Table 1 Frequently used protein cleavage reagents

Ancrod

Arg-X, Arg-Gly

Bromelain

C-terminal to Lys, Ala and Tyr

Chymotrypsin

C-terminal to hydrophobic residues, e.g., Phe, Tyr, Trp. Less sensitive with Leu, Met, Ala

Clostripain

C-terminal to Arg residues

20

Collagenase

N-terminal to Gly (X-Gly) in Pro-X-Gly-Pro

Elastase

C-terminal to amino acids with small hydrophobic side chains

Endoproteinase Arg-C

C-terminal to Arg residues

20

Endoproteinase Asp-N

N-terminal to Asp and Cys

10

Endoproteinase Glu-C

C-terminal to Asp and Glu

10

Endoproteinase Lys-C

C-terminal to Lys

20

Factor Xa

C-terminal to Arg in Gly-Arg-X

Ficin

uncharged or aromatic amino acids

Follipsin

Arg-X

20

Kallikrein

C-terminal to Arg in (Phe-Arg-X or Leu-Arg-X)

Pepsin

Broad specificity; preference for cleavage C-terminal to Phe, Leu, and Glu

7

Thermolysin

N-terminal to amino acids with bulky hydrophobic side chains, e.g., Ile, Leu, Val, and Phe 5

Thrombin

C-terminal to Arg

Trypsin

C-terminal to Lys and Arg

10

V8 protease

C-terminal to Glu, less active with Asp

Chemistry

Preferred cleavage site

Cyanogen bromide

Trp, (Met)

Formic acid

Asp – Pro

HCl

Asp-X, X-Asp

Hydroxylamine (alkaline pH)

Asn – Gly

N-bromosuccinimide (NBS) or N- chlorosuccinimide

Trp

2-Nitro-5-thiocyanobenzoate (NTCB)

Cys