BL483 Biochemistry Techniques

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BL483 Biochemistry Techniques -- Lecture 1 -- Protein Assay

5. Dealing with Pure Proteins

Although it is beyond the expt you will do in the lab this week, it is important to discuss the problem of determining the amount of a protein present in solution when it has been completely purified. The methods discussed in this lecture are really only effective for mixtures of proteins. Since all these methods (Folin/Lowry or Bradford) depend on the presence of specific amino acids (aromatic or lysine) in the proteins being assay, a pure protein may differ significantly from the standard protein being used to make the standard curve. So the amount of protein found may be off by 50% or more.

This is not a simple problem to overcome. If the protein contains a prosthetic group or cofactor bound to it, for example like iron in hemoglobin, then the amount of cofactor present can be used to quantify the amount of pure protein. If the protein contains no cofactor, then its UV spectrum can be determined and the peak absorbance in the near UV used to find the protein's unique extinction coefficient. Most proteins absorb near 280 nm and the extinction coefficients of many pure proteins can be found in Biochemical Handbooks and perhaps also on some WEB sites.

A protein's extinction coefficient for its UV peak (about 280 nm) can be calculated by quantifying the amount of protein in a solution of known absorbance by doing its amino acid composition or amino acid sequence. In the old days, the protein was dialyzed against deionized water until no salt was left and then the protein dried and weighed. This of course requires a lot of protein and today, more sensitive methods are used like the quantitative results from amino acid sequencing using an automated Sequencer based on the Edman method of N-terminal sequencing.

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