FLUOROMETRIC ANALYSIS OF THE LONG‐WAVELENGTH ABSORPTION BAND OF N‐7 METHYLATED GMP INTO THE CONSTITUENT BANDS OF THE TWO ELECTRONIC STATES

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Abstract
Steady-state measurements of fluorescence anisotropy are used to resolve the long-wave-length absorption spectrum of 7-methyl guanosine 5''-monophosphate (GMP) in pH 5 buffer at room temperature into component spectra that correspond to the electronic transitions I and II present in that spectral region. We have chosen this derivative of guanine because its fluorescence quantum yield is much greater than that of GMP. It is found that the data are adequately described by a model that involves emission exclusively from state I, with state II converting to it with 100% efficiency. The shape of the absorption spectrum of state II is virtually independent of the angle .theta. between the absorption transition dipole moments of states I and II, whereas that of state I is dependent on .theta.. We analyze the data on the basis of the premise that in the short-wavelength region state II is the predominantly absorbing state. This premise is based on studies of single-crystal polarized reflection and linear dichroism from stretched films. The spectral maxima for the two states are found to be at about 290 and 260 nm, respectively. There is also a weak band which is centered at about 245 nm. The oscillator strengths are found to be 0.07, 0.21 and .apprx. 0.04, for states I, II and that associated with the weak band, respectively. The importance of these findings with regard to the photophysical properties of nucleic acids and calculations of their CD spectra is discussed.