BIO 361 Lecture Notes - Lecture 5: Disulfide, Protein Sequencing, Cystine
27 Jun 2016
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Biochemistry professor sanford simon stony brook summer 2016. A protein composed of two different polypeptide chains linked by a disulfide bond) is separated by reducing the disulfide bond. Polypeptides are broken into smaller peptides using chemicals or enzymes. Peptide fragments are purified and the sequences are determined. Use overlapping sets of peptide sequences to reconstruct each polypeptide. Repeat fragmentation without breaking the disulfide bonds to identify the cys-containing sequence involved in the disulfide linkages. The first step is to determine the different types of subunits. Dansyl chloride reacts with primary amines (n-terminus or lysine side chain) to yield dansylated polypeptides that are hydrolyzed by acid. The n-terminal residue can be separated by chromatography and identified by fluorescent yellow color. Disulfide bonds must be cleaved to separate subunits. 2-mercaptoethanol (reducing agent) containing thiol bonds will exchange with cystine thiol bonds to cleave them and form two individual cysteine subunits. Iodoacetate is reacted with the cysteines to prevent re-formation of disulfide bonds.
