BS04@primeb.dundee.ac.uk (03/29/91)
To those who may be interested: I recently sent out a request for information concerning the use and handling of proteases. I was greatly helped by the response. In one reply there was a request for me to write a synopsis to the list. Well here it goes. I should say that I still have a lot to learn on the subject but I will tell you what I have picked up. I hope it will be some use...... It seems to me there are four groups of proteases one has to be carefull of (serine, cysteine, aspartic and metallo-proteases). The first two have recieved the greatest amount of research, then the metallo-proteases followed by the aspartic proteases. Needless to say there is a whole body science who see these proteins as the good guys but for me they are potentially ruinous and so have to be dealt with. There are two types of proteases (i) reversible and (ii) irreversible. The former only work when the inhibitor is maintained in the protein extraction medium/desalting medium. So care has to be taken to check that the inhibitor is not left out of desalting medium or chromatography medium, otherwise the proteases will then be free to do their worst. The latter tend to be very quick and once the protease is disabled that is the end of its destructive abilities. An important point needs to be made here. 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 ref. source). So it has to be added just before the cell breakage PMSF is not very soluble in water and should be kept at -20C in dry methanol/ propanol. The cocktail that I have come up with is: (i) PMSF Stock 200 mol/m3 (keep 4C methanol), use at 1 mol/m3, (ii) E64 Stock 1 mol/m3 (keep -20C, H20), use at 10 mmol/m3 (iii) EDTA Stock 500 mol/m3 (keep 4C), use at 5 mol/m3 These are used to combat serine, cysteine and metallo-proteases respectively. Still working on aspartic..... My original request was about whether I could make up aliquots of these inhibitors and freeze them. The basic answer was Yes... but care has to be taken selecting the correct solvent (see below for book reference). A word of warning..... alot of these chemicals are not very nice..... and lots of due care and attention should be applied.... I can recommend an excellent book on the subject: 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. Should tell all I have left out. It now remains for me to thank the following who answered my mail shot: DanJ (JHUHYG) Don Lehn Fred van Leuven Rob Beynon Shauna Farr-Jones Kathleen on Tom's account Bill Graziadei Bob Straubinger Martyn White Hope this has been informative to someone.... I know it has helped me. I hope I have got most of it right!!! Many Thanks Andy Johnston.