moving electrons between Complex III and complex IV of the electron transport chain. An overview of the electron transport chain is given below to aid in visualising this position.

(1)







There are a small number of characteristics of CytC that should be mentioned. Firstly it is a relatively small protein, with a mass of 12,000 Da and consisting of the order of 100 amino acids. Secondly it is a relatively soluble protein, compared to the other repsiratory cytochromes. Thirdly, whilst it is found within the mitochondrial membrane it is not intrinsically bound to it. These factors will become important when cell apoptosis is discussed.







Cardiolipin is a phospholipid molecule found within the innner mitochondrial membrane. Specifically it is a double phospholipid which contains four fatty acid tails. The phosphate groups are bound to a central molecule of glycerol at the 1 and 3 carbon positions. In the membrane it forms a bicyclic structure such as is depicted below












(2)

The DAG segment represents the residue of 2 fatty acid chains. The fatty acid chains are typically about 18 carbons in length and unsaturated. As has been mentioned cardiolipin is found primarily in the inner mitochondrial membrane where it's main function is structural. By mass it can form up to 20% of the membrane itself, arranging itself into a bilayer. However it also has a small number of other primary functions. It acts as a structural component of several of the electron transport proteins, most prominently complex IV, where the presence of cardiolipin enhances enzymatic activity. Also because of the bicyclic structure of cardiolipin it is able to act as a proton trap controlling and buffering the pH of the intermembrane space of the mitochondria.

Thus far we have discussed the focus molecules in a basic form. The area of interest for the study I am undertaking relates to a specific interaction between cardiolipin and CytC that occurs during apoptosis. A full description of the factors and processes of cell apoptosis is beyond my means and desires at this time. I shall however describe one event that occurs, within a context. Cell apoptosis, or programmed cell death. There are many factors that can trigger cell death from within the cell itself, including DNA damage or oxygen deprivation. There also exist a series of regulatory proteins including mammalian Bc12 which regulate the start of apoptosis. The most important way this is done is by controlling the release of CytC from the cellular membrane. The release of CytC is the first step in a cascade reaction that results in the closing down of cell function and the activation of executionor proteins. Within this process though a specific reaction between CytC and Cardiolipin takes place.

After CytC has been released from the mitochondrial membrane Cardiolipin begins a migration into the cytosol and the following interaction takes place. CytC and cardiolipin will bond loosely. This triggers a conformational change in the CytC complex, revealing a new active site and allowing for enzymatic activity of the protein. Specifically the process transforms the complex into a peroxidase enzyme. The enzymatic process also somehow alters the structure and nature of the cardiolipin such that, when they dissociate, the cardiolipin cannot reenter the mitochondrial membrane. This in turn generates a porous nature in the membrane which contributes to cell death. The exact mechanisms of this process are amongst the details we hope to study in this research project. For instance questions such as "Does the cardiolipin dissociate immidiately following enzymatic activity, or does it stay bound?" are interesting ones I hope to ask. However until I can perform basic voltammetry on CytC, Cardiolipin and the peroxidase complex I cannot be sure what areas will be of greatest interest to me.

Notes:

1) Image Taken from http://en.wikipedia.org/wiki/File:ETC.PNG

2) Image taken from http://en.wikipedia.org/wiki/File:Cardiolipin_bicyclic_structure.jpg

Voltammetry

This Blog entry is intended to describe the basic processes of Voltammetry, and the application of this method to the analysis of enzymes. This information is summarised from the following sources: Elements of Molecular and Biomolecular Electrochemistry (Jean-Michel Saveant, pgs 298 to 316), Electrochemical Methods Fundamentals and Application (Bard and Faulkener, 2nd Edition). An understanding of chemistry equivalent to a typical undergraduate is assumed.

In analytical chemistry there are a wide array of analytical methods. These include mass spectrometry, spectroscpic methods, NMR analsis and so forth. In many cases such methods are innapropriate to the analysis being attempted. For instance if a compound is found in a solution that is optically dense i.e coloured spectroscopic analysis may not be useful. In this situation the solution would need to be seperated for the analysis to be performed. However if an analyte is redox active or is a an ion then it can be directly analysed using electrochemical methods. In particular voltammetry is useful in defining the characteristics of redox active molecules. This method is described below.

The basic instrument of electrochemical analysis is the electrochemical cell. A scematic of a simple cell is provided below.

(1)

Every electrochemical cell will contain 2 electrodes, a cathode and an anode. It will also contain an electrolyte solution, and a device to detect either current or voltage. In many cases, if the electrolyte for each electrode cannot mix the solutions will be connected by a salt bridge. In the case of electrochemical analysis one electrode will be designated the reference electrode and placed within a sealed area. This electrode is of a known type whose electrochemical characteristics are well established e.g. a hydrogen electrode. The other electrode is designated the sensor or working electrode and is placed in direct contact with the solution which is to be analysed. Voltammetry uses a standard electrochemical cell in it's processes. In addition this cell is connected to a power supply and an ampmeter. In this form of analysis a known potential (voltage) is applied to the sensor electrode and the resultant current is detected. This potential is then altered and the changes to current are recorded and analysed.

To describe what occurs let us suppose that the following reaction takes place.

O+e- --> R

That is the oxidised form of the redox active molecule being analysed is reduced at the electrode surface. Via mass transport the analyte moves towards the elctrode. When in contact with it the analyte is reduced and then moves away via mass transport. The current detected is the rate of electron tansport. This rate will depend on the temperature, the rate of diffusion of particles in the solution etc and can be represented mathematically by the Cottrell equation presented here. (2)

Where I=the current detected in Amperes.

n=number of electrons transferred

F=Faraday Constant, 96,500 C/mol

A=Electrode surface area, cm3

cjo=Initial concentration of analyte, j, in oxidised form, mol/cm3

Dj= Diffusion coefficient for analyte j, cm2/s

t= Time, s

The oxidation of analyte at the electrode, whereby electrons are transferred from the analyte into the elctrode is also possible. Which occurs will depend on the electrode potential. Should the potential applied be more negative than the reduction potential of the analyte it will be reduced. Should it be more positive than the reduction potential it will be oxidised. Should a negative potential be applied to the already reduced form then no current will be detected, the reverse being true about the oxidation of the reduced form. Finally the reduction of the analyte at the electrode will result in a negative current, the oxidation of the analyte will yield a posotive current. Whilst many methods of varying the potential exist the one most important to this study is cyclic voltammetry. In this method the voltage is varied in a linear fashion from more positive than the reduction potential to significantly more negative and back again. The resultant current is then recorded and analysed. The potentials are chosen to ensure both complete oxidation and reduction during the experiment to allow for a more detailed analysis. Typically the data can be presented graphically, such as below.

(3)

As you can see the potential is shown along the x-axis and the current along the y-axis. The shape shown here is distinctive of a single electron transfer. A wealth of information can be gained from such a voltammagram including the reduction potential, electron stoichiometry and so on. This will be demonstrated in a later post as a voltammogram is interpreted.

To conclude a brief mention shall be given to voltammetry as conducted upon enzymes. It is known that enzymes can achieve a direct electon transfer with the electrode surface without excessive difficulty. A more important factor is the problem of diffusion and mass transport for such massive molecules. Such problems can be overcom by adhering the enzyme to a specific membrane which is present at the electrode surface. The conditions for this are clearly explained in "insert source". In summary though, if a membrane is selected which is very thin (typically monolayer) and allows free access of the active site of the enzyme to both the solution and the electrode surface an electrochemical analysis can be achieved. What this means is that the structure within the enzyme appropriate to electron transfer (such as the heme group of a cytochrome protein) must be placed to allow a free flow of electons between the group and the electrode. Thus the membrane must not restrict this movement which can be achieved by direct access or conformational changes within the enzyme. A further advantage of this method (in addition to removing mass transport issues) is that a relatively small amount of enzyme is required for the analysis. This allows for meaningful analysis of enzymes which are available only in small quantities or at high prices.

Notes:

1) Image taken from http://www.doitpoms.ac.uk/tlplib/pourbaix/images/electrode_potential.gif

2) Equation image taken from http://en.wikipedia.org/wiki/Cottrell_equation. Checked against various sources.

3) Image taken from http://en.wikipedia.org/wiki/File:Cyclovoltammogram.jpg.

The major analytical focus of my research project is to study the electrochemical profile of various proteins, specifically enzymes. As such it may be necessary to give a brief overview of what an enzyme is and some basic structural details. This information is summarised from "Biochemistry (Zubay, 3rd Edition)" pgs 199 to 212 and "Molecular Biology (Robert F. Weaver, 4th Edition) pgs 32 to 38 in addition to any specific references in the body of the text. This is a general overview and will not cover the more detailed aspects of enzyme kinetics. Instead a more simple mention of the structure and pupose of enzymes will be presented. This assumes a background knowledge of Chemistry equivalent to a typical undergraduate student and is intended to provide background information for those who have not studied biology.

Enzymes are a specific subset of protein molecules. These molecules are large organic molecules composed of a chain of amino acid groups bonded by a peptide bond. There are 20 distinct naturally occuring amino acids, an adequate list of which can be found online via wikipedia(1). All thes amino acids posess a carboxyllic acid group (COOH) and an amine group (NH2) bonded to a central carbon atom. Each amino acid also posseses a distinct side chain bonded to the central C atom e.g. CH3 in alanine. It should also be noted that all amino acids posess a chiral centre at the central carbon, and thus exhibit optical isomerism(2). Despite this the naturally occuring amnio acids exist in the S-Isomer, except Cysteine which exists in the R Isomer(3). Finally, an amino acid can exist either in an unionised or ionised state. When ionised it forms a zwitterion.

A generalised strucutral image of both the ionised and unionised forms are shown below.

(4)

As mentioned a protein itself is composed of a unique chain of covalently bonded amino acids. The exact length of this chain varies from protein to protein. Smaller ones may be composed of a few dozen amino acids, whilst larger proteins may contain thousands of individual amino acids. As the structure forms each amino acid is linked to the next via a peptide bond. Within the human cell this is usually an enzyme controlled reaction. A sample reaction to form a peptide bond is shown below.

(5)

Protein function depends greatly on the 3D shape of the molecule. A number of forces act on the molecule whilst it exists in it's straight chain form that drive it to form a specific structure. Several levels of organisation exist in protein structure, which will be summarised presently. The straight main chain is known as the primary structure.. After it is formed various interaction occur. A number of hydrogen bonds will form between nearby amino acid sub units of the protein, resulting usually in either a helix structure forming (known as an alpha helix) of a pleated sheet like structure (known a a Beta Sheet). In larger proteins different regions may exihibit one structure to another allowing both to exist. This level of organisation is called the secondary structure. The next level of organisation, the tertiary structure, is formed differently. This level of organisation exists due to the folding of the protein chain into a 3D shape. A number of forces exist whish contribute to this, and is largely dependent on the coposition of the side chains of the amino acids of the primary structure. The following 4 forces are some of the most important.

1) Hydrostatic forces. Various amino acid side chains present may be either hydrophobic or hydrophillic. Asumming an aqueous medium such as water this will cause the hydrophobic regions to be folded to the inside of the 3D shape and he hydrophillic regions to be found at the surface of the molecule.

2) Covalent bonding between amino acid side chains. It is possible for the constituents of the side chains to react with each other to form a covalent bond, forcing the molecule to fold to match it.

3) Ionic interaction/Dipole interaction. Many amino acid side chains exhibit either polar or fully ionic character. This results in repulsive and attractive forces altering the shape of the molecule.

4) Hydrogen Bonding. Further hydrogen between atoms brought into proximity due to protein folding will strengthen the strucure and cause further folding.

The Final level of organisation is known as the quaternary structure of a protein. This is where a number of seperate amino acid chains are bonded together to form a superstructure. Each sub unit is called a protein sub-unit. A schematic representation showng the connection between the structural levels is shown below.

(6)

Thus far I have spoken about protein structure in general. It is necessary though to refer specifically to enzymes. As mentioned before the structure of a protein is a major factor in it's function. Enzymes act as a biological catalyst allowing various reactions to take place more easily. Structurally this is possible due to the following. When the protein structure forms a specific region will exist possessing a distinct shape. This region is then able to interact with specific molecules in a certain way, often via hydrogen bonding. This interaction lowers the activation energy of a certain reaction which then can take place far more easily. In this respect they are very similar to inorganic catalysts. They differ from inorganic catalysts in the following ways.

1) They are highly specific. An enzyme will typically catalyse a very small number of reactions, usually of extremely similar molecules. By contrast inorganic catalysts such a platinum can often catalyse a vast range of reactions.

2) They are relatively unstable compared to inorganic catalysts. The function of an enzyme depends on it's 3D shape, specifically the shape of the active site of the enzyme. Small changes in the various forces that affect this, including temperature, pH and hydrostatic forces, can induce a change to this structure and deform the active site, limiting or even completely preventing enzyme activity.

3) Compared to simple inorganic catalysts enzymes are very large molecules. E.g Alcohol Dehydrogenase (an enzme that transforms alcohols into aldehydes or ketones) has a mass of 80 kDa.

There are many more aspects to enzyme activity, however this is hopefully a good basic introduction. In the next entry the basics of electrochemistry will be discussed, along with the application of these principles to the study of enzymes.

Notes.

1) The structural details were checked against "Biochemistry", Garrett and Grisham 3rd Edition and found to accurate. Link found at http://en.wikipedia.org/wiki/Proteinogenic_amino_acid

2) Excluding Glycine the simplest Amino acid, NH2CH2COOH. There is no chrial centre.

3) The side chain of cysteine contains an initial S atom, hence it exists in the R isomer.

4) Image Taken from http://en.wikipedia.org/wiki/File:Amino_acid_zwitterions.png.

5) Image taken from http://en.wikipedia.org/wiki/File:Amide_react.png

6) Image taken from http://en.wikipedia.org/wiki/File:Protein-structure.png

I am Peter Gearing, in my 3rd year as a student at the University of East Anglia student no 3169664.This Blog is being established for the purpose of recording and tracking the progress of my third year research project.

As of the current date (18/09/09) the complete details of the specific area of research being persued has not been decided on and thus only a more general outline of the project and the areas of study can be presented.In this entry I will outline some background information as the the type of research and the participants involved. The following entries will contain a more detailed summary of the scientific background relating to this project. After this as the specific area of research is decided the methodology will be entered, as well as significant results obtained during the study and from experimentation.

The supervisor for this project is Dr Julea Butt from the University of East Anglia. A title of the area of interest is "The use of Cyclic Voltammetry to study Immobolised Enzymes." What this means is that, using various techniques it is possible to take an enzyme filled solution and immobolise a certain fraction onto a surface. This surface containing the enzyme can then be subjected to any number of analytical techniques. However the one used in this study is Cyclic Voltammetry. This means that a potential difference (or voltage) is applied to the enzymes. This Voltage is then altered in a steady or cyclic fashion. The enyme will then be either oxidised or reduced, dependant on the Voltage applied. By measuring the resulting current a great deal of information regarding the enzymes can be recorded.

The next Blog entry will be a short mention of the biological function of Proteins. Following this will be an entry discussing the use of Cyclic voltammetry and the advantages of immobolised proteins. This should cover the background information necessary to understand this project and the area of interest.