Question

1) Why do we use Absorbances A215 and A280 to estimate protein concentration?

2) When is E(1%) at 280 =15 more appropriate to use in calculating the concentration of protein compared to E(1%) 280 =4.9

Answer 1

• Absorption at 280nm is due almost entirely to the aromatic amino acids: tryptophan and tyrosine.
• At 260 nm phenylalanine absorbs, and histidine, methionine, cystiene, and cystine absorb between 225 and 240 nm. At wavelengths less than 225 nm the peptide bond itself is the principal absorbing group, but the above amino acids also contribute.
• There are a number of reasons why these shorter wavelengths are not the most frequently used. At 192nm dissolved oxygen absorbs strongly, and thus measurements would have to be carried out in the absence of oxygen. 192 nm is below the working range of many spectrophotometers, since it requires a particularly good light source and stray radiation can be a problem. Wavelengths nearer 225 nm are used since, although the peptide bond absorbs less strongly, the interference by oxygen is limited.

• In general, the shorter the wavelength the larger the range of interfering substances. It is perhaps this reason that although A280 is less sensitive than some, and is dependent on the tyrosine and trypotphan content, it is one of the most used.
• The absorbance spectrum of tyrosine is pH dependent, but at 280nm both the protonated and unprotonated forms have similar absorption coefficients and so measurements do not have to be made at a particular pH.
• Some spectrophotometers used to measure column effluents use 206nm, which gives much greater sensitivity provided there is no interference.

(2)

At 280 nm, this value is approximated by the weighted sum of the 280 nm molar absorption coefficients of the three constituent amino acids, as described in the following equation: e = (nW × 5500) + (nY × 1490) + (nC × 125) • n is the number of each residue • Stated values are the amino acid molar absorptivities at 280 nm

The best extinction coefficient value is one that is determined empirically using a solution of the study protein at a known concentration.

Refer to the Pierce bulletin # TR0006.2 for additional information regarding the use of Pierce standards to calculate a “system-specific” extinction coefficient.