As a biochemist with extensive experience in the study of protein structure and function, I am well-versed in the intricacies of how proteins interact with light. The absorption of light at 280 nm by proteins is a phenomenon that is particularly significant in the field of biotechnology and molecular biology. This specific wavelength is often used in spectrophotometry to determine the concentration of proteins in a solution, as it is the most sensitive wavelength for detecting the presence of aromatic amino acids.

**The Absorption of Light at 280 nm by Proteins**

Proteins are composed of amino acids, which are the building blocks of these macromolecules. Among the 20 standard amino acids, there are a few that contain aromatic rings, namely tryptophan, tyrosine, and phenylalanine. These amino acids are unique because of their ability to absorb ultraviolet (UV) light. The absorption of light by these aromatic amino acids is due to the presence of conjugated double bonds within their aromatic rings, which allow for the transition of electrons to higher energy levels when they absorb photons of light.

The absorption maximum at 280 nm is primarily attributed to the presence of tryptophan, which has the strongest absorbance at this wavelength among the amino acids. Tryptophan's indole side chain has a high degree of conjugation, making it particularly effective at absorbing UV light. Tyrosine and phenylalanine also contribute to the absorbance at 280 nm, but to a lesser extent due to their lower molar absorptivities compared to tryptophan.

It is important to note that the absorbance at 280 nm is not solely due to the aromatic amino acids. The peptide bonds themselves can also absorb UV light, albeit at a different wavelength, around 200 nm. The absorption at 200 nm is due to the π to π* transitions within the peptide bond's amide group. However, the absorbance at this wavelength is less specific for proteins, as it can also be influenced by other components in the solution that contain peptide bonds or similar structures.

The use of 280 nm for protein quantification is advantageous because it is a wavelength where the contribution from nucleic acids is minimal. This allows for the specific measurement of protein concentration without interference from nucleic acids, which is particularly useful in applications such as the purification and analysis of proteins.

Moreover, the absorbance at 280 nm can also provide information about the protein's conformation. Changes in the protein's secondary or tertiary structure can affect the accessibility of the aromatic amino acids to the solvent, which in turn can alter the absorbance at 280 nm. This property is used in techniques such as circular dichroism (CD) spectroscopy to study protein folding and conformational changes.

In summary, the absorption of light at 280 nm by proteins is a complex process that involves the interaction of light with the aromatic amino acids and peptide bonds within the protein. This phenomenon is not only useful for protein quantification but also provides insights into the protein's structure and conformation.

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Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm. Amino acids with aromatic rings are the primary reason for the absorbance peak at 280 nm. Peptide bonds are primarily responsible for the peak at 200 nm.read more >>