FBICH 410 1st Edition Lecture 11Outline of Last Lecture - Protein Purification- Protein Separation- Visualization TechniquesOutline of Current Lecture - Protein Sequencingo Determining total amino acid compositon of protein by hydrolysis of protein with 6M HCL 100Co Determine order of amino acid in chain- Sequencing steps o Separate subunitso Break disulfide bondso End group analysis- determine N and C terminuso Cleave protein and study composition and sequence of fragmentso 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 acetylateo Reduce- dithiothritol DTT- have to acetylate to prevent re-crosslinking- End group determination-o N terminus- react with primary amine- dansyl chloride or FDNB- determines first AAo C terminus- caroxypeptidase or exopeptidase- remove C residue one at a time - Cleave protein into fragments and determine sequence- typically 50 or so residueso 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 degradationo Reagent= phenylisothiodynateo Shortens by 1 residue allowing sequence determination but can typically only be repeated 50 timesThese 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
View Full Document
Unlocking...