Reagents For Protein Cleavage Flashcards
Carboxypeptidase B
N-side of C-terminal residue
Rn = Lys or Arg
Rn-1 cannot be Pro
Carboxypeptidase A
N-side of C-terminal residue
Rn = any amino acid
Except Lys, Arg, Pro
And Rn-1 cannot be Pro
Carboxypeptidase Y
N-side of C-terminal residue
Rn = any amino acid
Pro very slowly
Hydrazinolysis
All peptide bonds broken
All residues, except C-terminal residue converted to AA-hydrazide
Organic solvent used to remove AA-hydrazides from the free C-terminal AA
Phenylthiocyanate (PITC)
C side of N terminal residue
Rn = any amino acid
Edman’s reagent
Cannot be used when N-terminal of alpha-NH2 is blocked
Trypsin
C side of Rn
Rn = Lys or Arg
Rn+1 cannot be Pro
Cannot cleave when Lys or Arg reside at a terminus
Chymotrypsin
C side of Rn
Rn = Phe, Trp, or Tyr (slowly at Met, Leu, Asn)
Rn+1 cannot be Pro
Cannot cleave if Phe, Trp, or Tyr are at a terminus
S. aureus V8 protease
Staphylococcal protease
C side of Rn
Rn = Glu , Asp
Rn+1 cannot be Pro
Thermolysin
N side of Rn
Rn = Leu, Ile, Phe, Trp, Tyr or Val
Slowly at Ala
Pepsin
N side of Rn
Rn = fastest at Phe, Tyr, Trp & Leu
Optimal at pH 1-2
Will cleave at Asp & Glu
Cyanogen bromide
C side of Rn
Rn = Met
Iodosobenzoic acid
O-iodobenzoate
C side of Rn
Rn = Trp
Sanger’s reagent
1-fluoro-2,4-dinitro-benzene
FDNB
Acts as tag replacing one amino H with 1,3- dinitro benzene to N terminus residue
Removal of disulfide bonds by oxidation
Performic acid leads to cysteic acid residues (–CH2–SO3-)
Removal of disulfide bonds by reduction
Dithioreitol (DTT) leads to Cys residues
Iodoacetate converts Cysteines to carboxymethyl-C residues
