Measuring the Lifetime of Bonds Made between Surface-linked Molecules (*)
- From the INSERM U 387, Laboratoire d'Immunologie, Hôpital de Sainte-Marguerite, BP29, 13274 Marseille Cedex 09, France
- § To whom correspondence should be addressed. Tel.: 33-91-26-03-31; Fax: 33-91-75-73-28.
Abstract
It is not well known how the kinetic constants of association between soluble receptors and ligands may be used to predict the behavior of these molecules when they are bound to cell surfaces. Spherical beads were coated with varying densities of anti-rabbit immunoglobulin monoclonal antibodies and driven along glass surfaces derivatized with rabbit anti-dinitrophenol. Particle motion was analyzed. The velocity, attachment frequency, and duration of binding events were determined on individual particles. It was found that i) beads exhibited frequent arrests lasting between a few tenths of a second and more than one minute; ii) when antibodies were diluted, the median arrest duration remained fairly constant (≈1 s) whereas binding frequency varied as the first power of the antibody concentration, suggesting that most particle arrests were due to the formation of a single bond; iii) when the shear rate was increased 7-fold, the duration of transient binding events remained constant. The disruptive force exerted on attachment points was estimated to range between about 6 and 37 piconewtons; and iv) the distribution of arrest durations suggested that binding was not a monophasic reaction but involved at least one intermediate step. Therefore, transient binding events reflected the formation of unstable associations that are not detected with standard techniques.
Footnotes
-
↵* This work was supported by a grant from the Association pour la Recherche sur le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 We are grateful to Prof. Evan Evans for pointing out the importance of the difference of the forces experienced by the sphere and the bond. The problem of determining the force on a single cell exposed to a laminar shear flow on a surface was first addressed by Schmid-Schoenbein et al.(30).
-
↵2 Note that this finding may be dependent on our model. While this paper was being submitted, an analysis of the interaction between blood neutrophils and P-selectin-coated surfaces was reported(35). The lifetime of P-selectin-ligand bonds was estimated at about 1 s in accordance with our estimate of about 2 s for neutrophil-E-selectin interaction(17), but no transient state was detected.
-
- Received April 11, 1995.
- Revision received July 18, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
