Presence Of Multimers Research Articles

Affinity and Kinetic Analysis of Fcγ Receptor IIIa (CD16a) Binding to IgG Ligands

Binding of pathogen-bound immunoglobulin G (IgG) to cell surface Fc gamma receptors (FcgammaRs) triggers a wide variety of effector functions. The binding kinetics and affinities of IgG-FcgammaR interactions are hence important parameters for understanding FcgammaR-mediated immune functions. We have measured the kinetic rates and equilibrium dissociation constants of IgG binding to a soluble FcgammaRIIIa fused with Ig Fc (sCD16a) using the surface plasmon resonance technique. sCD16a interacted with monomeric human IgG and its subtypes IgG1 and IgG3 as well as rabbit IgG with on-rates of 6.5 x 10(3), 8.2 x 10(3), 1.1 x 10(4) and 1.8 x 10(4) m(-1) s(-1), off-rates of 4.7 x 10(-3), 5.7 x 10(-3), 5.9 x 10(-3), and 1.9 x 10(-2) s(-1), and equilibrium dissociation constants of 0.72, 0.71, 0.56, and 1.1 mum, respectively. The kinetics and affinities measured by surface plasmon resonance agreed with those obtained from real time flow cytometry and competition inhibition binding experiments using cell surface CD16a. These data add to our understanding of IgG-FcgammaR interactions.

Carbohydrate-recognition domains of galectin-9 are involved in intermolecular interaction with galectin-9 itself and other members of the galectin family

Galectin-9 (Gal-9) is a tandem-repeat-type member of the galectin family associated with diverse biological processes, such as apoptosis, cell aggregation, and eosinophil chemoattraction. Although the detailed sugar-binding specificity of Gal-9 has been elucidated, molecular mechanisms that underlie these functions remain to be investigated. During the course of our binding study by affinity chromatography and surface plasmon resonance (SPR) analysis, we found that human Gal-9 interacts with immobilized Gal-9 in the protein-protein interaction mode. Interestingly, this intermolecular interaction strongly depended on the activity of the carbohydrate recognition domain (CRD), because the addition of potent saccharide inhibitors abolished the binding. The presence of multimers was also confirmed by Ferguson plot analysis of result of polyacrylamide gel electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Moreover, this intermolecular interaction was observed between Gal-9 and other galectin members, such as Gal-3 and Gal-8, but not Gal-1. Because such properties have not been reported yet, they may explain an unidentified mechanism underlying the diverse functions of Gal-9.

Adsorption Kinetics of Lysozyme on Silica at pH 7.4: Correlation between Streaming Potential and Adsorbed Amount

The adsorption of lysozyme in phosphate buffer (10-2 M + NaCl 10-2 M) at pH 7.4 on silica was studied at different flow rates. We measured simultaneously as a function of time (i) the streaming potential between the extremities of the capillary (internal diameter, 0.530 mm; length, 22 cm) and (ii) the amount adsorbed at x0 = 14 cm from the entrance by means of 125I-radiolabeled proteins. We observed two regimes in the kinetics of adsorption, the first one being a transport-controlled process. They are separated at a critical interfacial concentration Γcr depending on the initial ζ0 potential of the interface, from 0.035 μg/cm2 at low density of charges (|ζ0| < 25 mV) to a mean value of 0.055 μg/cm2 for higher density of charges (26 mV < |ζ0| < 45 mV). A direct interpretation of those results concerns the electrostatic interactions: more molecules, which are slightly positively charged at pH 7.4, are adsorbed on the more negative surfaces. The ζ potential at the transition between both regimes (or final value) was dependent on the initial value ζ0: no charge reversal occurred when ζ0 = −25 mV, while the interface became slightly positive when ζ0 = −45 mV. While both adsorbed amount and streaming potential increased in the first regime, in the second regime streaming potential showed very small variations, if any, and interfacial concentration continued to increase. Hence, we focused mainly on the correlation between both quantities in the first regime. The study of the initial adsorption kinetics as a function of flow rate was in accordance with the presence of multimers (diffusion coefficient D = 4.0 × 10-7 cm2 s-1; adsorption constant ka ≈ 4.0 × 10-3 cm s-1) in the shear zone near the wall. With these parameters, we performed numerical simulations to estimate the ratio between average and local interfacial concentrations, 〈Γ〉/Γ(x0). The variations of Δζ/〈Γ〉 versus time were then deduced. The data are also presented as Δζ/ζ0 as a function of 〈Γ〉 and analyzed according to the recent model of M. Zembala and Z. Adamczyk (Langmuir 2000, 16, 1593) for uniformly charged spheres. At low density of charges (ζ0 ≈ −25 mV) we observed Δζ ∝ 〈Γ〉 almost over the whole first regime, while a slight departure from linearity occurred above 0.3Γcr for higher density of charges. A semiquantitative analysis suggests that the molecules are oriented at the interface with their more positive parts facing the solid surface, the conformation of the molecules being different at small and high charge densities of silica.

Shear of Telechelic Brushes

The forces between two surfaces bearing solvated, telechelic polymer chains (terminated at both ends with a dipolar group) exhibit a clear attractive regime. The range and magnitude of this attraction indicate the presence of multimers on each surface, and the formation of entropically favored bridges between them as the telechelic layers overlap. On sliding the surfaces a shear force close to that expected from the stretching of the polymer bridges is observed, suggesting that the frictional forces can be tuned by varying the interaction strength of the telechelic end groups. [S0031-9007(99)09413-2]