Insulin receptor‐insulin interaction kinetics using multiplex surface plasmon resonance. Issue 12 (11th November 2013)

• Record Type: Journal Article
• Authors: Subramanian, Kannan; Fee, Conan J.; Fredericks, Rayleen; Stubbs, Richard S.; Hayes, Mark T.
• Is Part Of: Journal of molecular recognition. Volume 26:Issue 12(2013:Dec.)
• Page Start: 643
• Page End: 652
• Publication Date: 2013-11-11
• Subjects: Molecular recognition -- Periodicals; Models, Molecular -- Periodicals; Molecular Conformation -- Periodicals; Molecular Sequence Data -- Periodicals; Molecular Structure -- Periodicals; Carrier Proteins -- Periodicals; 572.8
• DOI: 10.1002/jmr.2307
• Languages: English
• ISSNs: 0952-3499

Abstract

<abstract abstract-type="main"> <title> <x xml:space="preserve">Abstract</x> </title> <p>Type 2 diabetes affects millions of people worldwide, and measuring the kinetics of insulin receptor‐insulin interactions is critical to improving our understanding of this disease. In this paper, we describe, for the first time, a rapid, real‐time, multiplex surface plasmon resonance (SPR) assay for studying the interaction between insulin and the insulin receptor ectodomain, isoform A (eIR‐A). We used a scaffold approach in which anti‐insulin receptor monoclonal antibody 83–7 (Abcam, Cambridge, UK) was first immobilized on the SPR sensorchip by amine coupling, followed by eIR‐A capture. The multiplex SPR system (ProteOn XPR36™, Bio‐Rad Laboratories, Hercules, CA) enabled measurement of replicate interactions with a single, parallel set of analyte injections, whereas repeated regeneration of the scaffold between measurements caused variable loss of antibody activity. Interactions between recombinant human insulin followed a two‐site binding pattern, consistent with the literature, with a high‐affinity site (dissociation constant <italic>K</italic><sub>D1</sub> = 38.1 ± 0.9 nM) and a low‐affinity site (<italic>K</italic><sub>D2</sub> = 166.3 ± 7.3 nM). The predominantly monomeric insulin analogue Lispro had corresponding dissociation constants <italic>K</italic><sub>D1</sub> = 73.2 ± 1.8 nM and <italic>K</italic><sub>D2</sub> = 148.9 ± 6.1 nM, but the fit to kinetic data was improved when we included a conformational change factor in which the high‐affinity site was converted to the low‐affinity site. The new SPR assay enables insulin‐eIR‐A interactions to be followed in real time and could potentially be extended to study the effects of humoral factors on the interaction, without the need for insulin labeling. Copyright © 2013 John Wiley & Sons, Ltd.</p> </abstract>