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