Initial synthesis and characterization of an immobilized heat shock protein 90 column for online determination of binding affinities.
Heat shock protein 90 alpha (Hsp90alpha) was immobilized on aminopropyl silica via the N terminus to create the Hsp90alpha(NT) column or via the C terminus to create the Hsp90alpha(CT) column. Binding to the exposed C terminus on the Hsp90alpha(NT) column was characterized using frontal chromatography and the C-terminus ligands coumermycin A(1) (CA1) and novobiocin (NOVO). The calculated K(d) 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-Hsp90alpha complex: 22.2+/-8.8 microM(-1) s(-1) and 2.7+/-0.6s(-1), respectively. Binding to the exposed N terminus on the Hsp90alpha(CT) column was characterized using frontal chromatography. The K(d) 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 IC(50) values for inhibition of ATPase activity on the Hsp90alpha(CT) column. The IC(50) for GM was 2.80+/-0.18 microM, and the relative IC(50) 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.
Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
Michał P
MP
Marszałł
Ruin
R
Moaddel
Krzysztof
K
Jozwiak
Michel
M
Bernier
Irving W
IW
Wainer
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D002853:Chromatography, Liquid / Q000379:methods*; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D020128:Inhibitory Concentration 50; D009862:Online Systems
D018841:HSP90 Heat-Shock Proteins; D000251:Adenosine Triphosphatases