Ligand-binding curve

Figure 3.3 Kinetics of four site ligand binding curves (1) to (5) represent the zero-, mono-, di-, tri- and fully liganded tetramers. Curve (6) represents the total concentration of liganded sites, (a) is based on the reaction of identical and independent sites with statistical multiplication factors (see p. 68) given to the intrinsic rate constant it = Is , (b) is based on a cooperative system (identical sites) with intrinsic rate constants of 0.04 for the first step and 1.0 for the subsequent three steps of ligand binding. In this graph lines 2,3 and 4 are omitted because they all straddle the baseline.

As a prerequisite to the determination of the quantitative structure-function relationship it is necessary to define the parameters with which the system of hemoglobin and its ligands is described at the thermodynamic level. The ligand binding curve expresses the thermodynamic properties of the system at equilibrium and therefore this discussion is limited to the thermodynamic equilibrium. 

Usually non-cooperative and non-Unear binding isotherms were observed in alkaloid-B-DNA complexation and the data were fitted to a theoretical curve drawn according to the excluded site model [126] developed by McGhee and von Hippel [127] for a non-Unear non-cooperative ligand binding system using the following equation ... 

Mattera, R., Pitts, B. J., Entman, M. L., and Bimbaumer, L. (1985) Guanine nucleotide regulation of a mammalian myocardial muscarinic receptor system. Evidence for homo-and heterotropic cooperativity in ligand binding analyzed by computer-assisted curve fitting. J. Biol. Chem. 260,7410-7421. 

Fig. 3 SPR sensorgrams. Upper Binding curves of 20 pg/mL of BclA to immobilized PAA-mannose in the presence of (A) 1.95 pM-0.25 mM a-benzyl-mannoside (the best ligand from tested monosaccharides) and (B) 0.95 mM-25 mM D-galactose (non-binder). Bottom SPR sensorgrams for D-mannose binding to immobilized BclA. (C) Equilibrium steady state curves for D-mannose varying from 1 to 500 pM. (D) The corresponding binding curve derived from steady-state equilibrium values.

The actual Hill plot for Hb is far from a linear line with constant slope. The actual curve has a varying slope between one to three. Thermodynamically, Eq. (6.8.2) implies that all n ligands bind simultaneously to Hb. There is no provision in this model for intermediary occupancy states. Therefore, this model is thermodynamically unacceptable. This is true a fortiori when n, obtained by fitting the experimental data, turns out to be a nonintegral number. 

Fig. 6.4 In vitro effects of mutation on desensitization and internalization of the dopamine receptor. Shown here are effects of mutation on dose-dependent intracellular cyclic adenosine monophosphate (cAMP) accumulation (A and B) and binding curves (C and D) for artificial ligand (SCH 23390) using three constructs controls (wild type, A and C) and the Thr360Ala mutant (360, B and D). In the desensitization experiments, cells were preincubated with 10 oA/ dopamine (o) or vehicle ( ) for 20min, and increasing concentrations of dopamine (10 to 10 (iM) were added to assess cAMP accumulation. Note that loss of efficacy and potency seen in wild-type cells (A) disappeared with the Thr360Ala mutation (B). Conversely, internalization, assessed by decrease in SCH23390 binding (C) after pretreatment with lOpM dopamine (o, compared to vehicle ), was essentially unchanged by the Thr360Ala mutation (D)...

Therefore, plotting the ALIS MS response from a titration series versus the total ligand concentration yields a saturation binding curve that can be fit to this equation by nonlinear regression analysis to yield the of the ligand of interest. 

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