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Fermentation is the anaerobic energy acquiring process in which organic substances are oxidized by other substances. In fermentation, ATP is synthesized by substrate level phosphorylation.
In respiration, organic substances are oxidized by molecular oxygen or sometimes by inorganic substances such as nitrate and sulphate. Thus ATP is formed by compiling to electron transfer as well as by substrate level phosphorylation. In photosynthesis, light energy is changed into the energy necessary for life processes. The mechanism by which ATP is synthesized is photosynthesis are similar to those in respiration.
Thus in respiration and photosynthesis, ATP is synthesized coupled to electron transfer and electron transfer system is constituted of cytochromes. Cytochrome is heme protein whose function is initially related to the valency change of heme iron.(Ref.Yamanaka)
Cytochromes are the membrane-bound hemeproteins containing heme groups carry out the electron transport chain. Cytochromes are usually formed as monomeric protein or as subunits of enzymatic complexes catalyzing redox reactions. Cytochromes are found in the inner mitochondrial membrane, endoplasmic reticulum of the eukaryotes, in chloroplasts of plants, in photosynthetic microorganisms and in bacteria. (Refâ€¦.1)
(History). Discovered in 1884 by Macmunn and later on named by Keilin in 1920 as Cytochromes are still identified on the basis of the positions of their lowest energy absorption band in the reduced state like cyto605.
(Structure and Functions). The heme group is a highly conjugated ring system surrounded by a metal ion which readily interconvert between the oxidation states. In most of the cytochromes, the metal present is iron which readily interconvert between Fe+2 and Fe+3 oxidation states. Thus the cytochrome are capable to perform the oxidation & reduction.
As the cytochromes are held within the membrane, the resulting redox reactions are carried to get maximum efficiency.
A complete distinct family of cytochromes is known as the cytochrome P450 oxidase due to its characterstic Soret peak by the absorbance of light at a wavelength near 450nm while rducing iron sodium dithionite and complexed to CO2. Cytochrome P450 are mostly involved in steriodgenesis and detoxification.(Ref â€¦1)
(The ETC). The electron transport chain couples a chemical reaction between an electron donor like NADH and an electron acceptor (such as O2) to transfer the H+ across a membrane through a set of mediating biochemical reaction. The H+ are utilized to produce ATP, the main energy intermediate in living organisms. Electron transport chain are used to extract energy from sunlight (photosynthesis) and from redox reaction such as the oxidation of sugars in respiration.
In mitochondria, the conversion of oxygen to water, NADH to NAD+ and succinate to fumarate drives the transfer of H+ ion while some bacteria having electron transport chain similar to those of chloroplasts or mitochondria use different electron donors and acceptors.
The function of electron transport chain is to produce gradient. In all the living organism, a series of redox reaction is used to produce a transmembrane electrochemical potential gradient. The energy in the form of transmembrane electrochemical potential gradient can be harnessed to do useful work like the transport of molecules across membranes. It can be used to do mechanical work such as rotating bacterial flagella and also to produce ATP.
(Electron Transport Chain in Bacteria). In eukaryotes, NADH is the most important electron donor. In electron transport chain, complex I, III & IV are the proton pumps while cytochrome a and cytochrome c are mobile electron carriers which the molecular oxygen is the electron acceptor.
In prokaryotes, the situation is a little more complex, because of number of different donors and electron acceptors. In electron transport chain, the electron can enter the chain at three levels:
At the level of dehydrogenase
At the level of quinines
At the level of cytochromes resulting in the form of more positive redox potential
or less Gibb's free energy.
The bacteria use multiple electron transport chain often simultaneously. Electron may enter an electron transport chain at the level of a mobile cytochrome or quinine carrier. Most of the electron from inorganic donors enter the electron transport chain at the cytochrome level.
(Types of Cytochromes). There are five types of cytochromes mostly occurring between ubiqinol and oxygen in the electron transport chain. Each having different organization depending upon their light absorbing spectra. A these are classified on the basis of their light absorption characteristic so they are not named in a proper way. Instead they are named on the basis of their wavelength. (Ref. Jain & Jain)
Various cytochromes differ from each other in the nature of the prosthetic group and its mode of attachment to the apoprotein part. The prosthetic group of cytochromes b, c, c1 is iron protoporphyrin IX also named as heme or hemin. Hemin or heme is prosthetic group of many protein like myglobin, hemoglobin, catalase and peroxidase. In cytochrome b, the heme is not covalently bonded to the protein as occur in case of cytochrome c. (Ref. Jain & Jain)
The type of heme present in cytochrome b is named as heme b and the one present is cytochrome c and c1 as heme c. (Ref. Jain & Jain)
The cytochrome a and a3 have an iron-porphyrin prosthetic group called heme a. cytochrome a and a3 are the terminal members of the respiratory chain and exists as complex sometime called as cytochrome oxidase. The cytochrome aa3 complex, thus differs from cytochrome as it contains 2 moles of highly-bound heme a. Moreover, cytochrome aa3 also contain 2 essential copper atoms. (Ref. Jain & Jain)
Cytochrome c is best known among the cytochromes. It is the only electron transport protein which can be separated by gentle treatment due to its high solubility in water. It is a small molecule with iron-porphyrin group (heme c) covalently attached to its single polypeptide chain containing about 100 amino acid residues in most of the species. The iron atom is bonded to the surface atom of a methionine residue and due to the nitrogen atom of a histadine residue. (Ref. Jain & Jain)
Cytochrome c is an ancient protein since its amino acid sequence has many points of similarity in all eukaryotes, microbes, plants and animals.(Ref. Jain & Jain)
Cytochromes are thus an important constituents of the electron transport system in both respiration and photosynthesis. Cytochromes with different heme show different properties making it necessary to know the structure and properties of the heme in each case. (Ref. Yamanaka)
Objectives/Scheme of Work:
Protein purification protocols comprise lot of steps from the given substance/organism. To avoid the waste efforts and miscalculations, certain rules have been devised to purify the target protein. The key objectives for the said scheme of work focus upon certain points which can lead us towards the most versatile work in biochemistry. The objectives of the work can be summed up as;
No. of steps should be less
Purification strategy should be cheap.
Speed of work must be sufficient.
Reliable techniques and apparatus used in the scheme.
The scheme/protocol should be economical
Assay development for purification process
Yield of the process
An important step in the protein purification protocol is the selection of the source of material. There are certain sources of material utilized for the process like as;
Tissue culture cells &
Microorganisms (e.g. bacteria & fungi etc.)
Basic protocol of protein purification consists of following steps.
Growth of the cells (e.g. bacteria)
Removing of debris
Precipitation/Concentration of the target material.
Purification of the target protein &
Analysis/Characterization of the purified proteins
The raw material for the respiratory chain study is the pure bacterial culture. The bacterial culture is usually grown aseptically upon the nutrient broth (10% w/v) in the refrigerated incubator shaker at 370C. The shaker speed is optimized so that maximum biomass can be obtained from the media. The selection of the bacterial strain plays an important role in biomass production. The pure strain gives the maximum contamination free production of biomass during the fermentation. The growth of cell culture in the fermentor is also helpful in laboratories as bulk production of biomass can be obtained by optimizing the parameters.
The bacterial biomass is then suspended in suitable buffers. Buffers maintain the pH and ensure the denaturing of the bacterial proteins. For suspension the biomass, the stock solution of the buffers is prepared to adjust the pH up to required level. A range of buffers are used for the storage of bacterial biomass but the most suitable one is the phosphate buffers with EDTA i.e. 100mM phosphate buffer alongwith 0.5M EDTA.
Respiratory membrane proteins are mostly embedded beneath the bacterial cell membrane, different mechanical and non-mechanical cell disruptions techniques are employed like bead milling, homogenizer, French press ultrasonication etc. The most reliable and economical method is the ultrasonication. Although it is a mechanical method applied for the disruption of the bacterial cell biomass but it makes possible the degasification of the biomass. The selection for the disruption methods also depends upon the fact that those methods are more successful which leave less cell debris/unbroken cells. The ultrasonication is based upon the application of ultrasound waves with the help of a probe to disrupt the bacterial cell wall. Different frequencies of ultrasound waves are applied to get the maximum result while the whole process is carried at 40C to avoid the protein denaturation. Bacterial cell contain maximum protein alongwith the cell membrane and in cytoplasm in comparison to the plant cell have maximum portion of it cytoplasm in the form of vacuole.
In protein purification protocol, the chromatography needs such a sample which is free of contamination causing bed clogging and poor purification. As such particulate matter or cell debris leads to abrupt results with contamination. Consequently, differential centrifugation is applied to get the supernatant and pallets separated. In first step, cell biomass is centrifuged at 10000rpm and 15000rpm keeping the temperature at 40C respectively for 15 - 20 minutes. This gives rise to supernatant and unbroken cell separate. The supernatant is then again centrifuged at 35000rpm maintaining the temperature at 40C for 45 minutes.
The cell membrane pallets (reddish in coloration) are then dissolved in phosphate buffer and are subjected to ultracentrifugation at 40000rpm at 40C for 60 minutes maintaining the pH at 7.4 - 7.6. The supernatant is stored at -200C to keep the membrane protein stability.
A treatment of the biological membranes with salt solutions changes their pH and dissociates the other proteins. Thus to accomplish the task of separation of membrane proteins of the bacteria, these membrane protein fractions are solubilized with non - ionic detergent. The membrane pallets are solubilized in non - ionic detergent like Triton X - 100 20% W/V (stock solution) with final concentration of 3% alongwith EDTA.
The membrane proteins are precipitated with the addition of salts also named as salting out. The salting out procedure comprises the addition of an inorganic salt like (NH4)2SO4 causing precipitation by the removal of water of solution from hydrophobic areas upon the protein surface. It has an edge over the other inorganic salts like ammonium acetate due to its maximum solubility in water and cheapness. The concentration of the salts is usually expressed in terms of percentage saturation at 00C but experimentally it has been proved that salt precipitation between 35 - 50 % have shown better result. The precipitated membrane proteins are then centrifuged at 40C for 30 minutes at 4000 - 5000 rpm. The decanted supernatant is then suspended in the phosphate buffer.
The salt precipitation needs to be followed by the removal of salts from the solution. For this purpose, the membrane protein suspension is taken in double volume and in the dialysis tubing closed by double knots. It is suspended in 500 - 5000 ml capacity funnel/beaker depending upon the volume of the protein sample to be concentrated.