Binding and mobility of anti-dinitrophenyl monoclonal antibodies on fluid-like, Langmuir-Blodgett phospholipid monolayers containing dinitrophenyl-conjugated phospholipids

The association of a fluorescently labelled anti-dinitrophenyl monoclonal antibody (ANO2) with Langmuir-Blodgett monolayers composed of three different binary mixtures of phosphatidylcholine and dinitrophenyl-conjugated phosphatidylethanolamine has been characterized. Quantitative fluorescence microscopy measurements demonstrated that measurable amounts of antibodies bound to the monolayers only at high molar fractions of dinitrophenyl-conjugated lipid (greater than or equal to 5 mol%). Fluorescence pattern photobleaching recovery measurements showed that the apparent translational diffusion coefficients and mobile fractions of a fluorescent lipid were high for all monolayer compositions and that the antibody translational mobility was measurable but slow and depended on the two-dimensional antibody density. The results demonstrate that the ANO2-binding characteristics of Langmuir-Blodgett monolayers containing dinitrophenyl-conjugated phospholipids are substantially different from those of similar model systems but that the ANO2 antibodies, when bound, display similar diffusive behavior.     

Relaxation processes on a picosecond time scale in hemoglobin and poly(L-alanine) observed by millimeter-wave spectroscopy

Broadband measurements of the millimeter-wave and far-ir absorption (10–10⁴ GHz) of lyophilized hemoglobin are reported. Additionally, the absorption of poly(L -alanine) and crystalline L -alanine at 70 GHz was measured for comparison. All measurements were extended over the temperature range from liquid helium to room temperature. For the millimeter range, this was attained by using the novel oversized-cavity technique. It was found that the millimeter-wave absorption of the materials increased nearly exponentially with temperature and increased as ν^1.2–ν² with frequency. The far-ir absorption of hemoglobin showed broadbands with almost no temperature dependence. The frequency and temperature dependence of the millimeter-wave absorption is quantitatively described as due to three distinct relaxation processes on a picosecond time scale occurring in asymmetric double-well potentials. These processes are most probably assigned to the NH ?OC hydrogen bonds of the peptide backbone.     

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.     

Interaction of antibodies with Fc receptors in substrate-supported planar membranes measured by total internal reflection fluorescence microscopy

A procedure for constructing substrate-supported planar membranes using membrane fragments isolated from the macrophage-related cell line J774A.1 is described. Total internal reflection (TIR) fluorescence microscopy is employed to demonstrate that fluorescently labeled Fab fragments of a monoclonal antibody (2.4G2) with specificity for a murine macrophage cell-surface receptor for IgG (moFc gamma RII) bind to the planar model membranes. These measurements show that the planar membranes contain moFc gamma RII and yield a value for the association constant of 2.4G2 Fab fragments with moFc gamma RII equal to (9.6 +/- 0.4) x 10(8) M-1 and indicate that the surface density of reconstituted moFc gamma RII is approximately 50 molecules/microns 2. In addition, TIR fluorescence microscopy is used to investigate the Fc-mediated competition of unlabeled, polyclonal murine IgG with labeled 2.4G2 Fab fragments for moFc gamma RII in the planar membranes. These measurements indicate that the reconstituted moFc gamma RII recognized by 2.4G2 Fab fragments also retains the ability to bind murine IgG Fc regions and yield a value for the association constant of polyclonal murine IgG with moFc gamma RII equal to (1-5) x 10(5) M-1. This work represents one of the first applications of TIR fluorescence microscopy to specific ligand-receptor interactions.     

Picosecond relaxations in model substances for proteins: A millimeter-wave investigation on crystalline alkyl amides

The dielectric absorption at millimeter-wave (mm-wave) frequencies (50–150 GHz) of N-methylacetamide (NMA), N,N-dimethylacetamide (DiNMA), and N,N-dimethylacrylamide (DiNMAcry) is measured. Measurements are performed using the oversized-cavity technique in the temperature range from liquid helium to room temperature. Additionally, a mm-wave interferometeric measurement at room temperature is made. NMA and DiNMAcry exhibit monotonic increases of the absorption coefficient with temperature as well as with frequency. For DiNMA a monotonic increase of the absorption coefficient with frequency is also found, while the absorption coefficient as a function of temperature shows a pronounced maximum at approximately 30 K. At this maximum the absorption coefficient of DiNMA exceeds those of NMA and DiNMAcry by about two orders of magnitude. The dielectric behavior of the three substances can be described by relaxation processes in asymmetric double-well potentials. For the low-temperature relaxation in DiNMA the double well could be established by two possible positions of the molecule in the crystal that are separated by a rotational movement. Hydrogen bonds and long side chains may hinder these relaxational movements in NMA and DiNMAcry, respectively, and thereby account for their comparatively lower absorption. The results are compared with similar results recently obtained on proteins and synthetic biopolymers.     

Diversity of Insects in Nature protected Areas (DINA): an interdisciplinary German research project

Biodiversity and Conservation - Insect declines and biodiversity loss have attracted much attention in recent years, but lack of comprehensive data, conflicting interests among stakeholders and...     

Binding of IgG to MoFc gamma RII purified and reconstituted into supported planar membranes as measured by total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy (TIRFM) has been combined with functional reconstitution of the mouse IgG receptor moFc gamma RII in substrate-supported planar membranes to quantitatively probe IgG-moFc gamma RII interactions. MoFc gamma RII was purified from the macrophage-related cell line J774A.1 using affinity chromatography with Fab fragments of the anti-moFc gamma RII monoclonal antibody 2.4G2. Purified moFc gamma RII was reconstituted into liposomes by detergent dialysis, and the liposomes were fused on quartz substrates to form supported planar membranes containing moFc gamma RII. TIRFM measurements showed that fluorescently labeled 2.4G2 Fab specifically bound to the planar membranes, confirming the presence of moFc gamma RII. The receptor density in the planar membranes was sufficiently high to allow direct detection of bound, fluorescently labeled polyclonal and monoclonal mouse IgG with TIRFM, demonstrating that moFc gamma RII retained Fc-mediated IgG binding activity after planar membrane formation and permitting direct measurement of bound IgG as a function of the IgG solution concentration. Cross-inhibition measurements showed that polyclonal mouse IgG blocked the binding of labeled 2.4G2 Fab and that 2.4G2 Fab blocked the binding of labeled polyclonal IgG. This work provides a direct measure of the relatively weak IgG-moFc gamma RII association constant and demonstrates a new model system in which the chemical and physical properties of IgG-moFc gamma RII interactions can be quantitatively characterized as a function of membrane, antibody, and solution properties.