Fluorescence nitroxide molecules

A new photochrome-fluorescence-spin method for the simultaneous quantitative analysis of the redox status and viscosity of a medium has been developed [20], The method of the viscosity measurement is based on the use of double fluorescence-nitroxide molecules. 

A problem of the experimental measurement of local polarity in the vicinity of donor and acceptor centers incorporated into a protein (bovine serum albumin, BSA) was solved with the use of the dual fluorescence-nitroxide probe (Bystryak et al., 1986 Rubtsova et al., 1993 Fogel et al., 1994 Likhtenshtein, 1993, 1996 Likhtenshtein et al., 2001). In such a hybrid molecule, the photoactive chromophore fragment in the excited singlet state can... 

Very fast electron transfers from P+ to bacteriochlorophyl (Bchl) and from (Bchl)- to QA do not depend on media dynamics and occur via conformationally non-equilibrium states (Fig.3.18). The dual fluorophore-nitroxide molecules (D-A) are also convenient objects for analysing the activity-dynamics relationship. The marked irreversible photoreduction of the nitroxide fragment of the dual probe incorporated into the binding site of HSA only took place when the nanosecond dynamical processes around the probe traced by ESR and fluorescence methods were detected (Rubtsova et al., 1993, Fogel et al, 1994 Likhtenshtein, 1986 Lozinsky et al., 2002). Similar results were reported for another model protein system, i.e. a-chymotrypsin with spin labeled methionin-92 groups (Belonogova et al., 1997). In the latter enzyme, the excited tryptophan group serves as an electron donor. 

Lozinsky E., Shames A., and Likhtenshtein G.I. (2001) Dual fluorophore-nitroxide molecules Models for study of intramolecular fluorescence quenching and novel redox probes. In Recent Research Development in Photochemistry and Photobiology., V. 2, Transworld Research Network. Trivandrum, pp. 41-55. 

The X-ray structure of zinc naphthalocyanate has been determined with Zn—N bond lengths of 1.983(4) A.829 Pentanuclear complexes with a zinc phthalocyanine core and four ruthenium subunits linked via a terpyridyl ligand demonstrate interaction between the photoactive and the redox active components of the molecule. The absorbance and fluorescence spectra showed considerable variation with the ruthenium subunits in place.830 Tetra-t-butylphthalocyaninato zinc coordinated by nitroxide radicals form excited-state phthalocyanine complexes and have been studied by time-resolved electron paramagnetic resonance.831... 

The physical basis of the second type of approach rests upon the effect of the local electrostatic potential upon dynamic interactions at encounters with charged quenching molecules resulting in fluorescence (phosphorescence) (Vogel et al., 1986 Anni et al., 1994) or between a stable radical, e.g. nitroxide, and another charged paramagnetic species (Likhtenshtein et al., 1972 Likhtenshtein, 1976, 1988,1993). In such cases, the relaxation parameters, i.e. the life-time of the fluorescence (phosphorescence) chromophore or spin-spin and spin-lattice relaxation rates of paramagnetic species are dependent upon the frequency of encounters, and, therefore, on local electrostatic fields... 

To determine the shape of the hydrophobic barrier of bilayer membranes, fatty acids and PC molecules spin labeled with nitroxides at various positions along the lipid chains were diffused into vesicles and their solvent-sensitive isotropic coupling constants were measured [54]. Results are plotted in Figure 5 in terms of distance of the probe from the bilayer center. Also shown is the profile of the dielectric constant along the membrane normal evaluated from the fluorescence lifetime distribution of fluorescence probes in PC liposomes [55]. These data correlate well with results from neutron diffraction studies that map the positional distribution of water and lipid moieties along the bilayer normal [56]. 

The fluorescence-photochrome technique was first applied to study the molecular dynamics of a stilbene fiuorescence-photochrome molecule, SITC, attached covalently to the terminal amino group of sperm whale myoglobin [18]. The same myoglobin residue was also labeled with a spin label, 4-iodoacetamide-TEMPO. The kinetics of the stilbene trans-cis photoisomerization (k pp) and the rotational diffusion frequency of nitroxide radicals (v ) was monitored by fiuorescence and E S R... 

Lipid peroxidation caused a decrease in phospholipid molecule mobility both in the region of polar heads and in the region of acyl chains till the depth of at least 1.7 nm from water-lipid interface (Panasenko et al. 1991). Under relative high levels of oxidation (>6 pmol malondialdehyde/g LDL phospholipid) the polarity of lipid phase increased. The decrease in efficiency of tryptophan fluorescence quenching by nitroxide fragments incorporated into hydrophobic regions at the depth of 2 nm from water-lipid interface indicated that lipid-protein interaction was disturbed as a result of oxidation of LDL lipids. 

Recent studies indicate that like the surfactant monomers themselves, the solubilized molecules are not rigidly fixed in the micelle but have a freedom of motion which is dependent to some extent on the solubilization site. In fact, as discussed in Chapter 3, solubilized probes are used as indicators of the fluidity of their micellar micro-environment. In particular, a decrease in the polarization of the fluorescent radiation from fluorescent probes located in the micellar core is indicative of the molecular motion of these probes. Similarly, tumbling of solubilized nitroxide probes proceeds at a more rapid rate than can be accounted for by simple rotation of the micelle itself indicating that the solubilized probe undergoes dynamic motion within the micelle. Such motion produces a characteristic hyperfine pattern on e.s.r. spectra. 

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