| Enzymatic cleavage |
| Trypsin |
Arg, Lys |
Specific; often used first; bonds to proline are not cleaved; amino-ethylcysteine provides additional site |
| Endoproteinase Lys-C |
Lys |
Very high-specific activity; very effective in gel digests |
| Clostripain |
Arg |
Lys bonds remain intact; yields may be lower than with trypsin; cleaves Arg Pro |
| Armillaria mellea protein
|
Lys |
Splits on amino side; sometimes after Arg |
| Staphylococcus aureus protein
|
Glu |
Sometimes cleaves after Asp; also cleaves cysteic acid |
| Endoproteinase Asp-N |
Asp |
Cleaves amino-terminal to Asp; also cleaves cysteic acid |
| Asparaginylendopeptidase |
Asn-X |
pH 5.5-6.5; stable to denaturants |
| Chymotrypsin |
Trp, Tyr, Phe, Leu, Met |
Largely specific for hydrophobic residues; also useful for limited digestion applications |
| Thermolysin |
hydrophobic |
Splits on amino side; may be used at 60ºC |
| Pepsin |
Phe-Phe |
Relatively nonspecific; prefers aromatics or Leu; low pH optimum; no disulfide interchange occurs |
| Subtilisin, Elastase |
various |
Nonspecific; for providing small peptides |
| Thrombin, Bromelain |
Arg-Gly, Gly-Val |
Limited proteolytic activity; give large fragments |
| Chemical-Enzymic |
| Succinylation-trypsin |
Arg |
Lysine blocked; changes its polarity; citraconylation provides reversible modification |
|
| Chemical cleavage |
| Cyanogen bromide |
Met |
Converted to homoserine lactone; yields >70%; Ser, Thr bonds difficult; Trp-X can cleave |
| Dilute acid (pH 2) |
Asp |
Use formic acid for insoluble proteins and dilute; some amide cleavage; yields >60% |
| Formic acid (80%) |
Asp-Pro |
Yields of 40% with high specificity |
| HCl (6 M) |
multiple |
For random generation of small peptides |
| Hydroxylamine |
Asn-Gly |
Also breaks imide link to Gly; yields ~50% |
| DMSO/HCl/HBr |
Trp |
Several alternative reagents available; oxidation and modification of amino acids likely |
| NTCB |
Cys |
Splits on the amino side; Cys amino blocked |
| aCleavage occurs at the carboxyl end of the amino acid unless indicated otherwise.
|
