Quantitative TSA Also Allows the Calculation of the

Dissociation Constant. To confirm the apparent

dissociation constant calculated by the ANS quenching assay, we conducted a quantitative thermal shift assay using increasing concentrations of GW0742 (positive control) and extract 2.

Implementing various physical chemistry concepts to explain physiological phenomena has led to an understanding that the

dissociation constant of a drug--receptor complex ([K.sub.d]) is one of the most fundamental characteristics that describe the pharmacological properties of drugs, as well as the bioactivity of various toxins and biosimilars in general.

[2] Nonstandard abbreviations: DRI, dietary reference intake; [K.sub.d],

dissociation constant; fT4, free thyroxine; fT3, free triiodothyronine; TSH, thyroid-stimulating hormone; FSH, follicle-stimulating hormone; LH, luteinizing hormone.

[K.sub.i1-2] is the

dissociation constant for the complex E[I.sub.2] (formed by the reaction E + 2I [right arrow] E[I.sub.2]) and all other parameters have the meanings already specified above.

(II) Association/dissociation of Bb-cC (association constant [k.sub.2];

dissociation constant [l.sub.2]):

where, R is the gas constant, T is the absolute temperature and K is the same as the solubility-product constant for water-insoluble compound or the

dissociation constant for cation-chelate.

The

dissociation constant was calculated from the binding constant ([K.sub.d] = 1/[K.sub.b]).

Frontal affinity chromatography (FAC) thus established allowed us by a simple experiment to determine the most important parameter of specific interaction, that is, an equilibrium constant (

dissociation constant or association constant).

where, [[cAMP].sub.C1] and [[cAMP].sub.C2] are the cAMP concentrations in compartments 1 and 2, A[C.sub.basal] is basal adenylyl cyclase activity (cAMP synthesis), [V.sub.maX] is the maximal rate of cAMP hydrolysis by PDE, [K.sub.m1] is the Michaelis constant for PDE activity, [I] is the concentration of PDE inhibitor, KI is the inhibitor

dissociation constant, [k.sub.1] and [k.sub.2] are the cAMP flux coefficients between C1 and C2, and [V.sub.1] and [V.sub.2] are the volumes of C1 and C2.

Thus acid

dissociation constant of thiol group was not determined in our experimental conditions.

The

dissociation constant of the MREs was obtained in Origin 8 (OriginLab Corporation; Northampton, MA) using nonlinear regression analysis as previously described with the equation Y = ((Bmax*X)/([K.sub.d]+X))+NS* X where Bmax is maximum binding, [K.sub.d] is the

dissociation constant, and NS is nonspecific binding [32].

4'OH-PCB 12 had the highest-affinity binding in this study, and it exhibited a significantly lower

dissociation constant ([K.sub.d1] = 2.7 nM) than its sulfate (16.8 nM) and [T.sub.4] (4.7 nM).