Amino Acids. Commonly, the optical absorption of proteins is measured at 280 nm. At this wavelength, the absorption of proteins is mainly due to the amino acids tryptophan, tyrosine and cysteine with their molar absorption coefficients decreasing in that order.
Interpreting Nanodrop (Spectrophotometric) Results, Interpreting Nanodrop (Spectrophotometric) Results, Measuring protein concentration using absorbance at 280 nm, Nucleic acid quantitation – Wikipedia, 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. Secondary, tertiary, and quaternary structure all affect absorbance, therefore factors such as pH, ionic strength, etc. can alter the absorbance spectrum.
The ratio of absorbance at 260 nm vs 280 nm is commonly used to assess DNA contamination of protein solutions, since proteins (in particular, the aromatic amino acids) absorb light at 280 nm. [2] [6] The reverse, however, is not true — it takes a relatively large amount of protein contamination to significantly affect the 260:280 ratio in a nucleic acid solution.
Summary. Proteins absorb strongly at 280 nm due to three types of its constituent amino acids. The peptide bonds found in the amino acids also absorb at 205 nm. The UV absorption of protein can be used both to quickly image and acquire spectra of microscopic samples non-destructively.
The extinction of nucleic acid in the 280 -nm region may be as much as 10 times that of protein at their same wavelength, and hence, a few percent of nucleic acid can greatly influence the absorption. METHOD. 1) Centrifuge non clear protein solutons for 5 minutes, 14000 rpm, prior to taking any readings.
