The classic work of Christian Anfinsen in the 1950s on the enzyme ribonuclease revealed the relation between the amino acid sequence of a protein and its conformation. Ribonuclease is a single polypeptide chain consisting of 124 amino acid residues cross-linked by four disulfide bonds
Anfinsen's plan was to destroy the three-dimensional structure of the enzyme and to then determine what conditions were required to restore the structure.
AGENTS -
1. Urea or guanidinium chloride- Effectively disrupt the noncovalent bonds.
2.β-mercaptoethanol -The disulfide bonds can be cleaved reversibly by reducing them with a reagent such as β-mercaptoethanol . In the presence of a large excess of β-mercaptoethanol, a protein is produced in which the disulfides (cystines) are fully converted into sulfhydryls (cysteines).
EXPERIMENT-
A. When ribonuclease was treated with β-mercaptoethanol in 8 M urea, the product was a fully reduced, randomly coiled polypeptide chain devoid of enzymatic activity. In other words, ribonuclease was denatured by this treatment.
B. Anfinsen then made the critical observation that the denatured ribonuclease, freed of urea and β-mercaptoethanol by dialysis, slowly regained enzymatic activity . The sulfhydryl groups of the denatured enzyme became oxidized by air, and the enzyme spontaneously refolded into a catalytically active form. Detailed studies then showed that nearly all the original enzymatic activity was regained if the sulfhydryl groups were oxidized under suitable conditions.
C. A quite different result was obtained when reduced ribonuclease was reoxidized while it was still in 8 M urea and the preparation was then dialyzed to remove the urea. Ribonuclease reoxidized in this way had only 1% of the enzymatic activity of the native protein.
The reason is that the wrong disulfides formed pairs in urea. There are 105 different ways of pairing eight cysteine molecules to form four disulfides; only one of these combinations is enzymatically active. The 104 wrong pairings have been picturesquely termed "scrambled" ribonuclease.
D.Anfinsen found that scrambled ribonuclease spontaneously converted into fully active, native ribonuclease when trace amounts of β-mercaptoethanol were added to an aqueous solution of the protein The added β-mercaptoethanol catalyzed the rearrangement of disulfide pairings until the native structure was regained in about 10 hours. This process was driven by the decrease in free energy as the scrambled conformations were converted into the stable, native conformation of the enzyme. The native disulfide pairings of ribonuclease thus contribute to the stabilization of the thermodynamically preferred structure.
Anfinsen's plan was to destroy the three-dimensional structure of the enzyme and to then determine what conditions were required to restore the structure.
AGENTS -
1. Urea or guanidinium chloride- Effectively disrupt the noncovalent bonds.
2.β-mercaptoethanol -The disulfide bonds can be cleaved reversibly by reducing them with a reagent such as β-mercaptoethanol . In the presence of a large excess of β-mercaptoethanol, a protein is produced in which the disulfides (cystines) are fully converted into sulfhydryls (cysteines).
EXPERIMENT-
A. When ribonuclease was treated with β-mercaptoethanol in 8 M urea, the product was a fully reduced, randomly coiled polypeptide chain devoid of enzymatic activity. In other words, ribonuclease was denatured by this treatment.
B. Anfinsen then made the critical observation that the denatured ribonuclease, freed of urea and β-mercaptoethanol by dialysis, slowly regained enzymatic activity . The sulfhydryl groups of the denatured enzyme became oxidized by air, and the enzyme spontaneously refolded into a catalytically active form. Detailed studies then showed that nearly all the original enzymatic activity was regained if the sulfhydryl groups were oxidized under suitable conditions.
C. A quite different result was obtained when reduced ribonuclease was reoxidized while it was still in 8 M urea and the preparation was then dialyzed to remove the urea. Ribonuclease reoxidized in this way had only 1% of the enzymatic activity of the native protein.
The reason is that the wrong disulfides formed pairs in urea. There are 105 different ways of pairing eight cysteine molecules to form four disulfides; only one of these combinations is enzymatically active. The 104 wrong pairings have been picturesquely termed "scrambled" ribonuclease.
D.Anfinsen found that scrambled ribonuclease spontaneously converted into fully active, native ribonuclease when trace amounts of β-mercaptoethanol were added to an aqueous solution of the protein The added β-mercaptoethanol catalyzed the rearrangement of disulfide pairings until the native structure was regained in about 10 hours. This process was driven by the decrease in free energy as the scrambled conformations were converted into the stable, native conformation of the enzyme. The native disulfide pairings of ribonuclease thus contribute to the stabilization of the thermodynamically preferred structure.
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