Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10533044 | Analytical Biochemistry | 2005 | 13 Pages |
Abstract
A simple protocol for generating a highly stable and active surface plasmon resonance (SPR) sensor surface of recombinant human hexahistidine cyclophilin A (His-CypA) is described. The sensor surface was sensitive and stable enough to allow, for the first time, the screening and ranking of several novel small-molecule (Mr â¼250-500 Da) ligands in a competition binding assay with cyclosporin A (CsA). It also allowed us to accurately determine the kinetic rate constants for the interaction between His-CypA and CsA. His-CypA was first captured on a Ni2+-nitrilotriacetic acid (NTA) sensor chip and was then briefly covalently stabilized, coupling via primary amines. The significant baseline drift observed due to dissociation of weakly bound His-CypA from the Ni2+-NTA moiety was eliminated, resulting in a surface that was stable for at least 36 h. In addition, immobilized protein activity levels were high, typically between 85 and 95%, assayed by the interaction between His-CypA and CsA. The mean equilibrium dissociation constant for CsA (KdCsA) binding to the immobilized His-CypA was 23 ± 6 nM, with on and off rates of 0.53 ± 0.1 μMâ1 sâ1 and 1.2 ± 0.1 (Ã10â2) sâ1, respectively. These values agree well with the values for the corresponding binding constants determined from steady-state and kinetic fluorescence titrations in solution.
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Martin A. Wear, Alan Patterson, Kirk Malone, Colin Dunsmore, Nicholas J. Turner, Malcolm D. Walkinshaw,