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R Schweitzer-Stenner

Publications and source records attributed to R Schweitzer-Stenner.

32 records · Page 2Linked to original sources
PubMed1991

Fluorescence resonance energy transfer on single living cells. Application to binding of monovalent haptens to cell-bound immunoglobulin E.

We have determined the specific binding of 2,4-dinitrophenyl (DNP)-haptens to two different monoclonal immunoglobulin (IgE) molecules bound to Fc epsilon-receptors on the cell surface of single, living rat basophilic leukemia cells subclone 2H3 cells. The measurements were performed at 4 degrees, 15 degrees, and 25 degrees C using a recently developed technique that permits the quantitative determination of fluorescence resonance energy transfer between two fluorophores on single cells in a microscope from the photobleaching kinetics of the donor fluorophore. We introduce here a method for performing binding studies on individual attached cells. At 25 degrees C, the titration studies yielded equilibrium binding constants Kint of 9 x 10(8), 8 x 10(8), and 8 x 10(7) M-1 for the monovalent haptens N-2,4-DNP-epsilon-amino-n-caproic acid, N epsilon-2,4-DNP-L-lysine, and N-2,4-DNP-gamma-amino-n-butyric acid, respectively. Our data indicate that the affinity constants for the first two haptens binding to IgE on adherent cells are 4 to 11 times larger than that of the corresponding values obtained by fluorescence quenching experiments with the same haptens and IgE molecules either in solution or bound to cells in suspension.

Animals↗
PubMed1989

The influence of structural variations in the F- and FG-helix of the beta-subunit modified oxyHb-NES on the heme structure detected by resonance Raman spectroscopy.

The dispersion of the depolarization ratio of two prominent Raman lines (1,375 cm-1 and 1,638 cm-1) of oxyhemoglobin-N-ethyl succinimide have been examined for pH values between pH = 6.0 and 8.5. Both exhibit a significant pH dependence. Calculation of the Raman tensor in terms of a fifth-order time dependent theory provides information about the pH-dependence of parameters reflecting symmetry classified distortions of the prosthetic heme group. To correlate these distortions with the functional properties of the molecule the following protocol was used: 1) An allosteric model suggested by Herzfeld and Stanley (1974) has been applied to O2-binding curves measured at different pH values between 6.5 and 9.0. From this calculation one obtains both, the energy differences between different molecular conformations and the equilibrium constants of oxygen and proton binding. 2) A titration model was formulated relating each conformation of a molecule to a distinct set of distortion parameters of the heme group. 3) The distortion parameters resulting from the analysis of our Raman data were assigned as an effective value due to incoherent superposition of the distortion parameters related to the different titration states. The application of this procedure yields an excellent reproduction of the pH-dependent effective distortion parameters of both Raman lines investigated. It is shown that the protonation of two tertiary effector groups located in the beta-subunits affect the symmetry of the heme in a contrary manner: the protonation of a His-residue (pK = 8.2, probably His(FG4) beta) causes a symmetric position of the proximal imidazole thus lowering the perturbations of the heme core. Further it influences the interaction between amino acid residues of the heme cavity and pyrrole side chains (probably Val(FG5) beta-vinyl (pyrrole 3) thus causing a decrease of the distortions related to the peripheral part of the heme. In contrast, the protonation of Lys (EF6) beta causes a tilt position of the proximal imidazole and an increase of asymmetric perturbations of the heme core, whereas the interaction between the pyrrole side chains and the heme cavity is weakened. Our results are consistent with stereochemical predictions of Moffat (1971) concerning the existence of a H-bond between His(FG4) beta and Cys(F9) beta.

Adult↗
PubMed1989

An extended Monod-Wyman-Changeaux-model expressed in terms of the Herzfeld-Stanley formalism applied to oxygen and carbonmonoxide binding curves of hemoglobin trout IV.

An extended Monod, Wyman, Changeaux (MWC)-model, the mathematical basis of which had been formulated by Herzfeld and Stanley (J. Mol. Blol. 82:231. 1974.) was used to fit oxygen and CO-binding curves of hemoglobin trout IV measured at different pH-values between pH = 8.0 and 6.0. From this calculation one obtains that even the fully liganded molecule exhibits a R----T quaternary transition upon approaching the acid pH-region. In the case of O2-binding, the cooperativity becomes negative below pH = 6.5. This can be related to the difference between the equilibrium constants of proton binding to the alpha- and beta-subunits. Furthermore, it can be shown that the interaction between the quaternary T----R- and the tertiary t----r-transitions is different for the alpha- and beta-subunits.

Animals↗
PubMed1989

Detection of the heme perturbations caused by the quaternary R----T transition in oxyhemoglobin trout IV by resonance Raman scattering.

The depolarization ratio dispersion and the respective excitation profiles of two structural sensitive Raman lines of oxyhemoglobin-trout IV (1,375 and 1,638 cm-1) have been measured at pH-values between 6.5 and 8.5. They were analyzed by employing a fifth order time dependent perturbation theory to calculate the polarizability tensor. This provides information about the pH-dependence of parameters reflecting symmetry classified distortions of the prosthetic heme groups. In order to correlate these distortions with functional properties of the molecule the following protocol has been employed: (a) a titration model was formulated relating each conformation of the molecule to a distinct set of distortion parameters the incoherent superposition of which provides the respective distortion parameter obtained from our Raman data. (b) The thermodynamic constants determining the equilibrium between these molecular conformations (i.e., the quaternary T and R-states, the low affinity t and the high affinity r-states of the distinct subunits, the pK-values of the Root- and Bohr groups) were obtained from a set of O2-binding curves that were analyzed in terms of an allosteric model suggested by Herzfeld and Stanley 1974. J. Mol. Biol. 82:231. The application of this procedure yields excellent reproduction of the pH-dependent effective distortion parameters of both Raman lines investigated. Thus established correlation between hemoglobin function (O2-binding) and structure (asymmetric perturbation of the hemegroup) provides some interesting insights into the molecular basis of the allosteric Root effect.

Animals↗
PubMed1988

Possible orientational constraints determine secretory signals induced by aggregation of IgE receptors on mast cells.

Three biologically active monoclonal antibodies (mAbs) specific for the monovalent, high-affinity membrane receptor for IgE (Fc epsilon R) were employed in analysing the secretory response of mast cells of the RBL-2H3 line to crosslinking of their Fc epsilon R. All three mAbs (designated F4, H10 and J17) compete with each other and with IgE for binding to the Fc epsilon R. Their stoichiometry of binding is 1 Fab:1 Fc epsilon R, hence, the intact mAbs can aggregate the Fc epsilon Rs to dimers only. Since all three mAbs induce secretion, we conclude that Fc epsilon R dimers constitute a sufficient 'signal element' for secretion of mediators for RBL-2H3 cells. The secretory dose-response of the cells to these three mAbs are, however, markedly different: F4 caused rather high secretion, reaching almost 80% of the cells' content, while J17 and H10 induced release of only 30-40% mediators content. Both the intrinsic affinities and equilibrium constants for the receptor dimerization were derived from analysis of binding data of the Fab fragments and intact mAbs. These parameters were used to compute the extent of Fc epsilon R dimerization caused by each of the antibodies. However, the different secretory responses to the three mAbs could not be rationalized simply in terms of the extent of Fc epsilon R dimerization which they produce. This suggests that it is not only the number of crosslinked Fc epsilon Rs which determines the magnitude of secretion-causing signal, but rather other constraints imposed by each individual mAb are also important.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Oligomerization and ring closure of immunoglobulin E class antibodies by divalent haptens.

Cross-linking of antibodies constitutes a widespread initiation signal for their respective effector functions. Cross-linking IgE-class antibodies provide the triggering signal to mast cells for their degranulation process. To obtain a quantitative insight into these cross-linking processes, the interactions between a DNP-specific monoclonal antibody of the IgE class and a series of divalent DNP haptens with spacers of different length and flexibility have been studied by fluorescence titration experiments. These were analyzed by employing the theoretical model developed by Dembo and Goldstein [Dembo, M., & Goldstein, B. (1978) J. Immunol. 121, 345-353] in a fitting procedure. Equilibrium constants that describe the aggregation and ring-closure processes caused by divalent hapten binding have been used as free parameters. The intrinsic binding constants were determined by fluorescence titrations with corresponding monovalent haptens. The main results are the following: (1) The divalent haptens with a short and flexible spacer [i.e., N alpha, N epsilon-di-(DNP)-L-lysine,meso-bis[(DNP-beta-Ala)amino]succinate, and bis[(DNP-tri-D-Ala)amino]heptane, having a maximal DNP-DNP distance of gamma = 14, 21, and 45 A, respectively] effect aggregation of the antibodies mainly into closed dimers. (2) The divalent hapten family with long and rigid oligoproline spacers di(DNP)-Ahx-Asp-(Pro)n-Lys with n = 24, 27, and 33 (i.e., gamma = 100, 110, and 130 A) causes aggregation of the antibodies predominantly into closed dimers and trimers. The corresponding equilibrium constants of the respective ring-closure processes decrease significantly with longer spacer length. (3) Evidence was found that intramolecularly monomeric ring closure of the IgE antibodies is caused by haptens containing oligoproline spacers with n = 37 or 42 (gamma = 130-150 A). The equilibrium constant of the ring-closure process increases with spacer length. This increase in stability indicates a difference in the imposed strain. Furthermore, the latter results imply that the distance between the two binding sites of the IgE molecule lies in the range dictated by the rigid oligoproline part of the respective hapten's spacer, i.e., 115-130 A. (4) Nearly all oligomeric ring-closure processes proceed relatively slowly with an approximate lower limit of a half-life of 5-10 s. This slowing down of the aggregation and ring-closure processes most probably reflects steric factors.

Calorimetry↗
PubMed1986

Characterization of the ion channel activity in planar bilayers containing IgE-Fc epsilon receptor and the cromolyn-binding protein.

Electric conductance was studied across micropipette-supported planar lipid bilayers, reconstituted with IgE-Fc epsilon receptor and the cromolyn-binding protein (CBP) isolated from membranes of rat basophilic leukemia cells (RBL-2H3). Currents were observed following the addition of aggregating agents, specific for either of the two proteins. The results show that the two proteins are necessary and sufficient for the opening of cation channels. Both aggregation of Fc epsilon receptor via IgE with a specific antigen and of CBP by anti-CBP induce channels with similar conductances and open-time distributions. In the presence of 1.8 mM calcium, the most frequently observed channels have a conductance of 1-2 pS. At 100 mM calcium conductance increased to 4-5 pS. Channels induced by antigen were susceptible to blocking by the anti-allergic drug cromolyn. These results suggest that CBP acts as the core of the cation channel and that the channel conductance and open-time characteristics are independent of the mode of aggregation.

Animals↗
PubMed1986

pH-dependent absorption in the B and Q bands of oxyhemoglobin and chemically modified oxyhemoglobin (BME) at low Cl- concentrations.

We have measured the optical absorbance in the maxima of the Q and B bands for oxyhemoglobin and oxyhemoglobin (BME) in dependence on the pH value of the solution in the region between pH 4.4 and pH 10. From the absorbance data optical titration curves are derived for both bands. These yield for oxyhemoglobin pK values 4.3, 5.3, 6.8, 7.8, and 9.0, whereas for oxyhemoglobin (BME) only one pK value at 4.3 is observed. These data are in good agreement to those derived recently from resonance Raman spectroscopy. The changes of the oscillator strengths in the Q bands are interpreted in terms of Gouterman's four-orbital model to arise from A1g-distortions of the heme group, resulting from changes of the heme-apoprotein interactions due to protonation processes of amino acid-side groups in the beta-chains. The difference between the sets of pK values in oxyhemoglobin and oxyhemoglobin BME is explained from the fact that the bifunctional reagent BME blocks important pathways of heme-apoprotein interactions. The fact that in any case increase of the Q band absorbance is accompanied by a corresponding increase in the B band absorbance leads us to the conclusion that the electronic structure of the B bands has to be described in terms of a six-orbital model, taking into account configurational interaction with the L and N bands.

Antisickling Agents↗
PubMed1986

Correspondence of the pK values of oxyHb-titration states detected by resonance Raman scattering to kinetic data of ligand dissociation and association.

The dispersion of the depolarization ratio of oxidation and spinmarker lines of oxyhemoglobin at low C1- concentration (less than 0.08 M) have been examined for different pH values in the acid and alkaline region. Interpreting the depolarization ratio dispersion curves by fifth order Loudon theory of the polarizibility tensor, we obtain tensor parameters depending linearly on symmetry classified distortions of the functional hemegroup. The pH dependence of these parameters are explained by assuming the influence of three titrable groups with pK = 7.8, 6.6, and 5.8 on the heme. Using these pK values, we are able to interpret the pH dependence of CO(O2)-dissociation and CO-association of the fourth hemoglobin subunit. We conclude from our measurements that the change of the Tyr HC2 beta-configuration induces heme-apoprotein interaction via the Tyr HC2 beta-Val FG5 beta H-bond, which are transduced to the heme via central and peripheral coupling.

Chlorides↗
PubMed1985

Heme-apoprotein interaction in the modified oxyhemoglobin-bis(N-maleimidomethyl)ether and in oxyhemoglobin at high Cl-concentration detected by resonance Raman scattering.

The dispersion of the depolarization ratio of oxidation and spin-marker lines of oxyhemoglobin-bis(N-maleimidomethyl)ether and oxyhemoglobin at high Cl- concentration (1 M) have been examined for different pH values in the neutral and alkaline regions. The oxidation marker line at 1375 cm-1 shows no pH-dependence in the physiological region for oxyHb-bis(N-maleimidomethyl)ether and a comparatively small variation for oxyHb at a Cl- concentration higher than 0.4 M. The spin-marker line at 1638 cm-1 exhibits a strong pH-dependence of depolarization ratio for high Cl- concentration, but a minor pH-induced variation for oxyHb-bis(N-maleimidomethyl)ether. Interpretation of these data yield the following conclusions: (1) The oxidation marker line monitors symmetry-lowering distortions of the heme group introduced by central coupling to the protein via the Fe-N bond, whereas the spin-marker line monitors peripheral coupling due to heme-protein interaction in the heme pocket. (2) At low Cl- concentrations (below 0.3 M) both types of coupling are present. These are induced by the salt bridge between His 146 beta and Asp94 beta and flexibility of the FG corner. (3) At high Cl- concentrations the salt bridge is missing, eliminating central coupling. (4) In oxyhemoglobin-bis(N-maleimidomethyl)ether, due to constraint of the bis(N-maleimidomethyl)ether bridging the FG corner and eliminating its flexibility and the missing salt bridge, both central and peripheral coupling are drastically reduced.

Adult↗
PubMed1985

Haem-apoprotein interactions detected by resonance Raman scattering in Mb- and Hb-derivates lacking the saltbridge His146 beta-Asp94 beta.

The dispersion of the depolarization ratio of oxidation- and spin-marker lines of sperm whale myoglobin derivatives (oxyMb, deoxyMb, ferric Mb-CN) and of ferric Hb-CN have been measured for different pH-values in the acid and alkaline region. No pH-dependence in the region above pH = 6.5 has been found. Below pH = 6.5, however, a significant pH-dependence of the oxyMb-oxidation marker line at 1,375 cm-1 exists. Additionally, a weak pH-dependence of the corresponding 1,355 cm-1 line of the deoxymyoglobin spectrum is observed. This effect can be explained assuming a titration of distal histidine, inducing a rupture of the ligand-imidazole H-bond in the case of oxymyoglobin. The pH-independent depolarization ratio disperson above pH = 6.5 in all systems investigated is explained by the lack of the haemoglobin saltbridge between His(HC3) beta and Asp(FG5) beta, which is essential for the cooperativity in the haemoglobin system.

Animals↗
PubMed1984

Investigation of pH-induced symmetry distortions of the prosthetic group in oxyhaemoglobin by resonance Raman scattering.

The depolarisation ratio and the excitation profiles of some prominent Raman lines of the oxyhaemoglobin spectrum (1,375 cm-1, 1,583 cm-1, 1,638 cm-1) have been measured as functions of the exciting laser frequency. The depolarisation ratio shows a complicated minimum-maximum structure in the preresonant region between Soret- and beta-band of the optical spectrum, which depends on the pH-value of the solution. These dispersion curves are interpreted by fifth-order Loudon theory of the polarizability tensor including static distortions of the haem group, which lower its symmetry from the ideal D4h-symmetry, and enhancement by a second, non-Raman-active phonon. The fitting constants needed to fit the experimental data are related to static distortions of A1g, B1g, B2g, and A2g symmetry types and thus give information on the symmetry lowering from D4h. The variation of the fitting constants with the pH-value of the solution is interpreted to be caused by protonation/deprotonation processes of titrable amino acid groups contributing to the alkaline and acid Bohr effect. The protonation changes the electrostatic interaction energies in the globular protein and destabilizes the salt bridge between His(HC3)beta and Asp(FG1)beta in the R-state. These processes induce distortions of the haem group via haem-apoprotein interactions. Our results give no indication for a dominant role of the covalent Fe2+-N [His(F8)] bond in this process. They are in agreement, however, with the allosteric model of Hopfield, which assumes all interactions to be evenly distributed all over the protein molecule.

Adult↗