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Initial synthesis and characterization of an immobilized heat shock protein 90 column for online determination of binding affinities

2007; Elsevier BV; Volume: 373; Issue: 2 Linguagem: Inglês

10.1016/j.ab.2007.11.001

1096-0309

Michał Piotr Marszałł, Ruin Moaddel, Krzysztof Jóźwiak, Michel Bernier, Irving W. Wainer,

Toxin Mechanisms and Immunotoxins

Heat shock protein 90α (Hsp90α) was immobilized on aminopropyl silica via the N terminus to create the Hsp90α(NT) column or via the C terminus to create the Hsp90α(CT) column. Binding to the exposed C terminus on the Hsp90α(NT) column was characterized using frontal chromatography and the C-terminus ligands coumermycin A1 (CA1) and novobiocin (NOVO). The calculated Kd values were 220 ± 110 nM (CA1) and 100 ± 20 nM (NOVO). Nonlinear chromatography was used to determine the association and dissociation rate constants associated with the NOVO–Hsp90α complex: 22.2 ± 8.8 μM−1 s−1 and 2.7 ± 0.6 s−1, respectively. Binding to the exposed N terminus on the Hsp90α(CT) column was characterized using frontal chromatography. The Kd values of the N-terminus ligands geldanamycin (GM, 90 ± 50 nM), 17-allylamino-17-demethoxygeldanamycin (17-AAG, 210 ± 50 nM), and radicicol (RAD, 20 ± 9 nM) were consistent with previously reported values. The effect of the immobilization on ATPase activity was investigated through the determination of IC50 values for inhibition of ATPase activity on the Hsp90α(CT) column. The IC50 for GM was 2.80 ± 0.18 μM, and the relative IC50 values were 17-AAG > GM > RAD, in agreement with previously reported values and indicating that immobilization had not affected ATPase activity or sensitivity to inhibition.