PROTEASE INHIBITORS

        This is an edited discussion of materials presented on proteases and their inhibitors from the internet posted on 29-March-1991 by "BS04@primeb.dundee.ac.uk", and additional notes on the use of inhibitors in our laboratory.

        There are four groups of proteases: serine, cysteine, aspartic and metallo-proteases. Proteases can be either reversible or irreversible. Reversible proteases react in the absence or above critical concentrations of their inhibitors. Often in the literature, cocktails of inhibitors are made up and little thought goes into what they are effective against, what concentrations are needed, and how long they are stable in an aqueous environment. A case in point is PMSF, a widely used serine protease. This has a half-life in water of 15-60 minutes (depending on your reference source). So it should be added just before the cell lysis. PMSF is not very soluble in water and should be kept at -20°C in dry methanol/propanol.

The cocktail that I have come up with is:

         (i) PMSF Stock 200 mM (keep 4°C methanol), use at 1 mM
         (ii) E64 Stock 1 mM (keep -20°C, H20), use at 10 mM
         (iii) EDTA Stock 500 mM (keep 4°C), use at 5 mM

These are used to inhibit serine, cysteine (papain, calpain, cathepsin B, cathepsin L), and metallo-proteases respectively. Still working on aspartic. These stocks may be made and frozen, but care has to be taken selecting the correct solvent. A good reference is Proteolytic Enzymes. Eds Robert J. Beynon and Judith S. Bond. Published by IRL Press at Oxford University Press, 1989. ISBN 0-19-963059-3 Pbk.

Addendum from our laboratory:
        In the absence of information about which class of protease(s) may be degrading a particular sample, we prepare a protease cockt ail from the following individual stocks:

Protease class Inhibitor Stock concentration Working concentration:
Serine proteases PMSF 100 mM in methanol 1 mM
Metalloproteases EDTA 500 mM in H2O 1 mM
Serine proteases Benzamidine 100 mM in H2O 1 mM
Acid proteases Pepstatin A (inhibits renin, cathepsin D, chymosin, protease B) 1 mg/ml in H2O 1 µg/ml
Thiol proteases (serine & cysteine proteases) Leupeptin 1 mg/ml in H2O 1 µg/ml

Other protease inhibitors that may be considered include aprotinin (inhibits serine proteases including trypsin, chymotrypsin, plasmin, trypsinogen, urokinase, kallikrein, human leukocyte but not pancreatic elastase - use at 100 - 500 micromolar); leupeptin (inhibits papain, calpain, trypsin, cathepsin B - use at 1 - 5 mM); AEBSF (inhibits serine proteases including trypsin and chymotrypsin - use at 10 - 100 mM); b< B>estatin (inhibits aminopeptidases including leucine aminopeptidase and alanyl aminopeptidase - use at 1 - 5 mM). Protease inhibitors dissolved in DMSO that will be added to live cells should be prepared so that the final DMSO concentration is less than 0.5%.
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Proper citation for data acquired from this document is: "Notes on Protease Inhibitors from a Bionet Newsgroup described In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/protease.html). 1997. Oklahoma City. Revision Date: October 2, 1997." Thank you to Sigma for providing certain specificities listed above.