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Abstract  

Bovine serum albumin (BSA) is a soft globular protein that undergoes conformational changes through several identified transition steps in the pH range 2–13.5. The ability to change conformation makes BSA capable of complexing different ligands from fatty acids to cations or drugs and carries them in the bloodstream. Microcalorimetric titration of BSA with NaOH solution was performed to measure the enthalpy of conformational changes. Two exothermic enthalpy changes were found in the course of the titration between pH 3 and 9.5, which can be identified with the E–F, and the F–N transitions. The enthalpy change at pH 3.5 (transition from the E to the F form of BSA, folding of intra-domain helices in domain I) is independent of the protein concentration. The second transition (F–N, folding of domain III) was observed at pH 4.8 for the 0.1% BSA solution, but it shifted to higher pH values as the protein concentration increased to 0.2% and 0.3%. The tightening of the protein structure with increasing pH was verified measuring intrinsic fluorescence of tryptophan residues. At even higher pH value, pH 10.5, fluorescence measurements revealed protein expansion. The BSA conformational changes were also measured by dynamic light scattering. The hydrodynamic diameter was smaller at the i.e.p. of BSA (5–7 nm at pH ~5) and larger at the two ends of the pH range (17.5 nm at pH 2 and 8.3 nm at pH 10).

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A sensitive and reproducible high-performance liquid chromatographic (HPLC) method for determination of talinolol (TAL) in rat plasma was developed and validated using pindolol (PIN) as an internal standard. Both TAL and PIN were separated on a Zorbax Eclipse XDB C18 column by gradient elution with 0.1% aqueous formic acid and acetonitrile as the mobile phase. Detection was performed using fluorescence measurement at λ ex 249 nm and λ em 333 nm. The method was validated and found to be linear in the range of 10–2000 ng mL−1. The limit of quantification was 10 ng mL−1 based on 100 μL of plasma. The variations for intra- and inter-day precision were less than 10%, and the accuracy values were between 92% and 102.9%. The extraction recoveries were more than 82%. The assay was successfully applied to an in-vivo pharmacokinetic study of TAL in rats that were administered a single oral dose of 10 mg kg−1 TAL. The maximum concentration (C max) and the area under the plasma concentration-time curve (AUC0–12) were 0.369 ± 0.024 μg mL−1 and 1.429 ± 0.027 μg h mL−1, respectively. The modulatory effect of apricot juice on P-glycoprotein-related efflux carriers was also investigated. Co-administration of apricot juice resulted in a significant increase of the amount of TAL in plasma (C max and AUC0–12 were 0.679 ± 0.021 and 2.357 ± 0.079, respectively; p < 0.05).

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Rapid analysis by coupling HPTLC with bioluminescence and mass spectrometry enables very fast response to bioactive substances in unknown samples. In this study marine sponges were screened for new bioactive compounds. After chromatographic separation of twelve methanolic marine sponge extracts the HPTLC plates were coated with bioluminescent bacteria ( Vibrio fischeri ) by a simple dipping procedure. If separated compounds were bioactive they inhibited or enhanced the bacterial luminescence and could be identified as dark zones on the luminescent background. This micro-biological detection revealed new compounds compared with physical (absorbance or fluorescence measurement) or chemical (microchemical derivatization) detection techniques. Effect-directed analysis turned out to be superior to target analysis in the search for natural products with a distinct effect. For identification of unknown bioactive zones the HPTLC system was coupled to a high-resolution mass spectrometer to obtain the exact masses of the unknowns. Thus, a Vibrio fischeri -bioactive zone was identified as avarone, a bioactive metabolite so far only known to be synthesized by the sponge Dysidea avara . This methodology proved very effective not only for detection but also for identification of unknown bioactive metabolites in sponges.

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Roy, S. S., Hajnoczly, G. (2008) Calcium, mitochondria and apoptosis studied by fluorescence measurements. Methods 46 , 213–223. Hajnoczly G Calcium, mitochondria and apoptosis

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Biehler, K., Haupt, S., Beckmann, J., Fock, H., Becker, T. W. (1997) Simultaneous CO 2 and 16 O 2 : 18 O 2 -gas exchange and fluorescence measurements indicate differences in light energy dissipation between the wild-type and the

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Acta Biologica Hungarica
Authors: Henryk Dębski, Magdalena Szwed, WiesŁaw Wiczkowski, Dorota Szawara-Nowak, Natalia Bączek, and Marcin Horbowicz

UV-B radiation in developing rye primary leaves as measured by ultraviolet-induced chlorophyll fluorescence measurements . Plant Cell Environ. 23 , 1373 – 1380 . 4. Cechin

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. Multiple effects of chromate on the photosynthetic apparatus of Spirodela polyrhiza as probed by OJIP chlorophyll a fluorescence measurements . Environ. Pollut. 115 : 49 – 64 . Arnon

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57 70 Von Wandruszka, R. , 1998. The micellar model of humic acid: evidence from pyrene fluorescence measurements. Soil Sci. 163 . 921

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.: Lymphocyte membrane potential and Ca 2+ -sensitive potassium channels described by oxonol dye fluorescence measurements. J Cell Physiol 125 , 72 (1985). Lymphocyte membrane potential and Ca 2+ -sensitive potassium channels described

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part to tight and rigid lipid packing, as suggested by our fluorescence measurements. In addition, the release of calcein from C 25,25 liposomes was less temperature sensitive compared to pure DPPC liposomes in the temperature range from 20 to 98 °C

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