Thanks to everyone who responded to my inquiry about protein concentration.
These were all valuable suggestions. I have collected the various responses
below to share with the group.
AAA METHOD IDEAS
How about post column ninhydrin detection?
I have done AAA on such a sample with the old fashion post-column,
ion-exchange, ninhydrin system with no problems.
1) Remove the heavy metal by pH adjustment and treatment with a chelation
resin (Chelex-100) or dialysis against EDTA/EGTA. Not sure of heavy metal
affinity of EDTA/EGTA. Then perform AAA on apo-protein.
2) Measure AAA using post-column derivatization
This protein sounds like a great candidate for amino acid analysis using
post-column derivatization techniques (Ninhydrin). The metal ions likely
will separate in the blow thru from the amino acids and should not interfere
with their chromatographic analysis.
Amino acid analysis should be the best method. I do vapor
phase acid hydrolysis,followed by derivatization with AccQTag,which contain
sodium borate. Separation is done on a C18 column. Metal chelators are
present throughout the procedure. I think that ,whatever holds the metal
will be destroyed by the acid hydrolysis,and picked up by the chelotor
after hydrolysis. Anyway chelation should not be very good under extreme
acid conditions. I selected AccQTag,because hardly anything interferes with
it. The
presence of extraneous non-protein material could give rise to unexpected
peaks,but I cannot foresee any major problem.
Any chance of leaching the protein off first? Also,
if this is a pure protein of some sort with known composition, I would look
at the quantification based on various individual amino acids & see how
well that corresponds to basing on total aa.
NITROGEN ANALYSIS
can you do a keldjhal (sp?) nitrogen analysis to get you in the ballpark?
A total nitrogen analysis (Kejdahl) could be done and then correlated to
protein. However the material requirement can be quite high. I believe
greater than 1 g.
We have used nitrogen analysis when we knew the protein sequence and the
nitrogen content of the protein to quantitate. We did this when the protein
was in an oil formulation and different instruments require different
amounts of sample. Antek instruments use the least amount of sample (they
also have a nitrogen specific HPLC detector if you have really small amounts
of sample). We weren't sample limited so we used a Leco nitrogen analyzer.
I authored the general chapter for protein analysis for the USP (this
chapter is currently out for comment but published in Pharmacopial Forum)
and I mentioned the use of nitrogen analyzers and the wet chemical technique
called Kjedahl.
OTHER METHOD SUGGESTIONS:
How about running a SDS-PAGE, and estimate the protein amount based on the
density of band.
Maybe SEC to get quantitative data via peak response vs a standard curve.
Use
known concentration BSA standards, inject, then make dilutions of
the product and injecting. Still would have to insure the UV peak response
in the sample is due to protein only, though, so maybe would require some
DAD scanning of metal-only injections, or adding a light scattering
detector.
If all else fails you might try quantitation using refractive index. I have
never used it myself but I understand that it is a concentration dependent
technique. Because it does not require absorbance of a particular
wavelength
like UV and colorimetric methods it is fairly universal. Below is a paper
that
uses it for detection of proteins during isoelectric focusing:
Wu, Jiaqui and Janusz Pawliszyn. Dual detection for capillary isoelectric
focusing with refractive index gradient and absorption imaging detectors.
Analytical chemistry 1994, 66, 867-873.
Nadine Ritter
BioReliance
Original question:
Does anyone know of a method to determine protein concentration in materials
that are chelated to heavy metals? Colormetric methods have been tried, but
exhibit high imprecision, and I have concerns about the accuracy of results
coming from a standard curve made with protein not chelated to metal. Amino
acid analysis was attempted but the heavy metal appeared to interfere with
the pre-column derivatization. An extinction coefficient is not yet
determined, so UV is not very useful yet. Ideas?
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