PMSF - (Oct/02/2001 )
Can anyone explain me with some detail the role of PMSF in molecular biology
PMSF is a general serine protease inhibitor. It is the most common inhibitor used in protein purification. Soluble in isopropanol.
PMSF is a highly toxic used in order to block serine proteases (like trypsin and chymotrypsin). It's an irreversible reaction. Can be used in protein purification, protein extraction...
Should be very carefully dissolved in isopropanol, and may be stored at -20°C for a year.
You can destroy PMSF by raising the pH. See Gold in Methods Enzymol XI, 706-708 (1967) or Fahrney/Gold JACS 85, 997, 1963. Its Km for chymotrypsin is 0.0056M at pH 6, and it reacts 50 times as fast with chymotrypsin as with trypsin.
Finally, its half life in water is above half hour.
PMSF at a maximum of about 1mM concetration is used in protein preparation to inhibit serine proteases. The first step in purifying an expressed protein is to rupture the cells by sonication. This results in release of the total proteins, some of which might be proteases. These proteases immediately start digesting the proteins including the expressed one, resulting in lower yields.
PMSF is to be added only in initial sonication step. Need not be nlcuded in all the buffers . It is toxic compound; needs careful handling.
PMSF is the most commonly used one as serine proteasesa re the predominant proteases. There are also aspartic proteases, cysteine proteases and metaaloproteases. EDTA is used to inactivate the latter. Some inhibitors are available for the fromer proteases. Protease inhibitor cocktail is available with Sigma; it inactivates all the proteases.
Precaution is necesary before including them during homogenization. you need to check in the crude protein preparation if the inclusion of these inhibiotors do not inactivate your protein ( activity test). My enzyme has a serine as nucleophile in the active site and is consequently inactivate by PMSF. So, I do not use it. There is one more case where another serine protease inhibioter ABESF was used with an enzyme with cysteine protease, it slightly inactivated the protein.
We can't seem to get PMSF into solution when we make it up in a 200 mM stock. We keep it at -20 and thaw it on ice. Any advice?
prepare 0.2M in ethanol. keep at -20°C
PSMF should be prepared in isopropanol or in ethanol as suggested by missele.
Normally, should be "thawed" as in such solutions, it shouldn't freeze !
For information : i prepare 100mM PMSF stock with no pb.
we prepare pmsf in methanol.