Fluorescence dichroism measurements

Amino acid sequences of eleven homologous sea anemone polypeptides have been elucidated. All possess three disulfide bonds. The six half-cysteine residues always occur in the same positions (7,8). Initial studies concerning the toxin secondary and tertiary structures relied upon circular dichroism, laser Raman, and, to a lesser extent, fluorescence spectral measurements (15—18). The circular dichroism spectra of the four toxins so far examined are essentially superimpos-able and thus indicate a common secondary structure. The only peak observed, a negative ellipticity at 203 nm, largely results from a non-regular ("random")... 

Ross WN, Salzberg BM, Cohen LB et al (1977) Changes in absorption, fluorescence, dichroism and birefringence in stained giant axons optical measurement of membrane potential. J Membr Biol 33 141-183... 

Figure 19.1. Schematic diagram of a general pump-probe-detect laser spectrometer suitable for picosecond electronic absorption, infrared (IR) absorption, Raman, optical calorimetry, and dichroism measurements. For picosecond fluorescence—a pump-detect method, no probe pulse needs to be generated.

Fluorescence measurements were performed with an SLM 8000 fluorometer. Quantum yields were obtained with quinine bisulfate as the standard (14,15). Excitation was at 272-280 nm, and the range of the integration used in the determination of 1/1(0) was 285-420 nm. Circular dichroism measurements were performed with a Jasco J-500C spectropolarimeter. 

The right panel in Figure 3 depicts 1/1(0) for (+)-catechin in mixtures of trifluoroethanol and 1-propanol that contain poly(L-proline) at a concentration of 0.90 mg/ml. In pure trifluoroethanol 1/1(0) is slightly less than one. At the opposite extreme of solvent composition, I/l(0) is greater than one and increases over a period of several days. The greatest sensitivity of 1/1(0) to solvent composition occurs near 25 75 trifluoroethanol 1-propanol, which is where the Form I - Form II interconversion is detected by circular dichroism. Measurements of the fluorescence of (+)-catechin in the mixed solvents, but in the absence of poly(L-proline), show no unusual dependence of emission on solvent. 

Apparatus. The spectrum and intensity of fluorescence were measured with a RF-540 spectrofluorometer (Shimadzu, Japan). An UV-260 (Shimadzu, Japan) spectrophotometer was employed in all absorption spectra recordings. The circular dichroism spectrometer JASCO J-810 (Hitachi. Japan) was used for measurement... 

Every property of an interface that can be optically probed can, in principle, be measured with the SFA. This may include information obtainable from absorption spectroscopy [55], fluorescence, dichroism, birefringence, or nonlinear optics [43], some of which have already been realized. 

Use of polarized light to excite fluorescence, and measurement of the state of polarization of the emitted light introduce another set of measurable parameters that can characterize structures and dynamics of molecules. The anisotropy of the polarization of fluoresence after excitation by linearly polarized light provides the rotational diffusion coefficient, or rotational correlation time, of the fluorophore. When there is fluorescence energy transfer, analysis of the anisotropy of both donor and acceptor can reveal the relative orientation, and the relative motion. Measurement of fluorescence after excitation by circularly polarized light provides the fluorescence-detected circular dichroism. This measurement characterizes the chiral environment of the ground state of the fluorophore. If the circular polarization of the fluorescence is measured, the circularly polarized luminescence is obtained. This measurement characterizes the chirality of the excited state. 

In conclusion, the method of fluorescence depolarization measurements on particles in compressed gels can be performed on chlorosomes and they lead to results, which are in good agreement with the theoretical predictions from [1], and results fromt linear dichroism measurements [2]. The results support the further use of the expressions in... 

The efficiency of energy transfer in photosynthesis depends on the relative orientations of the emission and absorption transition moments respectively of the donor and acceptor chlorophyll molecules. This is determined by the mutual orientation of the chromophores within the structure of the photosynthetic membranes. In order to gain a better insight into the molecular mechanism of energy transfer, it is necessary to characterize the directions of the transition moments in the frame of the chromophores. This can be achieved by combining the results of angle - resolved linear dichroism and fluorescence depolarisation measurements on macroscopically oriented lipid membranes with the results of emission-resolved fluorescence anisotropy data from isotropic systems (vesicles) (1). 

Figure 4. Order parameters S of chlorophyll a in DGDG membranes (1 250) as a function of the wavelength of excitation, as found from linear dichroism experiments. The continuous line is a theoretical fit based on fluorescence anisotropy measurements in castor oil.

Transketolase from Baker s yeast, easily split into TPP and apoenzyme, consists of 2 identical subunits of 70000 daltons. 2 TPP molecules are bound per mole of enzyme in a reversible manner. All coenzyme analogs tested turned out to be inhibitory to the enzymatic activity. A very high affinity was found for oxi-TPP and tetrahydro-TPP. There is evidence for a charge transfer complex between a tryptophan residue of apo-transketolase and TPP from fluorescence quenching and circular dichroism measurements. The chemical modification of the tryptophan residue at the coenzyme binding site supports this concept. The decrease of the magneto circular dichroism band at 292 nm after the reaction of apo-TK with the dimethyl (-2-hydroxi-5-nitrobenzyl) sulfonium chloride is regarded as direct evidence for the modification of a tryptophan residue. 

As described in section 4.3.2.3 the angle 0 between long molecular axis and surface normal can be determined from the dichroism of the first absorption band. Good quality films show 0 < 20°. This means that the molecules form a pin cushion type of aggregate. Also from fluorescence anisotropy measurements a high degree of order can be evaluated [172]. 

Thus, in our experiments, the orientation observed through fluorescence polarization is not directly related to the complete true stress, but depends on the deformation in the rubbery plateau zone. This result is rather different from that uhich is obtained with birefringence measurements in which stress and orientation are related through a constant coefficient. Also, infra-red dichroism measurements performed in our laboratory with films issued from the same polystyrene batch lead to the... 

Although it could be argued that this effect is specific to fluorescence polarization and originates from the perturbation introduced by the fluorescent label, the fact that the fluorescence polarization behaviour observed at 128 5 C for PAP 287 in a matrix of M = 200,000, is similar to that found from infra-red dichroism measurements for the matrix itself indicates that the contribution from the anisotropic medium is also involved to some extent in infra-red dichroism. Indeed the statistical unit of a polymer chain corresponds to an anisotropic object which can also be oriented by interaction with the strained surroundings. 

Unlike CD, the measurement of CPL is still mainly dependent on the use of custom-made instruments that have been designed, developed and improved by a limited number of research groups around the world over the last three decades [36,38,41,42,45,52,59-63]. However, the growing interest in developing chiral luminescent probes, and in particular Ln(in)-based systems, has resulted in the advertising and some availability of commercial instrumentation. The first commercial CPL spectrometer, which essentially consists of two CD spectrometers with the second one used as the emission spectrometer, was manufactured by JASCO Inc., the JASCO CPL-200. More recently, the company OLIS Inc. developed its Polarisation Toolbox to support fluorescence, polarisation of fluorescence, anisotropy, CPL, CD, and FDCD (fluorescence detected circular dichroism) measurements for its CD instrumentation. As of Febmary 2012, only CPL-related studies using the JASCO CPL-200 instrument have appeared in the hterature [64-67]. 

Figure 11 Order parameter (P2) as measured by fluorescence emission (filled circles) and absorption dichroism (open squares) and order parameter

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