کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10533044 | 961841 | 2005 | 13 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
A surface plasmon resonance-based assay for small molecule inhibitors of human cyclophilin A
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 345, Issue 2, 15 October 2005, Pages 214-226
Journal: Analytical Biochemistry - Volume 345, Issue 2, 15 October 2005, Pages 214-226
نویسندگان
Martin A. Wear, Alan Patterson, Kirk Malone, Colin Dunsmore, Nicholas J. Turner, Malcolm D. Walkinshaw,