Photophysical Studies on the Binding of Tetrasulfonatophenylporphyrin to Lens Proteins

Abstract

Previous studies have shown that mesotetra(p-sulfonatophenyl)porphine (TPPS) binds to lens proteins. This characteristic should increase the residence time of the sensitizer in the lens and therefore enhance the probability of inducing photooxidative damage to that tissue in vivo. Subsequent in vivo studies have verified that contention. The present studies were performed to determine the effect of such binding on the spectroscopy and photophysics of the porphyrins. It was found that the binding of TPPS (1) quenches the fluorescence of lens proteins, (2) causes a shift in the ground state absorption spectra, fluorescence excitation spectra and the triplet excited state spectrum of TPPS to longer wavelengths and (3) results in an increase in the triplet state lifetime of TPPS. In the presence of the isolated crystallins the average triplet lifetime increases in the following order: gamma less than beta less than alpha.

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“…2. The phenomenon of decreased intensity of the triplet excited absorbance of a photosensitizer in the presence of crystallin proteins has been observed previously ( 44 ). Normalizing the decay traces to the intensity at time 0 removed the effect of scattering observed in Fig.…”

“…The lifetimes (s −1 ) are the inverse of the observed decay rate (s −1 ). This nonlinear triplet lifetime extension is not unusual; other dyes have longer‐lived excited states when complexed with various biological macromolecules presumably because they can exclude O 2 , which would otherwise quench the triplet state of the sensitizer ( 44–47 ).…”

“…2 data are not background‐corrected. We chose to plot the actual decay traces rather than the background‐corrected decay traces because the decrease in total absorbance intensity has been observed in a very similar system by Roberts and co‐workers ( 44 ) while examining meso tetra( p ‐sulfonatophenyl)porphine (TPPS) and lens crystallins. The data are well fit to a monoexponential function as shown by the residuals of the curve fits in Fig.…”

“…Lakowitz and Weber ( 46 ) demonstrated that the fluorescence lifetime of ethidium bromide was dramatically (up to two orders of magnitude) increased when complexed with deoxyribonucleic acid. Roberts et al ( 44 ) suggested that the macromolecule surrounding the triplet excited dye locally blocks the transport of oxygen, producing hypoxic regions at the dye, thereby extending the triplet state lifetime of the dye, and other investigators have shown this phenomenon with various proteins and photosensitizers ( 45,47 ). This hypoxia is created by sensitizing the locally available O 2 to 1 O 2 , which reacts with the protein, leaving no free molecular oxygen.…”

“…This hypoxia is created by sensitizing the locally available O 2 to 1 O 2 , which reacts with the protein, leaving no free molecular oxygen. More time is necessary for O 2 to diffuse into the local environment of the sensitizer (where it can quench the excited state), thereby extending the triplet excited state of the dye ( 44,45,47 ).…”