Biochemistry of Proteins; Isolation of Ovalbumin and Enumeration of Thiol Groups

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  • Topic: Protein, Disulfide bond, Protein structure
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Biochemistry of Proteins; Isolation of Ovalbumin and Enumeration of thiol groups

Abstract
Thiol groups are important to protein folding and forming disulphide bonds that are essential to protein structure. Determining the number of thiol groups in a protein is important in determining the tertiary structure of the protein. The ovalbumin is the experiment was purified from egg white using centrifugation and ammonium sulphate precipitation and then the thiol groups identified using DTNB and spectroscopy. The ovalbumin was found to have one thiol group; from this we were also to infer that DNTB alkylates thiolgroups; whereas SDS keeps proteins denatured.

Introduction
A thiol group is a functional group containing a sulphur atom bonded to a hydrogen atom with the general formula –SH. Thiol groups are significant to biochemistry especially in the formation of cysteine from two cysteine monomers. When two thiol groups of cysteine are in close contact during protein folding, they can undergo an oxidation reaction. The disulphide bonds formed contribute to the tertiary structure of the protein. Thiol groups in the active site of an enzyme can form bonds with the enzyme's substrate in catalytic activity and cysteine residues in the active site of enzymes may react with heavy metal ions because of the high affinity between the two. This can deform and inactivate the protein, and is one mechanism of heavy metal poisoning. The experiment was carried out to establish the number of thiol groups in Ovalbumin. This was done so by isolating and purifying ovalbumin protein from a standard egg-white preparation. This is useful in deducing the number of disulphide bonds it contains and therefore the tertiary/quaternary structure of the protein.

Method
A sample of ovalbumin was purified from an egg-white standard preparation and the number of thiol groups per protein molecule determined. This was achieved by precipitating the ovalbumin out of the egg-white preparation using saturated ammonium sulphate to “salt out” the preparation and isolating the ovalbumin pellet through centrifugation. A standard curve of ovalbumin absorption at 280nm using the egg-white was then constructed, from this the concentration of ovalbumin in the preparation was determined using a spectrophotometer. The isolated ovalbumin was then reacted in four different reaction mixtures in cuvettes containing alternate mixtures Tris, DTNB, SDS and water, respectively, in order to establish the number of thiol groups. It is the reaction with DTNB that helps enumerate thiols. The number of thiol groups was determined through calculations that can be found in the appendix. These calculations included using Beer’s Law. All isolation procedures were carried out on ice, this is also true of the reagents (except SDS).

Results
Figure 1: Standard curve of Ovalbumin at A280

Figure 1 shows the standard curve of ovalbumin, with the regression line represented by a solid black line, the regression was found to be 0.9359 which represents that the data is concurrent with the line of best fit. Thus, the regression line can be used find the concentration of ovalbumin when the absorbance of the isolated ovalbumin is known. This curve can be seen to prove Beer’s Law as the more ovalbumin present in the solution, the higher the absorbance at 280nm. The extracted Ovalbumin was found to be too concentrated so the solution was diluted by 1000 and from figure 1 the absorbance at 0.901 corresponded to 0.8733mg/ml which is the concentration of the ovalbumin. 1.50.8733=1.72ml added to reaction mixture B and D.

Figure 2: The absorption spectra of four respective reaction mixtures over a 15 minute period.

Figure 2 shows the absorption spectra of the four respective reaction mixtures, with readings taken at one minute intervals over a 15 minute period. The curve for reaction mixture A is interesting as this is the control for reaction mixture B and thus should be a constant line....
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