Phosphorescence of protein-bound eosin and erythrosin. A possible probe for measurements of slow rotational mobility.

We used a pulsed dye laser working at 540 nm to excite triplet-state formation of eosin and erythrosin, either bound or unbound to bovine serum albumin, in aqueous solution anaerobically at pH 8 and 20-22 degrees C. Delayed emission from radiative transitions of the triplet state was readily detectable, both as delayed fluorescence and as red phosphorescence. Detection of the triplet state by measurement of phosphorescence at 645 nm upwards was at least 100-fold more sensitive than by absorbance measurements of ground-state depletion at 500 nm. When immobilized in poly(methyl methacrylate), the phosphorescence of eosin and erythrosin was polarized with an anisotropy parameter [Jablonski (1961) Z. Naturforsch. A16, 1-4] of about 0.25. The phosphorescence of erythrosin is sufficiently intense to be distinguishable from the long-wavelength end of fluorescence under conditions of continuous rather than pulsed excitation. Our observations suggest that phosphorescence depolarization of eosin or erythrosin probes could be used as a highly sensitive method of measuring rotational relaxation times in region from 10(-5) to 10(-3) s, such as those of the uniaxial rotation of membrane proteins.