TABLE 2.

Kinetic constants of native EGFP and mutant EGFPi to lipid A in different diluents and before removal of lipopolysaccharide

DiluentaEGFPi G10EGFPi G12EGFP (G0)S3Δ
KD (10−6 M−1)FU/μMbKD (10−6 M−1)FU/μMKD (10−3 M−1)FU/μMKD (10−6 M−1)
Water3.10c410.7 ± 11.50.4415.3 ± 2.52.59d605.1 ± 150.30.59
Tris-HCl, 50 mM, pH 7.34.18394.4 ± 4.20.9014.7 ± 0.21.81653.7 ± 3.50.011
150 mM NaCl in Tris-Cl, pH 7.39.58426.3 ± 9.60.5514.3 ± 0.50.10599.6 ± 0.30.83
Phosphate-buffered saline (150 mM NaCl, pH 7.3)24.4456.0 ± 16.22.6816.5 ± 3.10.31570.4 ± 124.6e
Sodium acetate, 50 mM, pH 6.00.27288 ± 4.80.1114.8 ± 0.31.79554.7 ± 10.40.003
Dialyzed extract (no lipopolysaccharide removal) Tris-HCl, pH 7.3289360 ± 4.666.512.5 ± 0.428.3782.3 ± 3.4
  • a To compare the effect of other parameters such as buffer, salt, and the presence of lipopolysaccharide all sensorgrams were run at the same pH except for water.

  • b Unit fluorescence (FU) was measured with a spectrofluorimeter (LS 50B; Perkin Elmer) with three independent measurements. Fluorescence in water and phosphate-buffered saline was highly variable, especially for native EGFP (G0).

  • c The equilibrium kinetic constant of complex lipid A-EGFPi, KD = kd/ka, was calculated from the surface plasma resonance rate constants. Association (ka) and dissociation (kd) rate constants were determined from experiments on E. coli lipid A monolayer immobilized on the HPA biosensor chip at 25°C with the BIACORE 2000. Kinetic constants were calculated from the sensorgrams by fitting with a 1:1 Langmuir model; all constants were derived from an average of five independent runs at five different concentrations (0.5 to 6 μM).

  • d The KD of native EGFP (G0) was mainly attributed to the dissociation from nonspecific interaction of G0 extract.

  • e —, not applicable.