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. Author manuscript; available in PMC: 2011 Mar 8.

Published in final edited form as: J Phys Chem B. 2010 Sep 2;114(49):16125–16130. doi: 10.1021/jp105502c

In all cases binding of Na⁺ obeys a two-step mechanism, with a fast phase completed within the dead time (<0.5 ms) of the spectrometer, followed by a single-exponential slow phase. The k_obs for the slow phase decreases with increasing [Na⁺] (see also Fig. 2). Experimental conditions are: 100 nM enzyme, 50 mM Tris, 0.1% PEG8000, pH 8.0, at 15 °C. Continuous lines were drawn using the expression b-aexp(−k_obst) with best-fit parameters: (A) a=0.000±0.000 V, b=8.239±0.001 V (0 mM Na⁺); a=0.000±0.000 V, b=8.470±0.001 V (3.25 mM Na⁺); a=0.175±0.002 V, k_obs =120±4 s⁻¹ , b=8.942±0.001 V (12.5 mM Na⁺); a=0.392±0.001 V, k_obs=96±1 s⁻¹ , b=9.340±0.002 V (200 mM Na⁺). (B) a=0.000±0.000 V, b=8.112±0.001 V (0 mM Na⁺); a=0.000±0.000 V, b=8.601±0.001 V (20 mM Na⁺); a=0.261±0.002 V, k_obs=64±4 s⁻¹ , b=9.391±0.001 V (120 mM Na⁺); a=0.488±0.001 V, k_obs=60±1 s⁻¹ , b=9.915±0.002 V (400 mM Na⁺). (C) a=0.000±0.000 V, b=8.485±0.001 V (0 mM Na⁺); a=0.162±0.001 V, k_obs=31±1 s⁻¹ , b=8.501±0.001 V (curve in the middle not labeled, 25 mM Na⁺); a=0.458±0.001 V, k_obs =27±1 s⁻¹ , b=8,608±0.001 V (300 mM Na⁺). The concentration of Na⁺ was changed in all cases by adding NaCl without keeping the ionic strength constant. The amplitude of the slow phase for factor Xa and activated protein C becomes less pronounced in the presence of Ca²⁺ (data not shown), reflecting the positive linkage between Na⁺ and Ca²⁺ binding in these proteases ⁵¹,⁵²,⁵⁴-⁵⁸. Binding of Ca²⁺ likely shifts the E*-E equilibrium in favor of E. In the case of thrombin, Ca²⁺ has no effect on Na⁺ binding.

In all cases binding of Na⁺ obeys a two-step mechanism, with a fast phase completed within the dead time (<0.5 ms) of the spectrometer, followed by a single-exponential slow phase. The k_obs for the slow phase decreases with increasing [Na⁺] (see also Fig. 2). Experimental conditions are: 100 nM enzyme, 50 mM Tris, 0.1% PEG8000, pH 8.0, at 15 °C. Continuous lines were drawn using the expression b-aexp(−k_obst) with best-fit parameters: (A) a=0.000±0.000 V, b=8.239±0.001 V (0 mM Na⁺); a=0.000±0.000 V, b=8.470±0.001 V (3.25 mM Na⁺); a=0.175±0.002 V, k_obs =120±4 s⁻¹ , b=8.942±0.001 V (12.5 mM Na⁺); a=0.392±0.001 V, k_obs=96±1 s⁻¹ , b=9.340±0.002 V (200 mM Na⁺). (B) a=0.000±0.000 V, b=8.112±0.001 V (0 mM Na⁺); a=0.000±0.000 V, b=8.601±0.001 V (20 mM Na⁺); a=0.261±0.002 V, k_obs=64±4 s⁻¹ , b=9.391±0.001 V (120 mM Na⁺); a=0.488±0.001 V, k_obs=60±1 s⁻¹ , b=9.915±0.002 V (400 mM Na⁺). (C) a=0.000±0.000 V, b=8.485±0.001 V (0 mM Na⁺); a=0.162±0.001 V, k_obs=31±1 s⁻¹ , b=8.501±0.001 V (curve in the middle not labeled, 25 mM Na⁺); a=0.458±0.001 V, k_obs =27±1 s⁻¹ , b=8,608±0.001 V (300 mM Na⁺). The concentration of Na⁺ was changed in all cases by adding NaCl without keeping the ionic strength constant. The amplitude of the slow phase for factor Xa and activated protein C becomes less pronounced in the presence of Ca²⁺ (data not shown), reflecting the positive linkage between Na⁺ and Ca²⁺ binding in these proteases ⁵¹,⁵²,⁵⁴-⁵⁸. Binding of Ca²⁺ likely shifts the E*-E equilibrium in favor of E. In the case of thrombin, Ca²⁺ has no effect on Na⁺ binding.