At 09:50 AM 2/1/2001 +0000, Angela Merlo wrote:
>
>Hi everyone,
>I try to purify an enzyme (bezoate dihydrodiol dehydrogenase) from
>Rhodococcus opacus (Gram +).
>I noticed that the optimal pH for this enzyme is 9, so I started an
>anion exchange chromatography with buffer pH 8 because it was supposed
>that 9 is too high. The chomatogram was nice and i got activity in a
>fraction. The problem came afterwards when I tryed a Hydrofobic
>Interaccion Chrom. I precipitated the protein pool with (NH4)2SO4 and
>resuspended them with puffer pH8+salt. The activity was high so I did a
>HIC (phenyl sepharose) with decreasing concentration of TrisHCl 25mM pH8
>+ (NH4)2SO4 1M. I got a good chromatogram but no activity in any
>fraccion. Then I tryed a butyl Q-sepharose column and also didnĄt work.
>Last time I used a buffer pH 7,5 and I had again no activity.
>Do you have any idea where the problem can be?
>
>Thanks
>
>Angela
>
>
>
>
Angela,
Does this enzyme have a multi-subunit structure? Perhaps the work-up and
chromatography on HIC is causing dissociation of the subunits, eventhough
this mode of chromatography is considered "mild". Any cofactor(s) that may
have been lost during the purification? Try dialysis after the HIC step vs.
a buffer that your enzyme "likes" and add back any metals, etc. that may be
required for activity.
Regards,
Dan L. Crimmins, Ph.D.
Washington University School of Medicine
Department of Immunology and Pathology
Division of Laboratory Medicine
660 S. Euclid Ave., Box 8118
St. Louis, MO 63110
Phone 314-454-8514
Fax 314-454-5208
e-mail crimmins@pathbox.wustl.edu
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