DETERMINATION OF THE pk VALUES OF THE LUMIFLAVIN TRIPLET STATE BY FLASH PHOTOLYSIS

Abstract

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.

Distinction of 2e- and le- Reduction Modes of the Flavin Chromophore as Studied by Flash Photolysis

“…Keeping these precautions in mind we found that flashing 15 pM flavin for microseconds lead to 100% foi-mation of flavoquinone triplet (3Fl$x) at pH > 6 (A, , , = 690 nm, E = 4400 cm-I M-') and to the conjugate acid ('HFl : : ) at pH < 5 (A, , , = 670 nm, E = 4800 cm-' M-l) [13,17]. The entire transfer of flavin into the excited triplet state results from multiple excitation of FI,, by the highly intense flash light, since the actual lifetime of the excited flavin singlet (zf = 6.9 ns for 3-methyllumiflavin [18]) is much shorter than the flash duration ( Fig.…”

“…The common feature in light-excited and dehydrogenase flavins is apparently the induction of basic character either by an auxiliary basic protein group [30,31] or by the intrinsic basicity generated in the triplet 3Fl,*, [13] which both help to split a substrate KH into R -and H', formally.…”

“…Schreiner et al [13]) and the corresponding flavosemiquinone radical HF1 (I. = 580 nm, E = 5000 cm-' M-' ; cf.…”

“…It turns out that, roughly speaking, there is an equal chance of hydride uptake at C-9 and other positions. Since a pyridine nitrogen atom such as N-5 is, even in the very weakly basic ground state [29], not a good candidate for nucleophilic attack and since its basicity is even higher in the excited triplet state (pK z 5 [13]), we are led to assume that the residual half of the reaction will run through a C-4a intermediate and subsequent 4a, 1 prototropy.…”

Repair of UV Light Induced DNA Lesions: A Comparative Study with Model Compounds

“…This high pH values (pH > 10). [55] Under these conditions, significant deprotonation of the dimer unit occurs (pK a ϭ 10.7), [56] quantum efficiency allowed the group to obtain a pH profile of the chain reaction. This study revealed half maximum which led to the proposal that electron transfer from the deprotonated dimer to the oxidized, light-excited flavin dimer cleavage at pH ഠ 7.5 and maximum splitting efficiency at pH ഠ 8.5.…”