Binding constant extrinsic

An alternative way of viewing the concept of extrinsic binding constants is as follows. The experimentally measured binding constant for each site of a polymeric protein, or enzyme, will depend on the number of available sites on each molecule e.g., for the hemoglobin tetramer the first binding reaction is... 

Again, the statistical factors 4, 5, etc., arise because Xr and K-y are intrinsic association binding constants, yet the overall expressions for the complexes requires extrinsic parameters e.g., Kr = [RX]/[R][X] = [R4X2]/[R4X][X] because there are three unfilled sites in the R4X molecule and two in each R4X2 molecule (see also Example 9.12). The fractional saturation Y is again defined by Eq. (9.49) ... 

Static quenching is observed when two molecules bind one to the other forming a complex. Intrinsic fluorophore such as the aromatic amino acids or extrinsic fluorophores such as ANS or fluorescein can be used to follow the interaction between the two interacting molecules. Fluorescence studies will allow deriving the binding parameters of the complex such as the stoichiometry and the binding constant. In these types of studies, the fluorophore is bound to one of the two interacting molecules. Each time the two molecules form a complex, fluorescence parameters such as intensity, anisotropy or / and lifetime of the fluorophore are monitored. 

The goal of these studies is to measure the association constants of the LTF-LCA and STF-LCA complexes. LCA is given as a fluorescein-LCA complex (from Sigma). The extinction coefficient of the complex is 132.996 mM-1 cm-1 at 495 nm. Two molecules of fluorescein are bound to one molecule of LCA. Binding of the STF or LTF to LCA-fluorescein complex induces a variation in the fluorescence intensity of the extrinsic probe. LTF and STF concentrations can be determined spectrophotometrically at 280 nm with an absorption E1 % = 14.3 and 14.0, respectively. 

There are four sites available for binding 02, but only one from which bound 02 can dissociate. Thus, from the law of mass action, the (overall) extrinsic equilibrium constant is equal to 4k /k- = 4X. Similarly, the extrinsic constant denoted by X2 is given by 1K2, where K2 is the intrinsic constant, and by the same reasoning K% = and Ke4 = JK4. 

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