Info

(MS-binding)

Fig. 7.19 Correlation between pK values from competitive MS binding assays and radioligand binding assays.

nated after a range of different time periods and the marker subsequently quantified by LC-ESI-MS/MS. The resulting dissociation curve is depicted in Fig. 7.21. It shows the typical curve progression for a reversible binding of a ligand to its target [96, 101]. The dissociation rate constant calculated from these experiments was k_1 = 0.094 G 0.003 min"1.

Knowing the rate constants of the target-marker binding experiment gives a different way to determine the dissociation constant Kd independently from saturation experiments [see Eq. (4) in Section 7.2.1]. For this purpose, the association rate constant k+1 has to be calculated from kobs according to Eq. (8):

Kinetic Binding Study No711

XI min

Fig. 7.20 Kinetic MS binding study quantifying bound marker -association experiment. Total binding (■) of NO 711 (20 nM; incubation temperature 37 0C) to mGATl. Nonspecific binding (o) was determined as binding of NO 711 in the presence of 10 mM GABA. One representative example is shown. Data points represent each mean G SEM from triplicate values.

XI min

Fig. 7.20 Kinetic MS binding study quantifying bound marker -association experiment. Total binding (■) of NO 711 (20 nM; incubation temperature 37 0C) to mGATl. Nonspecific binding (o) was determined as binding of NO 711 in the presence of 10 mM GABA. One representative example is shown. Data points represent each mean G SEM from triplicate values.

Kinetic Binding Study No711

Was this article helpful?

0 0

Post a comment