Extrinsinc fluorophores

These bind covalently to lysines and cysteines of proteins, and absorb and fluoresce in the visible. The fluorescence lifetimes of fluorescein and rhodamine are around 4 ns, and their emission spectra are not sensitive to medium polarity. 

5-((((2 iodoacetyl)amino)ethyl)amino)naphthalene-l-sulfonic acid (IAEDANS or 1,5-IAEDANS) and DNS bind covalently to proteins, while l-anilino-8-naphthalene sulfonate (ANS) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) bind non-covalently to proteins and membranes. Dissolved in a polar medium such as water, TNS and ANS show a very weak fluorescence that increases with decreasing medium polarity. When bound to proteins or to membranes, their fluorescence increases, and their maximum shifts to the blue edge. The intensity increase and the shift to shorter wavelengths are increasingly important with the decrease in binding-site polarity. Table 7.3 lists the characteristics of several extrinsic probes. 

Puric and pyrymidic bases show a weak fluorescence in aqueous medium, which increases by a factor of 10 in a medium of pH 2 and by a factor of 100 at 77 K. The quantum yield of the nucleic acids depends largely on the temperature, and their fluorescence lifetime is weak. Fluorescence polarization is high, even at ambient temperature. At a low pH, the emission maximum is temperature-dependent. 

Puric bases fluoresce at least three times more than pyrimidic bases. Guanidine is the most fluorescent basis. In the native state, DNA and RNA show a very weak fluorescence, 

Reproduced from Cantor, R.C. and Schimmel, P.R. (1980). Biophysical Chemistry, W.H. Freeman, New York. Fluorescence 

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