Table 2.

Dissociation constants (K_d) and enthalpy (ΔH_app) and entropy (TΔS) for Pnt and Zn²⁺ binding to S100B

	Kd (μM), ITC	ΔHapp (kcal/mol)	TΔS (kcal/mol)	Kd (μM), fluorescence
Pnt
Ca2+-S100B (low salt)a,b	53±10c	−4.0±0.2	2.1±0.2d	39±3
Ca2+-S100B (high salt)a,b	50±5c	−4.6±0.4	1.5±0.4d	44±4
Zn2+,Ca2+-S100B (low salt)e,f	64±15	−5.0±0.1	1.0±0.2d	39±5
Zn2+,Ca2+-S100B (high salt)b,e	35±12c	−4.3±0.5	1.6±0.5d	40±5
Zn2+
Pnt-Ca2+-S100B (low salt)g	0.13±0.09	−7.6±0.5	2.1±0.4d	-
Ca2+-S100B (low salt)h	0.09±0.02h	−6.2±0.1h	3.8±0.1d,h	-

^aConditions included 10 mM TES, pH 7.2, 15 mM NaCl, and 10 mM CaCl₂, at 37 °C. For the high-salt conditions, 100 mM KCl.

^bA single dissociation constant (K_d) is reported even though two sites for Pnt binding were detected by ITC for these complexes (n = 1.9±0.2). As expected, the fluorescence data also are best fit with a two-site model (n_measured = 1.8±0.2) for both the Pnt-Ca²⁺-S100B and Pnt-Zn²⁺,Ca²⁺-S100B complex. That n_measured used to fit the fluorescence data is less than or equal to n as determined from ITC is also consistent with having two noninteracting Pnt sites. Thus, the simplest model that fits the data under these conditions is that two independent Pnt binding sites exist with indistinguishable affinities.

^cThe c values range from 3 to 5 for the ITC experiments, indicating that the binding data are appropriate for evaluating K_d values.

^dAn estimate for the entropic contribution to binding (TΔS) was calculated using the dissociation constant (K_d) and the apparent molar enthalpy of association (ΔH_app) for Pnt binding.

^eConditions included 10 mM TES, pH 7.2,15 mM NaCl, 10 mM CaCl₂, and 100–500 μM ZnCl₂, at 37 °C. For the high-salt conditions, 100 mM KCl was added. For the fluorescence titrations in the presence of Zn²⁺, the Zn²⁺-to-S100B ratio did not exceed 1:1 to avoid precipitation (see Materials and Methods).

^fThe ITC data for Zn²⁺,Ca²⁺-S100B under low-salt conditions was evaluated (K_d∼64±15), but due to large amounts of precipitation at high Pnt levels, the stoichiometry was not reliably determined, and this dissociation constant is only considered an estimate. At lower protein concentrations, the fluorescence data were more reliable (K_d = 39±5; n_measured = 1.8±0.1).

^gConditions included 10 mM TES, pH 7.2,15 mM NaCl, 10 mM CaCl₂, and 100–400 μM Pnt, at 37 °C with c values ranging from 700 to 1000.

^hConditions included 10 mM TES, pH 7.2, 15 mM NaCl, and 10 mM CaCl₂, at 37 °C, and the value is from Wilder et al.⁸