Time-resolved fluorescence depolarization of rhodamine B and octadecylrhodamine B in Triton X-100 micelles and Aerosol OT reversed micelles.
ABSTRACT Time-resolved fluorescence and fluorescence anisotropy experiments were conducted on rhodamine B and octadecylrhodamine B incorporated into aqueous Triton X-100 micelles and in sodium bis(2-ethylhexyl) sulfosuccinate entrapped water or glycerol in a hydrocarbon solvent (heptane or dodecane). The time-resolved fluorescence behavior of the dye molecules in the micellar media was compared with that of the dye in homogeneous solution, from which a qualitative estimate of the polarity of the probe environment in the micelles could be inferred. The anisotropy decay of the fluorescent probes was analyzed with a biexponential decay model yielding correlation times characteristic for overall and internal micellar motion. The overall micellar rotation could be clearly distinguished from the faster internal motion in small water droplets and in glycerol droplets in heptane, for which there is good agreement between calculated and observed micellar rotation times. The hydrodynamic radii of glycerol droplets in dodecane medium are larger than the corresponding radii of droplets in heptane.
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- "All other conditions are as in Fig. 2. See ''Materials and methods'' for other details problems in biology including their application as reaction media (Khmelnitsky et al. 1989; Carvalho and Cabral 2000) and in protein biotechnology (Melo et al. 2001). They offer certain inherent advantages in fluorescence studies over membranes since reverse micelles are smaller and optically transparent, have well-defined sizes, and are relatively scatter-free (Laane and Visser 1987; Visser et al. 1988). Reverse micelles represent a type of organized molecular assembly which offers the unique advantage of monitoring dynamics of molecules with varying states of hydration that is difficult to achieve with complex systems such as membranes. "
ABSTRACT: Tryptophan octyl ester (TOE) represents an important model for membrane-bound tryptophan residues. In this article, we have employed a combination of wavelength-selective fluorescence and time-resolved fluorescence spectroscopies to monitor the effect of varying degrees of hydration on the dynamics of TOE in reverse micellar environments formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. Our results show that TOE exhibits red edge excitation shift (REES) and other wavelength-selective fluorescence effects when bound to reverse micelles of AOT. Fluorescence parameters such as intensity, emission maximum, anisotropy, and lifetime of TOE in reverse micelles of AOT depend on [water]/[surfactant] molar ratio (w (o)). These results are relevant and potentially useful for analyzing dynamics of proteins or peptides bound to membranes or membrane-mimetic media under conditions of changing hydration.European Biophysics Journal 01/2006; 35(1):62-71. DOI:10.1007/s00249-005-0009-7 · 2.47 Impact Factor
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ABSTRACT: The subnanosecond fluorescence and motional dynamics of the tryptophan residue in the bacteriophage M13 coat protein incorporated within pure dioleoylphosphatidylcholine (DOPC) as well as dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) and dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bilayers (80/20 w/w) with various L/P ratios have been investigated. The fluorescence decay is decomposed into four components with lifetimes of about 0.5, 2.0, 4.5 and 10.0 ns, respectively. In pure DOPC and DOPC/DOPG lipid bilayers, above the phase transition temperature, the rotational diffusion of the protein molecules contributes to the depolarization and the anisotropy of tryptophan is fitted to a dual exponential function. The longer correlation time, describing the rotational diffusion of the whole protein, shortens with increasing temperature and decreasing protein aggregation number. In DMPC/DMPG lipid bilayers, below the phase transition, the rotational diffusion of the protein is slowed down such that the subnanosecond anisotropy decay of tryptophan in this system reflects only the segmental motion of the tryptophan residue. Because of a heterogeneous microenvironment, the anisotropy decay must be described by three exponentials with a constant term, containing a negative coefficient and a negative decay time constant. From such a decay, the tryptophan residue within the aggregate undergoes a more restricted motion than the one exposed to the lipids. At 20C, the order parameter of the transition moment of the isolated tryptophan is about 0.9 and that for the exposed one is about 0.5.European Biophysics Journal 05/1990; 18(5):285-293. DOI:10.1007/BF00188041 · 2.47 Impact Factor
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ABSTRACT: A highly fluorescent anthraniloyl (Ant) group was covalently attached to the active site of α-chymotrypsin (CT), probably at Ser195. Ant-CT is stable at neutral pH for months, allowing a detailed fluorescence study of Ant-CT as a model protein to investigate its physical properties in 0.1 M Tris/HCl, pH 8.2, and in reversed micelles of n-octane, 0.1 M Tris/HCl, pH 8.2, and sodium bis(2-ethylhexyl)sulfosuccinate (AOT). Steady-state fluorescence measurements of the progressive red-shift of the center of gravity of the emission band as function of degree of hydration, wo, defined as [H2O]/[AOT], indicate that the average polarity in the vicinity of the probe is approaching that of bulk water at wo > 12. Time-resolved fluorescence measurements of Ant-CT in water and in reversed micelles showed that the active site has different properties in reversed micelles compared to those in water. Some specific changes at very low water content (0.6 < wo < 5) can be observed, which correlate with enzyme activity measurements in the same wo region (unpublished results). These effects are, for instance, significant changes in the average fluorescence lifetime and the internal flexibility of the probe. The overall rotational-correlation time of the enzyme in AOT reversed micelles seems to be independent on wo (5 < wo < 29), which suggests that the enzyme creates its own micelle.12/1992; 211(1‐2):47 - 55. DOI:10.1111/j.1432-1033.1993.tb19868.x