Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue

[Roger Rowlett]

In general, most protein quantification methods have significant problems with idiosyncracy, because proteins are quite variable in composition and structure. The best method is to use is the absorption at 280 nm, but this is quantitatively useful only if the molar absorptivity is known (and the protein is pure, or at least purified away from UV-absorbing materials). We work extensively with metalloenzymes, and quantifying protein:metal ratios accurately is highly problematic. Here is my take: The Bradford (dye-binding) assay is quite dependent on protein composition (hydrophobic content and perhaps other amino acid composition). We see variances of apparent to actual protein concentration of 50% or more The Lowry-type assays based on molybdenum blue formation (this includes the BCA assay which is a variant of the classic Lowry assay) are also highly composition dependent. We see variances of apparent to acutal protein concentration of up to 100%, depending on the protein involved. The Biuret assay is dependent primarily on the peptide backbone content, and has small variation of response to composition. It is not very sensitive, however. The microbiuret assay, which is similar to biuret but with measurements made at 330 nm is tolerable. Protein must be homogeneous, of course. We see relatively little variation (20% or so) between apparent and true concentrations of protein using this method, but it is subject to a variety of interferences. Far UV absorption of proteins is nearly independent of composition, as absorption in this region is due almost entirely to peptide bonds. This requires purified protein, and the absence of other UV-absorbing materials, but is otherwise quite tolerant. Maximal consistency can be obtained by carrying out measurements in denaturing detergents. We have used 0.01-0.1% Triton X-100 with success. The asborptivity of 1mg/mL solutions is approximately 20.5 at 210 nm and around 34 at 205 nm. various corrections can be applied to correct for absorption of aromatics at 205 nm. See Scopes (1974) Anal Biochem 59, 277-282. The systematic variance of this method is generally small, maybe 20% or less. I have found that methods 3-4 frequently agree, and give good agreement with well-characterized metalloproteins where the metal can be very accurately measured using ICP-OES. Cheers. -- Roger S. Rowlett Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu