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H Stapelfeldt

Publications and source records attributed to H Stapelfeldt.

18 recordsLinked to original sources
PubMed2000

Three dimensional alignment of molecules using elliptically polarized laser fields

We demonstrate, theoretically and experimentally, that an intense, elliptically polarized, nonresonant laser field can simultaneously force all three axes of a molecule to align along given axes fixed in space, thus inhibiting the free rotation in all three Euler angles. Theoretically, the effect is illustrated through time dependent quantum mechanical calculations. Experimentally, 3, 4-dibromothiophene molecules are aligned with a nanosecond laser pulse. The alignment is probed by 2D ion imaging of the fragments from a 20 fs laser pulse induced Coulomb explosion.

Journal Article↗
PubMed1999

Pressure denaturation and aggregation of beta-lactoglobulin studied by intrinsic fluorescence depolarization, Rayleigh scattering, radiationless energy transfer and hydrophobic fluoroprobing.

beta-Lactoglobulin (beta-lg) in aqueous solution under pressure showed a marked depolarization of intrinsic fluorescence assigned to a gradually increased rotational diffusion of tryptophyl moieties in pressure-unfolded states. The corresponding change in anisotropy provided a new and more accurate method for determining denaturation volume which, for beta-lg in neutral aqueous solution with ionic strength 0.16 (NaCl) at 25 degrees C, was delta V degree = -73 (SE 3) ml mol-1, corresponding to half denaturation at 123 MPa. The pressure unfolding led to exposure of hydrophobic regions to the protein-water interface that could be probed by fluorescence intensity of a beta-lg-1-anilinonaphthalene-8-sulphonic acid (ANS) complex with 1:1 stoichiometry, as determined by Job's method of continuous variation. The unfolding of beta-lg impaired the binding capacity of the inner calyx, with a reduction in binding capacity of 50% at 50 MPa, as shown by decreasing cis-parinaric acid fluorescence, decreasing anisotropy and decreasing radiationless energy transfer from tryptophans to this probe with increasing pressure. The pressure-induced reversible exposure of hydrophobic groups to the protein-water interface may, at least partly, explain the initial aggregation reactions, evident from increased Rayleigh scattering from approximately 50 MPa, prior to irreversible pressure-induced gel formation of beta-lg. Using results from this and previous studies, we propose a three step pressure denaturation model for beta-lg for neutral solution at ambient temperature, including an initial pressure-melted state (up to 50 MPa) with partial collapse of the inner calyx and solvent exposure of the free thiol group, followed by a reversible denaturation with exposure of hydrophobic regions (half denaturation at 123 MPa) and with irreversible denaturation with thiol-disulphide exchange becoming increasingly important at higher pressures. Effects of pressure on beta-lg, as measured by fluorescence depolarization, were found for the reversible denaturation steps to be similar to the effects of chemical denaturants but different with respect to shift in ANS emission maxima.

Anilino Naphthalenesulfonates↗
PubMed1999

Spectrofluorometric characterization of beta-lactoglobulin B covalently labeled with 2-(4'-maleimidylanilino)naphthalene-6-sulfonate.

Bovine beta-lactoglobulin, genetic variant B, has been labeled with 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid through covalent attachment through the Cys-121 thiol group for the study of stepwise pressure denaturation of this whey protein by fluorescence spectroscopy. The labeling was performed under nondenaturing conditions with a factor of 5 excess of the fluorophore in dimethylformamide/water (1:10) to yield the whey protein highly labeled after chromatographic separation. MALDI-TOF mass spectroscopy confirmed labeling. The emission from the fluorophore, which is sensitive to the microenvironment, has been characterized for the labeled protein (aqueous pH 7.4 solution, 25 degrees C) and has a lambda(em,max) = 410 nm (lambda(ex,max) = 318 nm) with a fluorescence lifetime of 6.1 +/- 0.2 ns. Fluorescence anisotropy increases and fluorescence quantum yield (Phi(f) = 0.103 at 320 nm) decreases with increasing excitation wavelength. For increasing hydrostatic pressure, fluorescence quantum yield showed a minimum at approximately 50 MPa, corresponding to the pre-denatured "pressure-melted" state in which thiol reactivity previously was found to increase prior to reversible protein unfolding.

Anilino Naphthalenesulfonates↗
PubMed1998

Thiol Reactivity in Pressure-Unfolded beta-Lactoglobulin. Antioxidative Properties and Thermal Refolding.

Pressure treatment of beta-lactoglobulin (0.11 mM in aqueous 0.16 M NaCl, pH 7.61, at 15 degrees C for 30 min, up to 400 MPa investigated) induces antioxidative properties as shown for linoleic acid peroxidation in oil-in-water emulsions. The antioxidative properties obtained through pressure treatment are gradually lost at ambient pressure and paralleled by a decrease in thiol exposure and reactivity, as determined with Ellman's reagent, in an entropy-controlled (DeltaS() = -247 +/- 7 J mol(-)(1) K(-)(1)) first-order renaturation process (half-life of 3.1 h at 25 degrees C, pH 7.61, independent of pressure used for denaturation at least up to 250 MPa) with a modest temperature dependence (DeltaH() = 23 +/- 2 kJ mol(-)(1)). The reactivity of the thiol group toward Ellman's reagent was studied kinetically by stopped-flow spectrometry. The apparent second-order rate constant for this reaction at pH 7.61 and 25 degrees C changes from 5.7 x 10(2) L mol(-)(1) s(-)(1) for native beta-lactoglobulin to 1.6 x 10(5) L mol(-)(1) s(-)(1) for beta-lactoglobulin pressure-denatured at 200 MPa. Half-denaturation occurred at approximately 50 MPa. The degree of exposure of the thiol group corresponds to half-denaturation around approximately 140 MPa with a reaction volume, DeltaV degrees, for denaturation of -61 +/- 3 mL.mol(-)(1), a difference in half-denaturation pressure which may indicate that pressure denaturation is a stepwise process.

Journal Article↗
PubMed1996

Kinetics of formation of fluorescent products from hexanal and L-lysine in a two-phase system.

Kinetics of formation of fluorescent condensation products from hexanal and L-lysine (or its N-acetylated forms) including mass-transfer has been studied in a two-phase system consisting of lysine (or lysine derivative) in an aqueous phosphate buffer and a 1-octanol solution of hexanal as model for formation of fluorophores between protein and carbonyl compounds in peroxidizing biological systems. The initial rate of formation of fluorescent products in the aqueous phase was found to be proportional to the concentration of hexanal and lysine and to increase in both phases with increasing pH in the aqueous phase, in contrast to a higher-order dependence on hexanal in the octanol phase. At pH = 6.8, the temperature dependence of the appearance of fluorescent products corresponds to apparent energies of activation of 63 kj.mol-1 and 87 kj.mol-1 in the aqueous phase and the octanol phase, respectively. Fluorescent condensation products appeared faster in the octanol phase. However, by a kinetic analysis, the fluorescent products were shown to be formed in the aqueous phase, corresponding to the lower energy of activation and to the simple second-order kinetics, and subsequently distributed between the aqueous phase and the octanol phase. L-Lysine reacted faster than N alpha-acetyl-L-lysine which reacted faster than N epsilon-acetyl-L-lysine. Using fluorescence quantum yields, determined to be 1.4.10(-2) in octanol and 8.10(-3) in water at pH 6.8, an apparent partition coefficient of 17 (octanol/water) was determined for the condensation product of L-lysine. The steady-state fluorescence in the octanol phase was attributed to two components with fluorescence lifetimes at 25 degrees C of 0.7 +/- 0.05 ns and 5.1 +/- 0.2 ns, assigned to hexanal and the condensation product, respectively. The emission spectra were resolved in the two components using phase-sensitive detection, and the condensation product had emission maximum at 405 nm.

Aldehydes↗
PubMed1996

Effect of high hydrostatic pressure on the enzymic hydrolysis of beta-lactoglobulin B by trypsin, thermolysin and pepsin.

Hydrolysis of beta-lactoglobulin B (beta-lg B) by pepsin, a process slow at ambient conditions, is facilitated at a moderately high hydrostatic pressure such as 300 MPa, corresponding to an apparent volume of activation delta V# = -63 ml mol-1 at pH 2.5, 30 degrees C and gamma/2 = 0.16. Digestion of beta-lg by trypsin and thermolysin is likewise enhanced by pressure, and the pressure effect has been traced to pressure denaturation of beta-lg B, which by high-pressure fluorescence spectroscopy has been shown to have a large negative volume of reaction, delta V(o) = -98 ml mol-1, at pH 6.7, 30 degrees C and gamma/2 = 0.16. Pressure denaturation is only slowly reversed following release of pressure and the enhanced digestibility is maintained at ambient pressure for several hours.

Hydrolysis↗