Proteases are usually a bit resistant to their own activity, but
degradation will always happen at a particular rate, usually very fast
above 15 C, measurable at 4 C, slow at -20 C, and very slow at -80 C.
Always work fast and continuously, "until done" when dealing with
autocatalytic or unstable enzymes like proteases. If you must work with
non-purified fractions like cell lysate-supernatants, other classes of
proteases like serine proteases (typically the most abundant abiet a poorer
drug target and virulence factor), but I'm not sure what the situation is
for Entamoeba) may well be responsible for the degradation, since these are
often more abundant and proteases tend to work better on each other than on
themselves. PMSF inhibits serine proteases about 80% at 5 mM, but it's
carcinogenic. You will better know what you're working with if you do
purify the protein before analyzing its kinetics, making antibodies, or
pursuing other goals. You can usually find buffers in which the enzyme is
relatively inactive but in which it is not irreversibly denatured, by
looking at characteristics of related enzymes or that enzyme in particular.
For instance, if divalent cations are necessary, use a buffer with EDTA.
High salt stabilizes many enzymes without denaturing them. Another trick
is to store preparations of autocatalytic enzymes in substrate so it acts
on the substrate rather than itself; e.g. spike the enzyme preparation with
albumin, which can be rapidly removed by specific columns when you want to
assay the enzyme.
Good luck. You may also read some good general books on protein
biochemistry; I tend to like Methods in Enzymology chapters for concise and
authoritative descriptions of biochemical methods.
At 01:37 PM 10/9/98 +0000, divsin at my-dejanews.com wrote:
>I am working on cysteine proteinase of Entamoeba histolytica.
>I could not isolate it because it has autodegradative activity,found smear on
>SDS PAGE gel. I kept at 4oC temp and not exposes more then that.I do not want
>purify it but i wanted to do work without any loss of activity.
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>>Gerald McLaughlin, Ph.D.
Dept Pathology and Laboratory Medicine
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Indianapolis, IN 46202-5120
317-274-2651; FAX 317-278-2018
E-mail: gmclaugh at iupui.edu