Ultraviolet Absorbance of Aromatic Amino Acids - Commonly used protein assay methods
Reference: E. Layne. Methods in Enzymology 3: 447 (1957)
Quick Guide
How does it work?
- Monitors the absorbance of aromatic amino acids, tyrosine and tryptophan or if the wavelength is lowered, the absorbance of the peptide bond. Higher order structure in the proteins will influence the absorption
Detection Limitations
- 20 µg to 3 mg
Advantages
- Quick
- Sample can be recovered
- Useful for estimation of protein before using a more accurate method
- Well suited for identifying protein in column fractions
Disadvantages
- Highly susceptible to contamination by buffers, biological materials and salts
- Protein amino acid composition is extremely important, thus the choice of a standard is very difficult, especially for purified proteins
- Absorbance is heavily influence by pH and ionic strength of the solution.
General Considerations
- This is often used to estimate protein concentration prior to a more sensitive method so the protein can be diluted to the correct range
Quantitative Procedure
- Zero the spectrophotometer with a buffer blank
- Make a standard curve using your standard of choice in the expected concentration range, using the same buffer that your unknown sample is in.
- Take the absorbance values at 280 nm in a quartz cuvette
- Place sample into quartz cuvette (make sure concentration is in the range of 20 µg to 3 mg
- Take absorbance at 280 nm
Estimation Procedure
- Zero spectrophotometer to water (or buffer)
- Take the absorbance at 280 nm in a quartz cuvette
- Change wavelength to 260 nm and zero with water (or buffer)
- Take absorption at 260 nm in a quartz cuvette
- Use the following equation to estimate the protein concentration
[Protein] (mg/mL) = 1.55*A280 - 0.76*A260
Discussion
Determination of protein concentration by ultraviolet absorption (260 to 280 nm) depends on the presence of aromatic amino acids in proteins. Tyrosine and tryptophan absorb at approximately 280 nm. Higher orders of protein structure also may absorb UV light or modify the molar absorptivities of tyrosine and tryptophan and thus the UV detection is highly sensitive to pH and ionic strength at which measurement is taken. Many other cellular components, and particularly nucleic acids, also absorb UV light. The ratio of A280/A260 is often used as a criterion of the purity of protein or nucleic acid samples during their purification. The real advantages of this method of determining protein concentration are that the sample is not destroyed and that it is very rapid. Although different proteins will have different amino acid compositions and thus different molar absorptivities, this method can be very accurate when comparing different solutions of the same protein.
To make an accurate determination of protein concentration, you will have to produce a standard curve (A280) with known amounts of purified protein. You will also have to provide a blank that is appropriate for the sample and contains the same concentrations of buffer and salts as the sample. It is often convenient to dialyze the sample and measure the absorbance of the retentate (still in the dialysis sack) using the dialysate as the blank. Care must be taken to use quartz cuvettes, since glass absorbs UV light. A handy equation to estimate protein concentration that is often used is
[Protein] (mg/mL) = 1.55*A280 - 0.76*A260
However, it is also a good idea to always use a standard curve and suggested that you evaluate the agreement of the results using the above equation with results using a standard curve.
This method is the least sensitive of the methods discussed here. For increased sensitivity, the wavelength can be lowered to the range of 210 to 225 nm. This measures the amide bond in proteins. However it is much more subject to interference from many more biological components and compounds used to make buffer solutions.
If you don't know what the protein concentration of an unknown sample is likely to be, the ultraviolet method might be a good starting point. Prepare a standard curve for the absorbance at 280 and 260 nm. After you have the data for the standard curve, rezero the spectrophotometer with water. Place your samples into a dry 1 mL quartz cuvette and read the absorbance. If the A280 of your unknown sample is less than 2, you should probably not dilute your sample further. If the absorbance is >2, dilution will be required. When you are finished with the first measurement, the unknown can be returned to its original tube with minimal loss.
Comments
Post a Comment