Biomedical Engineering Reference
In-Depth Information
Measuring protein absorbance at lower wavelengths (205 nm) increases the sensitivity of the
assay considerably. Also, as it is the peptide bonds that are absorbing at this wavelength, the assay
is subject to much less variation due to the amino acid composition of the protein.
The most common methods used to determine protein concentration are the dye-binding proce-
dure using Coomassie brilliant blue, and the bicinchonic-acid-based procedure. Various dyes are
known to bind quantitatively to proteins, resulting in an alteration of the characteristic absorption
spectrum of the dye. Coomassie brilliant blue G-250, for example, becomes protonated when dis-
solved in phosphoric acid, and has an absorbance maximum at 450 nm. Binding of the dye to a
protein (via ionic interactions) results in a shift in the dye's absorbance spectrum, with a new ma-
jor peak (at 595 nm) being observed. Quantifi cation of proteins in this case can thus be undertaken
by measuring absorbance at 595 nm. The method is sensitive, easy and rapid to undertake. Also,
it exhibits little quantitative variation between different proteins.
Protein determination procedures using bicinchonic acid were developed by Pierce Chemicals,
who hold a patent on the product. The procedure entails the use of a copper-based reagent contain-
ing bicinchonic acid. Upon incubation with a protein sample, the copper is reduced. In the reduced
state it reacts with bicinchonic acid, yielding a purple colour that absorbs maximally at 562 nm.
Silver also binds to proteins, an observation that forms the basis of an extremely sensitive method
of protein detection. This technique is used extensively to detect proteins in electrophoretic gels,
as discussed in the next section.
7.4 Detection of protein-based product impurities
SDS polyacrylamide gel electrophoresis (SDS-PAGE) represents the most commonly used ana-
lytical technique in the assessment of fi nal product purity (Figure 7.1). This technique is well
established and easy to perform. It provides high-resolution separation of polypeptides on the
basis of their molecular mass. Bands containing as little as 100 ng of protein can be visualized
by staining the gel with dyes such as Coomassie blue. Subsequent gel analysis by scanning laser
densitometry allows quantitative determination of the protein content of each band (thus allowing
quantifi cation of protein impurities in the product).
The use of silver-based stains increases the detection sensitivity up to 100 fold, with individual
bands containing as little as 1ng of protein usually staining well. However, because silver binds to
protein non-stoichiometrically, quantitative studies using densitometry cannot be undertaken.
SDS-PAGE is normally run under reducing conditions. Addition of a reducing agent such as
β-mercaptoethanol or dithiothreitol (DTT) disrupts interchain (and intrachain) disulfi de linkages.
Individual polypeptides held together via disulfi de linkages in oligomeric proteins will thus sepa-
rate from each other on the basis of their molecular mass.
The presence of bands additional to those equating to the protein product generally represent
protein contaminants. Such contaminants may be unrelated to the product or may be variants
of the product itself (e.g. differentially glycosylated variants, proteolytic fragment, etc.). Further
characterization may include western blot analysis. This involves eluting the protein bands from
the electrophoretic gel onto a nitrocellulose fi lter. The fi lter can then be probed using antibodies
raised against the product. Binding of the antibody to the 'contaminant' bands suggests that they
are variants of the product.
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