Write the principle behind the UV absorption of protein at 280nm

The principle behind the UV absorption of proteins at 280nm primarily stems from the presence of aromatic amino acids, namely tryptophan (Trp), tyrosine (Tyr), and to a lesser extent, phenylalanine (Phe). These aromatic amino acids contain conjugated ring systems that absorb UV light, particularly around 280nm, due to the π-π* electronic transitions within their aromatic rings.

The absorption of UV light by proteins at 280nm can be attributed to the following factors:

1. Tryptophan Absorption: Tryptophan is the most significant contributor to protein absorption at 280nm. It has a characteristic absorption peak around 280nm due to the π-π* electronic transition of its indole ring system. The indole ring of tryptophan absorbs UV light most strongly at this wavelength.

2. Tyrosine Absorption: Tyrosine also contributes to protein absorption at 280nm, albeit to a lesser extent compared to tryptophan. Tyrosine's absorption peak is slightly shifted towards shorter wavelengths compared to tryptophan, around 275nm. However, its contribution to the overall absorbance at 280nm is still notable.

3. Phenylalanine Absorption: Phenylalanine, another aromatic amino acid, absorbs UV light weakly compared to tryptophan and tyrosine. Its contribution to the absorption at 280nm is minor but can still be relevant, especially in proteins with a high phenylalanine content.

The absorption of UV light by these aromatic amino acids is due to the excitation of π electrons within their conjugated ring systems. When UV light of appropriate energy (around 280nm) is absorbed by the aromatic rings, it promotes π electrons from the ground state (π) to an excited state (π*). This absorption results in a decrease in the intensity of transmitted light, as some of the incident photons are absorbed by the protein.

The Beer-Lambert Law describes the relationship between the absorbance (A) of a sample, the molar absorptivity (ε) of the absorbing species, the path length (l) of the sample, and the concentration ([c]) of the absorbing species: