Introduction Methionine and cysteine are both sulphur containing amino acids. Most proteins will contain one, or both of them at some point in the polypeptide chain. As such, many amino acids contain sulphur in some form, which is required in small amounts in the mammalian diet. Methionine has a thioether side chain, and cysteine’s contains a thiol group. These side chains exist as free thiols inside the cell, and are oxidised causing them to pair up and form disulphide bonds in an extracellular environment. Thiols are more reactive than hydroxyl groups and react easily with mercurails and heavy metal salts.
The reaction with p-chloro-mercuribenzoate (PCMB) can be used to measure thiol groups, as there are changes in the ultraviolet spectrum of the compound. As proteins absorb strongly in the ultraviolet spectrum it is better to use reagents with absorption peaks in the visible spectrum. 5,5′-bisdithio-2-nitrobenzoate (DTNB) is highly specific for free thiol groups. Usually neither reagent absorbs in the significantly in the visible region. Thionitrobenzoate has a maximal extinction at 412nm and is bright yellow. The yellow colour is due to the presence of S-.
The increase in extinction at 412nm can be used to measure reactive thiol groups. The aim of the experiment is to purify the protein ovalbumin from egg white by precipitation at high salt concentrations. The number of thiol groups will be determined, this will then be compared to a standard preparation of the purified protein. Method The method followed is as described in the laboratory manual. In estimating the concentration of the prepared ovalbumin 0. 01ml of the preparation was taken, rather than the suggested 0. 1ml.
This was because at the suggested concentration, the absorbance at 280nm was off the scale of the machine. In the purification of ovalbumin procedure 0. 25g of powdered ammonium sulphate were added per ml of filtrate. 27ml of filtrate was produced 27 x 0. 25 = 6. 75g 6. 75g of powdered ammonium sulphate were added Results There are two additional results sheets from the laboratory manual that are included after this page. The absorbance with SDS shows a rapid increase, before the graph begins to increase more slowly and uniformly.
The absorbance levels off at 0. 3nm. The absorbance with no SDS remains at a constant level of 0. 1nm for the entire experiment. The absorbance of the control solution is also constant throughout the experiment. This shows the absorbance of the compounds in the solution without the ovalbumin, by taking this figure away from the other recordings, it is possible to discover the absorbance for ovalbumin alone. The absorbance with SDS shows a rapid increase, before the graph begins to increase more slowly and uniformly. The absorbance levels off at 0. nm. The absorbance with no SDS remains at a constant level of 0. 01nm for the entire experiment. The absorbance of the control solution is also constant throughout the experiment. This shows the absorbance of the compounds in the solution without the ovalbumin, by taking this figure away from the other recordings, it is possible to discover the absorbance for ovalbumin alone. This graph shows the absorbance curves for both the purified ovalbumin and the standard ovalbumin against time, allowing direct comparison between the two.
Whilst both graphs have a very similar shape, the purified ovalbumin has a final absorbance level nearly three times that of the standard ovalbumin. Discussion Our standard ovalbumin and purified ovalbumin had differing numbers reactive thiol groups in the presence of SDS. This may well be due to the accuracy of purification of the ovalbumin. With contamination of the purified ovalbumin by other proteins from the original egg white compound the absorbance figures may have been altered.
This in turn would have led to inaccuracies in the calculation of the number of reactive thiol groups. Both figures 1 and 2 show differing, but constant absorbencies for ovalbumin without SDS present, this indicates that there is no reaction occurring. In the presence of SDS both solutions show a large increase in absorbency as the reaction time increases, showing it must be SDS that causes the reaction to take place. It is also shown by the fact that our standard ovalbumin and purified ovalbumin had 0 reactive thiol groups in the absence of SDS.
Sodium dodecyl sulphate is an anionic detergent, which denatures proteins by wrapping around the polypeptide backbone. Detergents are surfactants that contain a hydrophobic portion, which is soluble in oil-like solutions, and a hydrophilic portion, which is soluble in water. This characteristic results in the formation of stable micelles with hydrophobic cores in an aqueous solution. In this reaction, the presence of SDS denatures the ovalbumin by disrupting the electrostatic interactions and hydrophobic interactions, therefore breaking down the quaternary structure of the protein.
This allows the DTNB to bind to the free thiol groups, causing the increase in absorbance as the solution becomes more and more yellow in appearance. In order to determine if the denaturation of ovalbumin by urea or SDS is a reversible reaction, the denaturation agent must first be removed from the solution. Conventional methods for detergent removal include hydrophobic adsorption, gel chromatography, dialysis, ion-exchange chromatography, and precipitation techniques. Having removed the detergent, the protein will refold.
As shown by the Anfinsen experiment the polypeptide sequence determines the folding and therefore the three dimensional structure. As the polypeptide sequence is unaltered refolding can occur through the process of nucleation aggregation and compaction. In order to test that the protein was no longer denatured, the absorbency of the solution at 412nm could be measured and compared with the graph in figure 1 above, it should match the plot of standard ovalbumin in the absence of SDS.
To measure the concentration of DTNB in a solution of unknown concentration, the above experiment could be carried out using a known concentration of thiol groups. This would allow the concentration of DTNB to be calculated, as there is a 1:1 ratio of DTNB molecules (and therefore concentration) to the number of thiol molecules. The determination of the number of thiol groups by DTNB is carried out at pH> 7. 5 because the extinction coefficient is strongly pH dependent at pH values more acidic than 7. 5. With an altered pH the maximal extinction may be altered, meaning that the absorbency figures will be wrong.