CHMI-2227EL Lecture Notes - Lecture 8: Methionine, Chymotrypsin, Bromine
27 Jun 2018
School
Course
Professor
Biochemistry - Day 8 2018.02.09
Determining the Amino Acid Sequence of a protein:
Step 1
-Separation of chains
-Subunit interactions depend on weak forces
-Separation is achieved with
-Extreme pH:
-8 M urea or
-6 M guanindine HCl
-High salt concentration (usually ammonium sulfate)
Step 2
-Cleavage of Disulphide bridges: between or within
polypeptide chains
-1. Performic acid oxidation: converts all cysteine residues to
cystic acid residues
-2. Sulfhydryl reducing agents: 2-mercaptoethanol, -
dithiothreitol or dithioerythyritol,
-to prevent the cysteine residues from recombining, treat with
an alkylating agent like iodocetate to form a stable S-
carboxymethyl derivative (this is because the thiol group is
covered)
Step 3
-Identify the N- and C- terminal residues
-N- terminal analysis:
-2 methods are widely used for identifying the N-terminal amino acid
or a protein
-1. Labeling all amino groups
-2. Hydrolyzing the peptide to amino acids
-3. Determination of the labeled amino acid
-This is a sequential process
-Reagents used:
-1. Pehr Edman degradation using Edman’s
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Document Summary
Determining the amino acid sequence of a protein: Cleavage of disulphide bridges: between or within polypeptide chains. Performic acid oxidation: converts all cysteine residues to cystic acid residues. Sulfhydryl reducing agents: 2-mercaptoethanol, - dithiothreitol or dithioerythyritol, To prevent the cysteine residues from recombining, treat with an alkylating agent like iodocetate to form a stable s- carboxymethyl derivative (this is because the thiol group is covered) Identify the n- and c- terminal residues. 2 methods are widely used for identifying the n-terminal amino acid or a protein. Pehr edman degradation using edman"s reagent phenylisothicyanate (pitc) Pitc reacts with the n-terminal amino group of a peptide under alkaline conditions (ph =9) Ptc polypeptide is then treated with trifuoroacetic acid to release n-terminal amino acid residue as a thiazolinone derivative. Thiazolinone derivative is then treated with weak acid to give a stable product called phylthiohydantoin (ptc- derivative) This pth-derivative is then identi ed by chromatography.
