Ultraviolet Absorbance of Protein
Ultraviolet absorbance image of a protein crystal in solutionUltraviolet Absorbance Microscope
Jablonski Energy Level Diagram Depicting Absorbance and Fluorescence Transitions
Protein, including that in tissues and protein crystals, absorbs ultraviolet light quite strongly. Rather, it is the amino acids that make up the proteins that absorb the UV light. The strong absorbance of UV light by protein allows for rapid analysis of protein samples, including protein crystals, by microscopy and microspectroscopy.
Commonly, the optical absorbance of protein is measured at 280 nm. At this wavelength, the absorbance of protein is mainly due to the amino acids tryptophan, tyrosine and cysteine with their molar absorption coefficients decreasing in that order. Of course, the molar absorption coefficient of the protein itself at 280 nm will depend upon the relative concentrations of these three amino acids. Therefore, different proteins can have different absorption coefficients and even the wavelength of the maximum absorbance may differ. This fact can be used to help identify different types of proteins by relatively fast and simple optical tests.
Imaging Protein by UV Absorbance
Most commonly, protein crystals are imaged by their intrinsic protein fluorescence. This is mostly the fluorescence of tryptophan. As such, protein fluorescence requires very powerful ultraviolet light sources and very sensitive cameras because the fluorescent emission from proteins is so weak. However, the powerful UV light source can destroy the protein due to the long exposures required.
A much faster way to imaging proteins, either in cells, tissues or as crystals, can be done by using their strong absorbance of UV light as a contrasting mechanism. By using a ultraviolet microscope or microspectrophotometer equipped for UV imaging, the sample containing the protein is imaged with 280 nm light. The protein will absorb this light more strongly than the surround sample and will appear darker. See the picture above for an example of UV absorbance of a protein crystal in salt solution. This technique is very fast, exposing the protein to UV light for far less time.
Spectroscopy of Protein by UV Absorbance
CRAIC Technologies microspectrophotometers are used to acquire spectra of microscopic samples containing protein, such as individual protein crystals, by their UV absorbance. The microspectrophotometer consists of a UV-visible-NIR range microscope integrated with a spectrophotometer. As such, it is able to measure the UV-visible-NIR spectra of microscopic samples of tissue, protein crystals and other protein contain structures. By using absorbance, it is able to measure these samples quickly and non-destructively.
Microspectroscopy allows the scientist to learn more about the optical features and the chemical structure of the protein. Microspectroscopy also allows for the determination of the concentration of protein in a sample as the absorbance at 280 nm is proportional to the protein concentration.
If the protein sample does not have tryptophan or tyrosine, which absorb at 280 nm, it can easily measure the concentration by the Scopes Method. In this method, the protein concentration is determined by the absorbance at 205 nm in which the peptide bonds are analyzed directly.
DNA or RNA purity can also be determined by measuring the absorbance ratios of 260 to 280 nm. This is because the nucleic acids that make up DNA and RNA absorb strongly at 260 nm. A ratio of about 2.0 is considered "pure" for RNA while a ratio of about 1.8 is considered "pure" for DNA. Lower ratios indicate the presence of protein.
Protein absorbs strongly at 280 nm due to a number of its constituent amino acids. The peptide bonds found in the amino acids also absorb at 205 nm. The UV absorbance of protein can be used both to quickly image and acquire spectra of microscopic samples non-destructively. The spectra can also be used to determine protein concentrations and the relative amounts of protein to DNA or RNA.