Protein determination by UV absorption.
Protein Determination by UV Absorption
Near UV Absorbance
Near UV Absorbance is UV Absorbance at 280 nm: A simple spectrometer can quantify the amount of protein in a solution. Radiation absorption by proteins in the near UV depends on the content of Tyr and Tr (and to an extent on the amount of Phe and disulfide bonds). Thus the A280 varies greatly between different proteins (for a 1 mg/mL solution, from 0 up to 4 [for some tyrosine-rich wool proteins], although most values are in the range 0.5–1.5). This method has its advantages; it is simple, and the sample is recoverable. Be that as it may it also has disadvantages which include, interference from other chromophores, and the specific absorption value for a given protein must be determined. 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.
Far UV Absorbance
Absorption of the peptide bond is strongly in the far UV with a maximum at about 190 nm. The strong absorption of proteins at these wavelengths has been used in protein determination. Because of the difficulties caused by absorption by oxygen and the low output of conventional spectrophotometers at this wavelength, measurements are more conveniently made at 205 nm, where the absorbance is about half that at 190 nm. Most proteins have extinction coefficients at 205 nm for a 1 mg/mL solution of 30–35 and between 20 and 24 at 210 nm. Various side chains, including those of Trp, Phe, Tyr, His, Cys, Met, and Arg (in that descending order), make contributions to the A205. The advantages of this method include simplicity and sensitivity. The sample is recoverable and in addition there is little variation in response between different proteins, thus permitting near-absolute determination of protein. Disadvantages of this method include the necessity for accurate calibration of the spectrophotometer in the far UV. Many buffers and other components, such as heme or pyridoxal groups, absorb strongly in this region.
See also our Protein Concentration Protocol and Protein Concentration Protocols from other labs.
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