hydrolysis of hydrophobic proteins
David A Rintoul
drintoul at matt.ksu.ksu.edu
Thu Sep 3 08:34:26 EST 1992
this may not be the classis topic for this question, but I
am dealing with a biological molecule, at any rate.
We have purified a protein from lens fiber cell plasma membranes that
is giving us trouble. The protein is extremely hydrophobic; we
isolate and purify it by extracting the membranes with chloroform and
methanol (1/1) and run the extract over a lipophilic Sephadex column.
By SDS-PAGE, is is pure (no other self-respecting protein would hang
around for that kind of treatment!). Now we need to do an amino acid
analysis to quantitate the stuff, and to ensure that it corresponds to
the amino acid composition predicted from the cDNA sequence.
The approaches thus far have been to dry it down under nitrogen,
hydrolyze it in HCL, and assay the amino acids by HPLC (pico-tag or
ninhydrin absorbance). We tried hydrolyzing it for 24 hours, and the
analysis indicated a substantial amount of something which was vaguely
identified as unhydrolyzed protein. The amount of amino acid released
was also consistent with this diagnosis; it was about 40% of what we
expected. So, we tried hydrolyzing it for 72 hours, and the results
are the same (even worse).
Several other facts need to be considered as well. When we run the
protein on SDS-PAGE, we never boil it, since others have shown (and we
have corroborated) that it irreversibly aggregates at this
temperature. Could the temperature that we use for hydrolysis (105 C)
be the problem? If so, how do you hydrolyze proteins quantitatively
without heating them? The other fact is that after drying the protein
down from the solvent (1/1 chloroform/methanol), it cannot be
resuspended in solvent, indicating that is is also irreversibly
aggregated and insoluble in any solvent (chloroform, methanol,
mixtures of these, ethanol, trifluoroethanol etc).
So, the real questions are:
1. Can we do the hydrolysis on protein that has not been dried down,
but still contains a bit of chloroform and methanol?
2. Does anyone out there have experience with such hydrophobic
proteins? Any hints?
This analysis is the last thing we need to do before writing up the
paper, and it is getting frustrating that we seem unable to get it
done. Any clues or suggestions will be greatly appreciated.
P.S. We have considered SDS-PAGE and blotting to PVDF, followed
by AA analysis, but, since the purpose of this whole escapade
is to quantitate the protein from the AA content, I don't think
this is a good (quantitative) approach.
Dave Rintoul Internet: drintoul at matt.ksu.ksu.edu
Biology Division - KSU Latitude 39.18, Longitude -96.34
Manhattan KS 66506-4901 TCN: id2418
(913)-532-5832 or 6663 FAX: (913)-532-6653
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