Hi Folks,
I have a hemoglobin that is from a cyanobacterium. We are trying to do
NMR analysis for structural studies. I am concerned from initial scans
that I may have apo-protein (ie, globin with no heme) in my prep.
Although paramagnetic effects complicate spectra, I must try to get data
for the holo-protein. The hemoglobin is over-expressed in E. coli, so I
may be draining the heme pool from the bacterium OR a chromatography step
is the cause for the apo-protein presence. I have ruled out the later
through ABS 415 nm/ABS 280nm ratios using purified material. I am
currently trying to supplement the culture so I can provide adequate heme
supply to the bacterium. Or better yet, why not strip all heme out, and
then replace it quantitatively.
Many hemoglobins can be reconstituted with free hemin in an equimolar
ratio with apo-protein. This is documented in many instances. However,
I have had little success with the procedure. There are two
possibilities. One, I am just not performing the protocol properly or
two, the heme extraction step (acidify to pH 2, then 2-butanone
extraction), is somehow harming my protein, rendering it incapable of
accepting hemin in a reconstitution experiment. I am leaning towards the
former, as I know of no precedence of this extraction procedure harming
other hemoglobins (as in hemoglobin, myoglobin, leghemoglobin,
cytochromes, etc.). If the extraction is harming the protein, then NMR of
apo-protein is out of the question. I am trying some crystal preps, but
I am not basing my entire PhD thesis on trying to crystallize this
protein or else I will be graduating a decade from now. I keep thinking
I am missing something very simple (as is ususally the case). ANyone
have any thoughts?
Thanks in advance.
Marc V. Thorsteinsson
Department of Biochemistry
201 Engel Hall
Virginia Polytechnic Institute
Blacksburg, VA 24061
mthorste at vt.eduthor at bev.net
