Jan
Are you saying that the protein you are starting with is not colored (heme
is gone or damaged) or that the native protein is only weakly colored (low
extinction coefficient at the Soret wavelength)?
The former is more disturbing than the latter. For some heme proteins, like
peroxidases, you can bleach the heme. DTT and some sulfide containing
compound can do this quite easily and I have watched in horror as my brown
crystals turned clear (and lost diffraction). If the heme ligand is a
histidine, you might knock out a non-covalently bound heme during
purification on a Ni-/Co-chelation column. However, the heme is usually
tightly bound in most, but not all, heme proteins under non-denaturing
conditions.
If the problem is the latter, cyanide, carbon monoxide, and other heme
ligands can shift the Soret band to a different lambda and, occasionally,
enhance the extinction coefficient.
Normally, crystallizing heme proteins is not a problem with respect to the
heme behavior if the heme is in its resting ferric state. Just avoid
detrimental heme ligands and possible reductants (unless you want the
ferrous state) and oxidants. But to answer your question better, more
information is needed.
Cheers,
Michael
*****************************************
R. Michael Garavito, Ph.D.
Professor of Biochemistry
Michigan State University
East Lansing, MI 48824-1319
517-355-9724 (Office)
517-353-9334 (Fax)
[EMAIL PROTECTED]
*****************************************
Hello everybody,
I wonder if anybody has experience with heme (or to be more precise: heme b) containing proteins which Xtals do not look red under the microscope. How might the technique for crystallization (e.g. sitting drop, hanging drop) influence the intensity of the color? Many thanks!
Jan
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