Binding of 8-anilino-1-naphthalenesulfonate to lecithin:cholesterol acyltransferase studied by fluorescence techniques

The solvatochromic fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) has been used to study the hydrophobicity and conformational dynamics of lecithin:cholesterol acyltransferase (LCAT). The ANS to LCAT binding constant was estimated from titrations with ANS, keeping a constant concentration of LCAT (2 microM). Apparent binding constant was found to be dependent on the excitation. For the direct excitation of ANS at 375 nm the binding constant was 4.7 microM(-1) and for UV excitation at 295 nm was 3.2 microM(-1). In the later case, not only ANS but also tryptophan (Trp) residues of LCAT is being excited. Fluorescence spectra and intensity decays show an efficient energy transfer from tryptophan residues to ANS. The apparent distance from Trp donor to ANS acceptor, estimated from the changes in donor lifetime was about 3 nm and depends on the ANS concentration. Steady-state and time-resolved fluorescence emission and anisotropies have been characterized. The lifetime of ANS bound to LCAT was above 16 ns which is characteristic for it being in a hydrophobic environment. The ANS labeled LCAT fluorescence anisotropy decay revealed the correlation time of 42 ns with a weak residual motion of 2.8 ns. These characteristics of ANS labeled LCAT fluorescence show that ANS is an excellent probe to study conformational changes of LCAT protein and its interactions with other macromolecules.     

Förster energy transfer from nonexponentially decaying donors

Necessary modifications to the expression for the F?rster energy transfer rate are discussed when fluorescence decay of the donor in the absence of acceptor is nonexponential. Discrete and continuous models of the nonexponentiality are taken into account. No general solution of the problem is found. It is, however, suggested that in many of the biochemical problems the most appropriate modification of the transfer rate can be that which is based on the assumption of the same constant value of the radiative decay rate for all donor molecules. The effect of the assumed form of the F?rster energy transfer rate on the recovered values of the distance distribution and dynamics parameters of some exemplary bichromophoric systems is examined.     

Red blood cells do not attenuate the SPCE fluorescence in surface assays

We describe the positive effect of surface plasmon-coupled fluorescence emission (SPCE) on the detection of a signal from a surface immunoassay in highly absorbing or/and scattering samples. A model immunoassay using fluorescently labeled anti-rabbit antibodies that bind to rabbit immunoglobulin on a silver surface was performed, and the signal was detected in the presence of various highly absorbing and/or scattering solutions or suspensions, such as hemoglobin solution, plastic beads, and red blood cells. The results showed that a highly absorbing solution consisting of small molecules (dye, hemoglobin) attenuates the SPCE signal approximately 2-3-fold. In contrast, suspensions with the same absorption containing large particles (large beads, red blood cell suspension) attenuate the SPCE signal only slightly, approximately 5-10%. Also, a suspension of large undyed, highly scattering beads does not reduce the SPCE signal. The effects on the immunoassay signal of the sample background absorption and scattering, the size of the background particles, and the geometry of the experimental set-up are discussed. We believe that SPCE is a promising technique in the development of biosensors utilized for surface-based assays, as well as any assays performed directly in highly absorbing and/or scattering solutions without washing or separation procedures. Figure Red blood cells (unlike hemoglobin) do not attenuate the SPCE fluorescence in surface assays.     

Application of headspace solid-phase microextraction followed by gas chromatography-mass spectrometry to determine short-chain alkane monocarboxylic acids in aqueous samples

In this study, a procedure was developed to determine short-chain alkane monocarboxylic acids (SCMAs) in aqueous samples using headspace solid-phase microextraction (HS-SPME) followed by gas chromatography (GC) coupled with mass spectrometry (MS). A Stabilwax-DA capillary column (30 m × 0.32-mm inner diameter, 0.50-μm film thickness) was used for GC separation and a 60-μm poly(ethylene glycol) fiber was used to isolate SCMAs from water and introduce them into the gas chromatograph. Parameters of HS-SPME, analyte desorption, and GC-MS analysis were selected and an analytical procedure was proposed. Limits of quantitation were on the order of about 0.2 mg L^-1. As an example of the application of the procedure, SCAMs were determined in municipal wastewater at different steps of treatment.     

The lock-in and reorientational phase transitions in metallic superlattices

We show that in the metallic magnetic superlattices, along with the bilinear RKKY-like exchange, there is significant biquadratic contribution arising from intrinsic and fluctuational mechanisms. Within the phenomenological approach, we postulate a particular form of free energy density functional and study the thermodynamic behaviour of the system under consideration. The model permits a transparent interpretation of the phase transitions, observed in metallic multilayers, with multiple oscillation periods and biquadratic exchange effects taken into account.     

The impact of a carbon nanotube on the cholesterol domain localized on a protein surface

The influence of a single walled carbon nanotube on the structure of a cholesterol cluster (domain) developed over the surface of the endothelial protein 1LQV has been investigated using the classical molecular dynamics (MD) simulation technique. We have observed a substantial impact of carbon nanotube on the arrangement of the cholesterol domain. The carbon nanotube can drag out cholesterol molecules, remarkably reducing the volume of the domain that settles down on the protein.     

Fluorescence Anisotropy Controlled by Light Quenching*

We demonstrated that fluorescence anisotropy can be effectively decreased or increased in the presence of light quenching, depending on relative polarizations of excitation and quenching pulses. For parallel light quenching, anisotropy decreases to 0.103 and z-axis symmetry is preserved. In the presence of perpendicular light quenching, the steady-state anisotropy of a pyridine-2-glycerol solution increases from 0.368 for an unquenched sample to 0.484 for a quenched one. We show that the angular distribution of transition moments loses z-axis symmetry in the presence of perpendicular light quenching. In these cases we used more general definitions of anisotropy. Induced by light quenching, anisotropy can be applied in both steady-state and time-resolved measurements. In particular, the systems with low or no anisotropy can be investigated with the proposed technique.     

Distillation preconcentration and clean-up of aqueous samples for direct aqueous injection-gas chromatographic determination of volatile polar organic pollutants

The applicability of distillation to concentrate and clean up heavily loaded aqueous samples for the analysis of volatile polar organic compounds by means of direct aqueous injection-gas chromatography was studied. Recoveries for acetone, acetonitrile, acrolein, acrylonitrile, butanone, 1,4-dioxane, ethyl acetate, and 3-pentanone were in the range of 60.6 to 73.4% with relative standard deviations of 3.6 to 5.5%. The corresponding enrichment factors were in the order of 200. The recovery did not depend on the concentration in the studied range of 0.70 to 89 μg/kg. The detection limits with the mass selective detector operating in the selected ion monitoring mode were in the order of 0.1 μg/kg. The method was successfully applied to treated waste water from a pharmaceutical factory. The content of the above analytes in the real sample ranged from below 0.1 to ca. 83 μg/kg. Received: 16 December 1996 / Revised: 9 April 1997 / Accepted: 16 April 1997     

2′-Deoxyisoguanosine and Base-Modified Analogues: Chemical and Photochemical Synthesis

The synthesis of 2′-deoxyisoguanosine ( 2 ), and the pyrrolo[2,3-d]pyrimidine and pyrazolo[3,4-d]pyrimidine 2′-deoxyribonucleosides 3 and 4 is described. Condensation of the imidazole precursor 5 with benzoyl isocyanate followed by reaction with ammonia gave 2 . Its N(7) regioisomer was obtained from 6 . Compound 2 was also prepared by the photochemically induced conversion of 2-Chloro- and 2-bromopurine 2′-deoxyribofuranosides 9a and 10 , respectively, in aqueous solution, The photo reaction was further used for the synthesis of the compounds 3 and 4 starting with the amino-chloro-2′-deoxynucleosides 9b and 9c , respectively.     

Fluorescence intensity and anisotropy decays of the intrinsic tryptophan emission of hemoglobin measured with a 10-GHz fluorometer using front-face geometry on a free liquid surface

We measured the intensity and anisotropy decays of the intrinsic tryptophan emission from hemoglobin solutions obtained using a 10-GHz frequency-domain fluorometer and a specially designed cuvette which allows front-face excitation on a free liquid surface. The cuvette eliminates reflections and stray emissions, which become significant for low-intensity fluorescence such as in hemoglobin. Three lifetimes are detectable in the subnanosecond range. The average lifetime of hemoglobin emission is ligand dependent. The measured values of average lifetimes are 91, 174, and 184 ps for deoxy-, oxy-, and carboxyhemoglobin, respectively. Fluorescence anisotropy decays of oxy-, deoxy-, and carbonmonoxyhemoglobin can be fitted with up to three correlation times. When three components are used, the floating initial anisotropyr _o is, in each case, higher than the steady-state anisotropy of tryptophan in vitrified solution. For deoxy hemoglobin it is close to 0.4. The data are consistent with an initial loss of anisotropy from 0.4 to about 0.3 occurring in the first 2 ps.