How do xanthophylls protect lipid membranes from oxidative damage?

Here, we address the problem of the antioxidant activity of carotenoids in biomembranes. The activity of lutein and zeaxanthin in the quenching of singlet oxygen generated by photosensitization was monitored in lipid vesicles using a singlet oxygen-sensitive fluorescent probe and with the application of fluorescence lifetime imaging microscopy. The antioxidant activity of xanthophylls was interpreted on the basis of electron paramagnetic resonance oximetry results showing that xanthophylls constitute a barrier to the penetration of molecular oxygen into lipid membranes: to a greater extent in the 13-cis configuration than in all-trans. These results are discussed in relation to the trans-cis photoisomerization of xanthophylls observed in the human retina. It can be concluded that photoisomerization of xanthophylls is a regulatory mechanism that is important for both the modulation of light filtration through the macula and photoprotection by quenching singlet oxygen and creating a barrier to oxygen permeation to membranes

An Overview of Lutein in the Lipid Membrane

Lutein, zeaxanthin, and meso-zeaxanthin (a steroisomer of zeaxanthin) are macular pigments. They modify the physical properties of the lipid bilayers in a manner similar to cholesterol. It is not clear if these pigments are directly present in the lipid phase of the membranes, or if they form complexes with specific membrane proteins that retain them in high amounts in the correct place in the retina. The high content of macular pigments in the Henle fiber layer indicates that a portion of the lutein and zeaxanthin should not only be bound to the specific proteins but also directly dissolved in the lipid membranes. This high concentration in the prereceptoral region of the retina is effective for blue-light filtration. Understanding the basic mechanisms of these actions is necessary to better understand the carotenoid–membrane interaction and how carotenoids affect membrane physical properties—such as fluidity, polarity, and order—in relation to membrane structure and membrane dynamics. This review focuses on the properties of lutein

Azadioxatriangulenium:a long fluorescence lifetime fluorophore for large biomolecule binding assay

Of the many optical bioassays available, sensing by fluorescence anisotropy have great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is in the order of 20–200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatics dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecules assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immuniglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time by more than 75 %, and a change in the steady-state anisotropy increase of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay for detecting binding events involving biomolecules of far larger size than what is possible with the other red emitting organic dyes

Photothermal Microscopy: Imaging of Energy Dissipation From Photosynthetic Complexes

An idea of a photothermal imaging microscopy (PTIM) is proposed, along with its realization based on a dependence of fluorescence anisotropy of dye molecules on heat emission in their nearest vicinity. Erythrosine B was selected as a fluorophore convenient to report thermal deactivation of the excited pigment–protein complex isolated from the photosynthetic apparatus of plants (LHCII), owing to the relatively large spectral gap between the fluorescence emission bands of chlorophyll <i>a</i> and a probe. Comparison of the simultaneously recorded images based on fluorescence lifetime of LHCII and fluorescence anisotropy of erythrosine shows a high rate of thermal energy dissipation from the aggregated forms of the complex and, possibly, thermal energy transmission along the protein supramolecular structures. Relatively high resolution of this novel microscopic technique, comparable to the fluorescence lifetime microscopy, enables its application in a nanoscale imaging and in nanothermography

Single Molecule Detection Approach to Muscle Study: Kinetics of a Single Cross-Bridge During Contraction of Muscle

D166V point mutation in the ventricular myosin regulatory light chain (RLC) is one of the causes of familial hypertrophic cardiomyopathy (FHC). We show here that the rates of cross-bridge attachment and dissociation are significantly different in isometrically contracting cardiac myofibrils from right ventricle of WT and Tg-D166V mice. To avoid averaging over ensembles of molecules composing muscle fibers, the data was collected from a single molecule. Kinetics were derived by tracking the orientation of a single actin molecule by fluorescence anisotropy. Orientation oscillated between two states, corresponding to the actin-bound and actin-free states of the myosin cross-bridge. The cross-bridge in a wild-type (healthy) heart stayed attached and detached from thin filament on average for 0.7 and 2.7 s, respectively. In FHC heart, these numbers increased to 2.5 and 5.8 s, respectively. These findings suggest that alterations in myosin cross-bridge kinetics associated with D166V mutation of RLC ultimately affect the ability of a heart to efficiently pump the blood