scissors bich

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Author:
alyspins
ID:
106410
Filename:
scissors bich
Updated:
2011-10-04 16:12:51
Tags:
endopeptidases protein squencing
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Description:
names and specitifity of a variety of endopeptidases; protein sequencing
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  1. Trypsin
    • Rn-1= positively charged residues: Arg, LYS;
    • Rn NOT Pro
    • highly specific
  2. Chymotrypsin
    • Rn-1= bulky hydrophobic residues: Phe,Trp, tyr
    • Cleaves more slowly for Rn-1=Asn, His, Met, Leu
    • Rn NOT pro
  3. Elastase
    • Rn-1= small neutral residues: Ala, Gly,
    • Ser, Val
    • Rn NOT Pro
  4. Thermolysin
    • Rn=Ile, Met, Phe, Trp, Tyr, Val
    • Rn-1 NOT Pro
    • Occasionally cleaves at Rn=Ala, Asp, His, Thr
    • heat stable
  5. Pepsin
    • Rn =Leu, Phe, Trp, Tyr
    • Rn-1 NOT Pro
    • Also others; quite nonspecific;
    • pH optimum =2
  6. Endopeptidase V8
    Rn-1=Glu
  7. CNBr
    Rn-1=Met
  8. dansyl chloride
    • bonds with primary amine
    • help determine N-terminus
  9. FDNB
    • bonds with primary amine
    • help determine N-terminus
  10. Carboxypedptidase
    C-terminus determination
  11. 8 steps for sequencing protein
    • 1) Seperate subunits
    • 2) break disulfide bonds
    • 3) Amino Acid Composition
    • 4)End group analysis
    • 5) "Molecular Scissors"
    • 6)repeat 5 w/ differenet scissors
    • 7) sequence each purfied peptide fragment with Edman Degradation
    • 8) reconstruct sequence
  12. How do you seperate subunits of protein?
    use: extreme pH; 8M urea; 6M guanidine HCl; high salt concentration
  13. Break disulfide bonds
    • 1) reduce by DTT or 2-mercaptoethanol, block with iodoacetate OR
    • 2)oxidize by performic acid
  14. Amino Acid composition
    acid and enzymatic hydrolysis of polypeptides then amino acid compostion analyzed by HPLC
  15. Amino Acid grp determination
    • N-terminus: Dansly Chloride & FDNB
    • C-terminus: carboxypeptidase or other exopeptidase
  16. cleave chains into smaller fragments
    Trypsin, Chymotrypsin, elastase CNBr
  17. sequence peptides produced from cutting in 5&6
    use Edman Degredation: -PHENYLISOTHICYANATE
  18. Reconstruction
    comapre and align overlapping peptide pieces
  19. what cleaves large argomatics and where?
    Pepsin cleave at N terminus; Chymotrypsin cleaves at C terminus (Rn-1)

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