ABSTRACT
The separation of haemoglobin and cytochrome C can be achieved through various chemical techniques such as electrophoresis, ultracentrifugation, and chromatography. These techniques depend on differences in the physicochemical properties of the proteins, such as size, charge, and solubility, to separate them into distinct fractions.
Based on the net charge of proteins, ion exchange chromatography is a commonly used technique for separation. The sample mixture is applied to a column packed with a resin containing charged groups, and proteins are separated based on their degree of binding to the resin. By altering the pH and salt concentration of the buffer, the proteins can be eluted from the column in a controlled manner, allowing for their separation.
Size-exclusion chromatography is another technique to separate proteins based on their size. The sample mixture is applied to a column packed with resin containing pores of different sizes. The proteins are separated based on their ability to enter and exit the pores. Smaller proteins pass through the pores and take longer to elute, while larger proteins are excluded and elute more quickly.
Electrophoresis separates proteins based on their size and charge. In gel electrophoresis, the sample mixture is applied to a gel matrix, and an electric field is applied to separate the proteins based on their size and charge. Proteins can be visualized using methods such as Coomassie Blue staining or Western blotting. Ultracentrifugation is a technique that separates proteins based on their density and size. The sample mixture is at high speeds, and the proteins are fractionated based on their sedimentation rate, allowing for their separation.
The choice of technique depends on the specific properties of the proteins being separated and the intended application of the separated proteins.
INTRODUCTION
Haemoglobin and Cytochrome C are two important proteins in living organisms involved in different physiological functions. Haemoglobin is a protein found in red blood cells that carries oxygen from the lungs to other body parts. At the same time, Cytochrome C is a protein involved in the electron transport chain in mitochondria, which generates ATP, the cell’s energy currency. Chromatography separates the mixture based on its chemical and physical properties.
The Bradford assay is a method commonly used for protein quantification, based on the dye Coomassie brilliant blue to proteins. The assay is highly sensitive and can be used with many proteins, making it a popular choice in protein research. In protein separation, it can determine the protein concentration in a sample before and after separation, allowing for the quantification of individual proteins. For example, if a mixture is separated using gel electrophoresis, the amount of each protein in the sample can be determined by staining the gel with Coomassie Brilliant Blue and using the Bradford assay to measure the amount of dye bound to each protein band. This allows for the relative abundance of each protein to be determined. The Bradford assay can be used to optimize protein separation conditions. By measuring the protein concentration before and after separation using different techniques, such as different types of chromatography or centrifugation, the most effective method for separating a particular protein can be determined.
However, this method measures the total protein content in a sample and does not provide information on the identity or purity of individual proteins. For this reason, it is combined with other methods, such as spectrometry, to identify and characterize individual proteins.
METHODS
- Dithionite-Citrate-Bicarbonate (DCB) method.
This is the method illustrated in Fig 2. It uses dithionite to reduce the iron in haemoglobin to the ferrous state (Fe2+), which causes the haemoglobin to lose its characteristic red colour and become colourless. However, Cytochrome C is not affected by dithionite and remains oxidized. After reduction with dithionite, the sample is treated with a citrate buffer to stabilize the reduced haemoglobin and prevent its re-oxidation. Bicarbonate is added to the mixture to raise the pH and cause the haemoglobin to re-oxidize, regaining its characteristic red colour.
- Ion exchange chromatography
This method uses an ion exchange resin column with a charged functional group (such as DEAE or CM). The sample containing both haemoglobin and Cytochrome C is loaded onto the column, and the proteins are separated based on their different charge properties.
Ion exchange chromatography using a CM Sephadex column can separate haemoglobin and cytochrome. The CM Sephadex is first equilibrated with a buffer of appropriate pH and ionic strength in this method. The protein mixture containing haemoglobin and cytochrome C is applied to the column, and the proteins are separated based on the different charges. After loading the sample onto the column, the resin is washed to remove unbound proteins. The proteins are then eluted from the column by increasing the ionic strength of the buffer or changing the pH. Haemoglobin can be eluted first, followed by cytochrome C.
- The Ferricyanide treatment method.
It is used to separate haemoglobin from cytochrome C. this is based on their differential reactivity towards Ferricyanide. The Ferricyanide solution and sodium phosphate buffer are prepared according to desired concentrations and pH. It is then mixed with the haemoglobin and cytochrome C protein sample with the Ferricyanide solution in a 1:1 ratio. Incubation of the mixture for 5-10 minutes then occurs. Sodium phosphate buffer is added to the mixture, stopping the reaction and bringing the pH to a suitable range for further separation. Centrifuge the mixture to remove any insoluble material. The supernatant is applied to a cation exchange column (CM Sephadex or DEAE Sepharose) to separate the haemoglobin from cytochrome C based on their differential charge properties. Then elute the proteins from the column using a salt or pH gradient.
RESULTS AND DISCUSSION
Fig 1 shows UV-vis spectra for ferric haemoglobin and myoglobin in the pH range of 5-13. This pH range indicates the presence of two hexacoordinated states in these proteins. At low pH (5-6), the spectra show a high-spin ferric heme with a water molecule as the sixth ligand. This is due to the protonation of the distal histidine residue, which weakens its interaction with the heme iron and allows a water molecule to occupy the sixth coordination site.
The UV-vis spectra indicate two states to be present in these proteins, both of which are hexacoordinated. The low pH form features a water molecule ligated to a high spin (S=1/2 iron). At pH 13, there is a tendency for the spectra to lose some intensity without changing the shape. The spectra show a hexacoordinated ferric heme with a hydroxide ion as the sixth ligand (Oh et al., 2022). This unexpected behaviour may be due to the formation of unstable six-coordinate species at extreme pH values. This may indicate some instability in the protein structure at such high pH values, which could lead to the denaturation of the proteins. This is not expected at such extreme pH. Globins in ferrous and ferric forms are stable and do not denature at pH values as high as 12. However, extreme pH values can still affect the protein structure and stability. It is important to carefully control the pH conditions during protein analysis to ensure that the protein structure remains intact and the results are accurate.
As in Fig 2, a graph of UV-vis spectra of ferrous deoxy-haemoglobin at different pH values (11-13) in the top panel and spectra of oxy and carboxymonoxy haemoglobin and ferrous cytochrome c in the bottom panel could be used to investigate the effect of pH and oxygenation state on the absorbance spectrum of these hemoproteins. Hemoproteins such as haemoglobin and cytochrome c contain a heme group responsible for their absorbance properties. The heme group comprises a porphyrin ring with a central iron atom that can bind to various ligands such as oxygen, carbon monoxide, or protons. Changes in the oxygenation state or pH of the protein can alter the electronic and chemical properties of the heme group, which can, in turn, affect the absorbance spectrum. If the peak shifts to a higher wavelength (i.e. to the right on the x-axis) as the pH increases, this might indicate changes in the protonation of the protein or the heme group.
In the bottom panel, if a peak shifts to a higher wavelength as the haemoglobin becomes more oxygenated, this might indicate changes in the electronic structure of the heme group as it binds to oxygen. If a peak shifts to a higher wavelength as haemoglobin binds to carbon monoxide, this might indicate changes in the electronic and chemical properties of the heme group as it binds to a different ligand.
Ferricyanide treatment exploits the fact that haemoglobin can reduce Ferricyanide to Ferricyanide, while cytochrome C cannot. This difference in reactivity can selectively remove haemoglobin from a protein mixture, allowing for the subsequent separation from a protein mixture and the subsequent separation of the two proteins by ion exchange chromatography.
Since haemoglobin and cytochrome C have different net charges at a given pH, they bind differently to the CM Sephadex resin. Haemoglobin has a net negative charge at pH 7.4 and will bind to the positively charged CM resin. Cytochrome C, on the other hand, has a positive charge at pH 7.4 and will not bind to the CM resin. After loading the sample onto the column, the resin is washed to remove unbound proteins. The proteins are then eluted from the column by increasing the ionic strength of the buffer or by changing the pH. Haemoglobin can be eluted first, followed by Cytochrome C. Ion exchange chromatography using a CM Sephadex column is a powerful tool for protein separation, as it allows for the separation of proteins based on their charge properties. Optimizing the pH, buffer conditions, and salt concentration is important to achieve the best separation and resolution of the proteins of interest.
The pH 13 dithionite-induced six coordinated species is not observed in myoglobin, indicating that it is not a general feature of all globins or heme proteins. Forming a six-coordinate species in response to dithionite treatment is not unique to heme proteins. It has also been observed in other heme-containing systems, such as heme enzymes and heme-containing model compounds. The absence of the six coordinated species in myoglobin is likely due to the myoglobin protein’s specific structural and electronic properties. Myoglobin has a compact tertiary structure with a relatively small pocket for the heme group, which may hinder the formation of the six coordinated species. The heme iron in myoglobin has a relatively low affinity for ligands such as dithionite, which may also contribute to the lack of observation of the six coordinated species.
CONCLUSION
In conclusion, the choice of method for separating haemoglobin and Cytochrome C depends on the specific experimental goals and the properties of the proteins. Each separation method has advantages and disadvantages, and the selection of the appropriate technique will depend on the researcher’s specific needs. However, separating haemoglobin and cytochrome C is a common technique in biochemistry and molecular biology. The several methods for separating these two proteins include; electrophoresis, ultracentrifugation, and chromatography. Chromatography involves separating the proteins based on their interactions with a stationary phase, while electrophoresis separates proteins based on their charge and size. Ultracentrifugation is a technique that separates proteins based on their density.
References
Oh, Y., Nguyen, N., Jung, H.J., Choe, Y. and Kim, J.G., 2022. Changes in Cytochrome C Oxidase Redox State and Hemoglobin Concentration in Rat Brain During 810 nm Irradiation Measured by Broadband Near-Infrared Spectroscopy. Photobiomodulation, Photomedicine, and Laser Surgery, 40(5), pp.315-324.
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