|
Advantages of Ultraviolet Absorbance of Protein
Ultraviolet absorbance image of a protein crystal in solution Intrinsic Fluorescence of Protein Science of Microspectrophotometers Design of Microspectrophotometers Ultraviolet Absorbance Microscope Ultraviolet Absorbance Microspectrophotometer
Absorbance microspectra™ of a protein crystal
There are two main methods for detection of proteins and protein crystals. One is to image or acquire spectra of the intrinsic fluorescence of the proteins. The second is to image or acquire the absorbance spectra of the proteins. Of the two, the second is better especially for the identification and analysis of protein crystals. 1. Speed Protein usually absorbs strongly at 280 nm. This means that images and spectra can be acquired quickly. On the other hand, the intrinsic fluorescence of proteins is weak meaning that exposures must be long. 2. Damaging UV light Weak fluorescence means that protein samples must be exposed to damaging UV light for longer periods of time. Absorbance imaging and spectroscopy is completed rapidly so UV exposure is short and limited. 3. Tryptophan required for fluorescence The residue tryptophan has the highest quantum yield of the amino acids that fluoresce. If it is not present in a high enough concentration, there is no detectable fluorescence. With absorbance, tryptophan is just one amino acid that absorbs. Additionally, the peptide bonds between the amino acids also absorb! 4. Fluorescence can be quenched Impurities, other amino acids, even the proteins structure can conspire to quench fluorescence. 5. Contaminants easily distinguished Salt crystals and other contaminants are easily and rapidly distinguished from protein crystals by UV absorbance imaging and spectroscopy. 6. Determination of protein concentration UV absorbance can be used to determine the concentration of the protein in a crystal. 7. Analysis of DNA and RNA UV absorbance can be used to analyze DNA and RNA samples and crystals. Neither DNA nor RNA fluoresce. 8. Determining the contamination of DNA and RNA samples The contamination of DNA or RNA crystals by protein can be rapidly and safely measured in a non-destructive fashion. This is not possible using protein fluorescence.
|
