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