Lumiflavin was prepared by photolysis of an alkaline aqueous solution of riboflavin (Knowles and Roe, 1968). As a solvent we used triply distilled water, as buffers (0,2 M) (Schwabe, 1958): pH 1-3'7 sodium acetate, hydrochloric acid pH 3·7-5·6 sodium acetate, acetic acid pH 6-12 potassium dihydrogen phosphate, sodium hydroxide pH 13 potassium chloride, sodium hydroxide (E. Merck. Darmstadt, Germany).
RESULTSDependence of the lumiflavin triplet -triplet absorption spectrum on pH. Figure I shows the transient change in absorption when lumiflavin soJutions of pH = 2, pH = 7, and pH = 13 are flashed. From this the triplet-triplet absorption spectra of the acid (pH = 2), neutral (pH = 7), Kramer and Maute, 1972). The flash energy could be varied between 300 J and 900 J. The half duration of the flash was about 4-6 JlS (3001) and 6 JlS (900 J). The cuvette was surrounded by a Kodak-Wratten filter No. 2A which absorbs light of wavelength shorter than 400 nm. Using the flash apparatus described above (kinetic spectrophotometry) the spectra were recorded point-by-point at intervals of 10 nm (half-width value 2·5 nm) in the spectral region from 300 to 780 nm; the spectra were corrected for stray light.The high buffer concentration ensures that the protolytic equilibria in the triplet state are really established.Oxygen was removed by bubbling nitrogen (>99,9%) through the solutions for 45 min. The results with solutions treated in this way were identical to those with solutions degassed by the 'freezepump-and-thaw' procedure.The flash apparatus was the same as described earlier (Fischer, 81 S. SCHREINER, U. STEINER and H. E. A. KRAMER* Institut fuer Physikalische Chemie der Universitaet Stuttgart, Germany•Author to whom correspondence should be addressed.
DETERMINATION OF THE pK VALUES OF THE LUMIFLAVIN TRIPLET STATE BY FLASH PHOTOLYSISConsequently one has to assurne the existence of at least three different protolytic forms for the excited states, too. Kavanagh and Goodwin (1948) found a marked change of f1uorescence of lumiflavin and riboflavin in the regions of pH 2-4 and 9-10'5. The pK values of the lumiflavin triplet are still unknown. For f1avins, there are only a few investigations of the triplet state with respect to variation of the pH (Shiga and Piette, 1964; Lhoste et ai., 1966;Katan et al., 1971).In this paper we present the results of our investigations on the triplet-triplet absorption spectra of the acid ('LfH2+), the neutral ('LfH) and the basic ('Ln forms of the lumiflavin triplet as detected by flash photolysis.Abstract-The triplet-triplet absorption spectra in aqueous solution of the acid ('UH,+), the neutral ('UH) and the basic ('Ln forms of lumiflavin (6,7,9-trimethylisoalloxazine) were measured by flash photolysis. The pK. values of the corresponding protolytic equilibria of the lumiflavin triplet were found to be 4,4, ± 0·1 and 9·8 ± 0·15.
