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/
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:
Fred van Leuven
Kathleen on Tom's account
Hope this has been informative to someone.... I know it has helped me. I hope I
have got most of it right!!!