Endoproteinase Lys-C
 

    Specificity : cleaves at K-X bonds. Some activity at N-X bonds has been reported

    pH optimum : 8.5-8.8

    Stability :  enzyme is active in 0.5% SDS and 5 M urea.

    Recipe : dissolve the substrate in 100 mM ammonium bicarbonate at 10 g/l. Addthe enzyme at 1:50 and incubate for 2 hours. Add the same amount of enzyme again and incubate for 22 hours.

    References : Perides, G., Kuhn, S. Scherbarth, A. and Traub, P. Probing of the structural stability of vimentin and desmin-type intermediate filaments with calcium activated proteinase, thrombin and lysine-specific endoproteinase Lys-C. Eur. J. Cell Biol. 43(1987)4 50-458.
 
 

    Endoproteinase Asp-N
 

    Specificity : cleaves at the C-terminal side of X-D or X-cysteic acid bonds. Cleavage at X-E bonds has been reported.

    pH optimum :  7.0

    Stability : the enzyme is active in 2 M urea. The activity of the enzyme drops off rapidly away from the optimum pH. Freeze-thawing cycles are not recommended for enzyme solutions.

    Recipe :  dissolve the substrate in 50 mM phosphate buffer (pH 7.0). Add the enzyme 1:20 and incubate for 4-18 hours at 37 C.

    Reference : Bentz, H., Chang, R-J., Thompson, A.Y., Glaser, D.B. and Rosen, D.M. Amino acid sequence of bovine osteoinductive factor. J. Biol. Chem. 265(1990)5024-5029.
 
 

    Endoproteinase Glu-C  (V8 protease)
 

    Specificity : cleaves at E-X and D-X bonds in phosphate buffers and at E-X bonds in ammonium bicarbonate buffers. Cleavage does not occur if X=P.

    pH optimum : 4.0-8.0

    Stability :  enzyme is active in 0.2% SDS, 1 M guanidine hydrochloride and 4 M urea. Some activity is retained at up to 100 C. The enzyme is unaffected by the presence or absence of divalent cations and can be used in the presence of EDTA.

    Recipe : for cleavage of E-X bonds: Dissolve the substrate in 100 mM ammonium bicarbonate at 10 g/l. Add enzyme 1:30 and incubate for 2-18 hours at 37 C. For cleavage of E-X and D-X bonds: Dissolve the substrate in 100 mM phosphate buffer (pH 7.8) at 10 g/l. Add enzyme 1:30 and incubate for 2-18 hours at 37 C.

    Reference : Tomasselli, A.G., Frank, R. and Schiltz, E. The complete primary structure of GTP:AMP phosphotransferase from beef heart mitochondria. FEBS Lett.
202(1986)303-307.
 

    Pepsin
 

    Specificity : cleaves preferentially C-terminal to F,L and E. It does not cleave at V, A or G. Other residues may be cleaved, with very variable rates.

    pH optimum : 2 - 4

    Stability : active in 4 M urea and 3 M guanidine HCl. Enzyme is stable at 60 C. Pepsin is irreversibly inactived at pH > 6.

    Recipe : doissolve the substate in 10 mM HCl at 1-10 g/l and adjust the pH to 2.0. Add pepsin at 1:100 and incubate for 1 hour at 25 C. Pepsin acts very quickly at room
temperature. Be very careful handling pepsin. If it contaminates a stock solution with 0.1 % TFA, it will remain active.

    Reference : Konigsberg, W., Goldstein, J. and Hill, R.J. the structure of human haemoglobin VII. The digestion of the beta chain of human haemoglobin with pepsin. J. Biol. Chem.
238(1963)2028-2033.
 
 

    Trypsin
 

    Specificity : trypsin cleaves very specifically at R-X and K-X bonds. If X=P, no cleavage occurs. Main trypsin preparations contain some chymotrypsin activity.

    pH optimum : 7 - 9

    Stability : trypsin retains activity in 0.1% SDS, 1 M guanidine HCl and 30% ethanol. Trypsin is autolytic, although 20 mM calcium chloride has been reported to slow autolysis. Trypsin is irreversibly inactived at pH > 11. Trypsin is not very stable above 40 C.

    Recipe : dissolve the substrate in 100 mM ammonium bicarbonate at 1-10 g/l. Add the enzyme at 1:50-100 and incubate for 1-4 hours at 37 C. Much longer incubations can be
used if the enzyme preparation has low chymotrypic activity.

    Reference : Eggerer, J. Hysteretic behaviour of citrate synthase. Site-directed limited proteolysis. Eur. J. Biochem. 143(1984)205-212.
 

  Chymotrypsin
 

    Specificity : cleaves on the C-terminal side of F, Y, W and L. Some cleavage reported C-terminal to M, I, S, T, V H, G and A.

    pH optimum  : 7.5 - 8.5

    Stability : functions in the presence of 2 M guandidine hydrochloride and 0.1% SDS. Store lyophilized or in 1 mM HCl and 2 mM calcium chloride.

    Recipe : dissolve protein substrate at up to 10 g/l in 100 mM ammonium bicarbonate. Add enzyme solution to give a 50:1 ratio of substrate to enzyme. Incubate for at least 4 hours at 37 C.

    Reference : Spackman, D.H., Stein, W.H. and Moore, S. The disulfide bonds of ribonuclease. J. Biol. Chem. 235(1960)648-659.
 

    Pronase
 

    Specificity : pronase is a mixture of endo- and exo-proteinases. It cleaves almost any peptide bond.

    pH optimum : 7 - 8. Different components of the mixture may have different optima.

    Stability : pronase requires calcium ions. It retains activity in 1% SDS and 1% Triton X. Some components of the mixture are very stable to urea and guandinium HCl, but complete digestion will not occur.

    Recipe : dissolve 0.1 micromoles/microliter of substrate in 50 mM ammonium bicarbonate with 5 mM calcium chloride. Add enzyme solution to give a 100:1 ratio of substrate to enzyme. Incubate for 24 hours at 37 C. Addition of 1:20 aminopeptidase M may be necessar y to acheive complete digestion.

    Reference : Garner, M.H., Garner, W.H., and Gurd, F.R.N. Recognition of primary sequence variations among sperm whale myoglobin components with successive proteolysis
procedures. J. Biol. Chem. 249(1974)1513-1518.
 

    Endoproteinase Arg-C
 

    Specificity : cleaves C-terminal to R. Some R-X bonds are resistant, but the systematics are unknown. Some cleavage at K-X bonds has also been observed.

    pH optimum : 7.5 - 8.5

    Stability : enzyme remains active in 0.1% SDS, but activity drops with time. Enzyme is denatured by 4 M urea. Purified enzyme autolyzes to form a smaller, also active form of the enzyme.

    Recipe : dissolve the substrate in 1% ammonium bicarbonate. Add the enzyme at 1:50 and incubate for 8 hours at 37 C.

    Reference : Poncz, L. and Dearborn, D.G. The resistance to tryptic hydrolysis of peptide bonds adjacent to N,N-dimethyllysyl residues. J. Biol. Chem. 258(1983)1844-1850.
 

    Aminopeptidase M
 

    Specificity : cleavage does not occur for X-P bonds or at N-blocked amino acids.

    pH optimum :  7.0 - 9.0

    Stability :  enzyme is stabel between pH 3.5 to 11.0.

    Recipe : dissolve substrate at 1-10 g/l in 50 mM ammonium bicarbonate. Add enzyme at 1:50 and remove times aliquots to obtain a sequencing ladder.

    Reference : Wachsmuth, E.E., Fritze, I., and Pleiderer, G., An amiopeptidase occuring in pig kidney. An improved method of preparation. Physical and enzymic properties. Biochem. 5(1966)169-174.
 

    Elastase
 

    Specificity : not very specific. Cleavage at C-terminal side of G, A, S, V, L and I.

    pH optimum :  8.0 - 8.5

    Stability : enzyme is active in 0.1% SDS. The enzyme rapidly autolyzes. 0.1 mM calcium ions stabilize the enzyme.

    Recipe : dissolve the substrate at 1-10 g/l in 100 mM ammonium bicarbonate. Add elastase at 1:50 and incubate for 4 hours at 37 C.

    Reference : Grunnet, I. and Knudsen, J. Medium-chain fatty acid synthesis by goat mamary-gland fatty acid synthetase. The effect of limited proteolysis. Biochem. J. 209(1983)215-222.