S.Pakalnis, V.Sitas, H.Schneckenburger, R.Rotomskis
Picosecond absorption spectroscopy of biologically active pigments NADH, FMN and fluorescence marker Rhodamine-123

A variety of organic chemicals has been found to be useful in photobiology, either as fluorescent probes for investigations of specific biological sites, or as photosensitizers, where they are initiating specific chemical reactions resulting in therapeutic effects. After the discovery of Rhodamine-123 (Rh-123) as a specific fluorescent marker for mitochondria [1,2] and its activity of photosensitization [3,4], the nature of the primary photochemical processes and action mechanisms of Rh-123 in vivo still are not completely elucidated [5-7]. Moreover, microscopic energy transfer spectroscopy in vitro was established using mixed solutions of reduced nicotinamide adenine dinucleotide (NADH) and the mitochondrial marker Rh-123 [8]. NADH and flavin molecules are both involved in redox reactions occuring within the cytoplasm or organelles, e.g., mitochondria. If electron transfer within the respiratory chain, which is located at the inner mitochondrial membrane, is inhibited, oxidation of NADH and flavins is impeded. Thus, the respiratory function is related to the redox state of NADH and flavin molecules, whose fluorescence intensity is dependent on oxygen. However, the fluorescence intensity changes are relatively small due to superposition of fluorescence bands of mitochondrial NADH with cytoplasmatic NADH and nicotinamide adenine dinucleotide phosphate. Therefore, an almost selective method for detection of mitochondrial NADH was developed utilizing energy transfer from excited NADH to acceptor molecules - Rh-123 selectively accumulated in mitochondrial membranes. To achieve a complete understanding of the functions of Rh-123 in biological objects, it is essential to investigate the relaxation of excitation energy in biopigments (NADH and flavin mononucleotide (FMN)) as well as the mitochondrial marker Rh-123. In this paper we report on a direct study of the excited state dynamics of NADH, FMN, and Rh-123 in aqueous solutions by means of picosecond transient absorption spectroscopy.

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