F BICH 410 1st Edition Lecture 11 Outline of Last Lecture Protein Purification Protein Separation Visualization Techniques Outline of Current Lecture Protein Sequencing o Determining total amino acid compositon of protein by hydrolysis of protein with 6M HCL 100C o Determine order of amino acid in chain Sequencing steps o Separate subunits o Break disulfide bonds o End group analysis determine N and C terminus o Cleave protein and study composition and sequence of fragments o Repeat with different cleave sites and reconstruct fragment sequence Composition is how many of each amino acid Sequence is amino acid order Ways to break polypeptide chemical acid or enzymatically hydrolyze peptidase o Can use extreme pH 8M urea 6M guanidine HCL or salt conc Reduce or oxidize disulfide bonds o Oxidize performic acid don t have to acetylate o Reduce dithiothritol DTT have to acetylate to prevent re crosslinking End group determinationo N terminus react with primary amine dansyl chloride or FDNB determines first AA o C terminus caroxypeptidase or exopeptidase remove C residue one at a time Cleave protein into fragments and determine sequence typically 50 or so residues o PROTEOLYTIC ENZYME Trypsin cuts C terminal side of ARG LYS Chymotrypsin cuts C side of aromatic AA Cyanogenbromide cuts c terminus of Met Elastase cuts C side of small neutral AA GLY ALA Proline prevents cutting Sequencing Edmans degradation o Reagent phenylisothiodynate o Shortens by 1 residue allowing sequence determination but can typically only be repeated 50 times These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute Repeat cleaving and sequencing to allow reconstruction of polypeptide seq and match up overlapping fragments
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