At 9:50 AM +0000 2/1/01, Angela Merlo wrote:
>Hi everyone,
>I try to purify an enzyme (bezoate dihydrodiol dehydrogenase) from
>Rhodococcus opacus (Gram +).
Angela-
Is your enzymatic activity inhibited by transition metals??
We are working on two enzymes which are inhibited by traces of
transition metals. We have found it crucial to purify all buffers,
and ammonium sulfate, by passing them through Chelex resin (BioRad)
to remove traces of transition metals. Check the specification of you
AR grade inorganic chemicals, and then calculate the likely
concentration of Pb, FE, etc. One of my graduate students did so-
with his apparently pure kinase, the concentration of Pb could have
been higher than the concentration of enzyme.
Another possibility is to add EDTA to your solution, but this
is much less desirable because it is also likely to strip Zn, Ca, and
other metals which are required for the activity of many enzymes.
For example, one of the enzymes we are purifiying is a
metalloprotease whose activity is almost completely inhibited by EDTA.
David
-- David A. Schooley Dept. of Biochemistry/330 Univ. of Nevada Reno, NV 89557 schooley@unr.edu tel: (775) 784-4136; fax (775) 784-1419 NOTE NEW AREA CODE: Mandatory after 5/15/99
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