Protein ligand equilibria

Let v5 be the number or moles of solute molecules adsorbed per area of surface. The subscript on v refers to the case of adsorption on a solid surface as contrasted with v used in the case of protein-ligand equilibria 4-47 49). Let [A] be the equilibrium solute concentration. 

The effect of pH on protein-ligand equilibria is discussed and the equations are applied to the binding of succinate, D-tartrate, L-tartrate, and meso-tartrate by the catalytic site of fumarase. 

Before discussing the effect of pH on protein-ligand equilibria, it is necessary to discuss an aspect of acid dissociations that was too advanced for Chapter 1. Consider a protein A that has two acid groups. The acid dissociation constants are defined by... 

Visser, A. J. W. G., and Lee, J. (1980). Lumazine protein from the bioluminescent bacterium Photobacterium phosphoreum. A fluorescence study of the protein-ligand equilibrium. Biochemistry 19 4366-4372. 

Using sub-ambient temperatures for preparing the protein-ligand equilibrium mixtures and for centrifugation of the GPC spin column, the dissociation rate constant decreases and the off-rate diminishes, thereby expanding the kinetic window observable with GPC spin column screening to even weaker binders with Kd values >20 pM. 

Equilibria in Solution The stability of a protein-ligand complex in solution is measured in terms of the equilibrium constant and the standard free energy of association based on it. For association of species P and L in solution to form a complex PL, i.e., for... 

The Determination of Equilibrium Dissociation Constants of Protein-Ligand Complexes by NMR... 

In a mixture of protein and ligand there are three species present in equilibrium the free ligand (L), the free protein (P) and the protein-ligand complex (PL). Assuming one binding site, the equilibrium concentrations [P], [L] and [PL] are described by ... 

In the isotope edited/ filtered spectra of a protein-ligand complex, the species actually observed is generally the complex itself. This is an important difference from transferred NOE or saturation difference techniques, where the existence of an equilibrium between free and bound species - and a certain rate of exchange between them - is essential (Chapts. 13 and 16). The general conditions for isotope filtering/editing are therefore identical to those required for standard protein NMR sample concentrations are usually limited by availability and solubility of the components to the order of 1 mM. Considerably lower concentrations will reduce the sensitivity of the experiments to unacceptable levels,... 

Fig. 2.3 Plots of the concentration of the protein-ligand complex present at equilibrium [CJ (pM, shown as pM) as a function of the binding constant (pM), with various initial concentrations of protein [P]q and ligand [1]. Note that the [CJ values are the concentrations of the protein-ligand complex just prior to the GPC spin...

The expression for the equilibrium concentration of the protein-ligand complex [C], described above using Eq. (5), can also be re-written in terms of the total initial protein concentration [Pj such that ... 

The protein-ligand binding affinity is usually expressed as the equilibrium dissociation constant, K, which is described by the following relationship between the concentrations of free receptor [ ], free ligand [S], and the receptor-ligand complex [ES ... 

ALIS measures the MS response of the ligand following its dissociation from the protein-ligand complex. Therefore, the magnitude of the MS response corresponds to the equilibrium concentration of the receptor-ligand complex concentration [ S] times the compound s MS calibration factor Cms, which depends on the ionization efficiency and other molecular properties of the ligand ... 

It should also be noted that when the rate of change in the protein-ligand complex concentration is zero (by definition, when the system is at equilibrium), this equation reduces to the equilibrium expression below, with the binding affinity constant defined as the ratio of the dissociation rate koff to tho association rate kon-... 

As mentioned above in a qualitative sense, it can be seen from this equation that, for a given association rate constant kon, lower value of dissociation rate constant koff yields a smaller value of Ka and hence a higher equilibrium concentration of the desired protein-ligand complex. 

Next to the detection of enzyme inhibition, ESI-MS can also be used to monitor protein-ligand interaction, employing an assay format similar to fluorescence-based receptor assays. Using a similar continuous-flow analytical screening system as shown in Fig. 5.2, a competitive assay can be set up using ESI-MS to measure the interaction of the analyte(s) with an affinity protein such as an antibody, receptor or enzyme [28]. Figure 5.10 shows the equilibrium reactions that form the basis of the assay concept. In a first step, the sample was injected into a con-... 

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