Biochemistry of Proteins. Isolation of Ovalbumin: Characterisation of Thiol Groups and Separation by Gel Filtration

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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
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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

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