Biomedical subjects

H V Hsieh

Publications and source records attributed to H V Hsieh.

4 recordsLinked to original sources

PubMed1995

Dissociation kinetics between a mouse Fc receptor (Fc gamma RII) and IgG: measurement by total internal reflection with fluorescence photobleaching recovery.

Total internal reflect with fluorescence photobleaching recovery (TIR-FPR) has been used to examine the dissociation kinetics between monomeric mouse IgG and a mouse Fc receptor (moFc gamma RII) reconstituted into substrate-supported planar membranes. IgG1, IgG2a, and IgG2b exhibited similar dissociation kinetics, whereas IgG3 did not bind. The fluorescence recovery curves for the IgG-moFc gamma RII interactions were best described by two reversible components (1.4 s-1, 66% and 0.06 s-1, 18%) and an irreversible component ( < 0.01 s-1, 16%). The kinetic parameters for a mouse anti-dinitrophenyl (DNP) IgG1 antibody were equivalent in the absence and presence of saturating amounts of DNP-glycine, demonstrating that possible allosteric changes which might occur in IgG1 upon hapten binding do not appreciably affect the kinetic characteristics of moFc gamma RII binding. The fluorescence recovery curves for polyclonal mouse IgG Fc were similar to those for intact IgG, showing that decreasing the size of the IgG 3-fold does not alter the dissociation rate. The dissociation kinetics of IgG1 decreased considerably in a low ionic strength buffer, indicating that the IgG1-moFc gamma RII interaction has significant electrostatic components.

Animals↗

PubMed1994

Theory for measuring bivalent surface binding kinetics using total internal reflection with fluorescence photobleaching recovery.

Total internal reflection with fluorescence photobleaching recovery (TIR-FPR) is a method for experimentally examining coupled diffusion and reaction kinetics at surfaces. In a previous work (Thompson et al. 1981. Biophys. J. 33:435-454), a theoretical basis for interpreting TIR-FPR data was described for monovalent ligands that undergo a reversible reaction with monovalent surface sites in a single step. Here, the theory for TIR-FPR has been extended to two different surface binding mechanisms that involve sequential, bivalent surface attachment. Methods for obtaining the intrinsic surface association and dissociation kinetic rates from measured fluorescence photobleaching recovery curves are described. The new theory should be applicable to the association of bivalent protein ligands such as antibodies with supported planar model membranes.

Animals↗

PubMed1993

Total internal reflection fluorescence microscopy: application to substrate-supported planar membranes.

The use of total internal reflection illumination in fluorescence microscopy (TIRFM) is reviewed with emphasis on application to fluorescent macromolecules that specifically and reversibly bind to planar model membranes supported on glass or quartz substrates. Several methods for characterizing macromolecular motion and organization are discussed: the measurement of equilibrium binding curves to obtain values for equilibrium binding constants; the measurement of fluorescence photobleaching recovery curves to obtain values of kinetic rate constants and surface diffusion coefficients; and the measurement of fluorescence intensities as a function of the evanescent field polarization to characterize orientational order. Applications to cell-substrate contact regions are summarized and future directions of TIRFM are outlined.

Chemical Phenomena↗

PubMed1992

Direct measurement of the weak interactions between a mouse Fc receptor (Fc gamma RII) and IgG1 in the absence and presence of hapten: a total internal reflection fluorescence microscopy study.

Total internal reflection fluorescence microscopy (TIRFM) has been used to directly measure the weak dissociation constants of IgG with a mouse IgG receptor (moFc gamma RII) that has been purified and reconstituted into substrate-supported planar membranes. Dissociation constants were measured for three different mouse monoclonal anti-dinitrophenyl (DNP) IgG1 antibodies and for polyclonal mouse IgG, in the absence and presence of saturating amounts of hapten (DNP-glycine). The dissociation constant for polyclonal mouse IgG was 3 microM, which agrees well with previous results. The dissociation constants for the three monoclonal antibodies with moFc gamma RII ranged from 2 microM to 3 microM and were not statistically different, suggesting that changes in moFc gamma RII dissociation constants which may exist within the IgG1 subclass are less than the error of the TIRFM measurements (approximately 20%). The measured IgG1-moFc gamma RII dissociation constants were not different for individual monoclonal antibodies in the absence or presence of saturating concentrations of DNP-glycine, directly showing that possible allosteric changes which might occur upon hapten binding and affect the equilibrium characteristics of Fc receptor binding are small. This work demonstrates a new approach for quantitatively examining the effects of solution components on weak receptor-ligand interactions.

Animals↗