Angela,
Do you track your enzyme with gel electrophoresis to verify that you
actually have it, and have you checked the column void for presence of
enzyme and activity?
Wilson
At 08:24 AM 2/1/2001 -0600, Dan Crimmins wrote:
>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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