Binding equilibrium, analysis

Binding of heme by isolated N-domain causes a change in sedimentation coefficient consistent with a more compact conformation and leads to the more avid association with the C-domain (125). Sedimentation equilibrium analysis showed that the Kd decreases from 55 pM to 0.8 pM (Fig. 5) (106). In addition, the calorimetric AH (-1-11 kcal/mol) and AS (-1-65 kcal/mol K) for the heme-N-domain-C-domain interaction and the AH (-3.6 kcal/mol) and AS (-1-8.1 kcal/mol K) derived from van t Hoff analysis of ultracentrifuge data for the interaction in the absence of heme indicate that hydrophobic interactions predominate in the presence of heme and a mix (e.g., hydrophobic and van der Waals forces) drives the interaction in the absence of heme. However, FTIR spectra (Fig. 6) indicate that little change in the secondary structure of domains or intact hemopexin occurs upon heme binding (104). 

Equilibrium dialysis is an analytical technique which is usually performed with commercial apparatus. In this technique, the two dialysis chambers, separated by a membrane, are filled respectively with macromolecule solution and with ligand, which is usually radioactively labelled. After equilibrium has been reached, samples are removed from the two chambers the concentration of free ligand is determined from one sample, and free plus bound ligand from the other. Parameters characterising the binding equilibrium can be determined by appropriate analysis of the data. 

Under conditions where cAttot cBitot, terms involving the total concentration of A do not occur in the analysis (as shown above), and it is therefore not possible to use Eqn. 9.24 to analyse the stoichiometry of the binding equilibrium. However, even under these experimental conditions, it is possible to obtain information about the number of binding sites, provided the binding constants of the two processes are sufficiently different in magnitude. 

D Rosen, MY Okamura and G Feher (1980) Interaction of cytochrome c with reaction centers of Rhodopseudomonas sphaeroides R-26 Determination of number of binding sites and dissociation constants by equilibrium analysis. Biochemistry 19 5687-5692... 

Fig. 3 Set of sensorgrams (model) suitable for equilibrium analysis (left) and binding isotherm with indicated equilibrimn sensorgram results (right). Model of pseudo first-order reaction. Parameters ft = I nM cm, K = 10 M fca = 4.5 x 10 M s ...

Fig. 3 Synthetic phosphopeptides representing sequences around Ros Y2267 and pY2327 were analyzed for binding of the JV-terminal SH2 domain of SHP-1 by surface plasmon resonance. Representative experiments are shown (left panel). The data were fitted to determine the kinetic constants ka and k. Signals at equilibrium were also fit using an equilibrium analysis to determine the affinities of the interactions (right panels). Reproduced from [10] with permission from the Company of Biologists 2004...

Cassman and King have reported cooperative binding of NADH to beef heart s-MDH (67). They also found evidence for the existence of s-MDH monomer from sedimentation equilibrium analysis at low protein concentrations (67). The fluorescence studies of the binding of NADH to beef heart s-MDH were interpreted in terms of a model in which both monomer and dimer bind NADH with cooperative binding to the dimer (67). The dissociation constant for the first site was about 25 /Jkf and for the second site about 0.18 iiM. 

Resonant mirror biosensing is a superb technique for the analysis of receptor-ligand interactions in real time. Therefore, association constants may be determined directly from kinetic constants This may sound a little strange, but let us consider the binding equilibrium shown in Scheme 7.4. By definition, equilibrium is reached when the rate of association (M s ) of receptor with ligand is equalled by the rate of receptor-ligand complex dissociation diss (s )- Hence... 

Na-K ATPase is composed of two different subunits, one of about 100000 (a-subunit) and one of about 50000 (yS-subunit), as shown by sodium dodecyl-sulfate (SDS) gel electrophoresis [75,76]. Since both subunits are glycoproteins and therefore bind different amounts of SDS as compared to normal proteins, their SDS gel electrophoretic mobility, and hence their apparent molecular weights can deviate considerably. Recently the molecular weights of the separated subunits of Na-K ATPase from rabbit kidney outer medulla have been determined more accurately by sedimentation equilibrium analysis in the absence of detergents [77]. The value for the a-subunit thus determined is 131000 (120600 for its protein part) and 61 800 for the /8-subunit (42 800 for its protein part). 

To briefly recount the different methods which are commonly employed for titration analysis, we can begin by considering a simple 1 1 binding equilibrium between a host species (H) and a guest species (G) to form a host-guest complex (HG), as shown in Eq. (8.1) ... 

The theoretical treatment of the model of Aniansson and Wall is not directly applicable to the time constants obtained in ultrasonic experiments, but this gap has been bridged.The amplitude is predicted to be zero at the c.m.c. then increases with concentration to a broad maximum and thence slowly decreases, as observed for sodium dodecyl sulphate. An analysis of amplitudes in P-jump kinetics implies the possibility of a third relaxation process due to a change in electrolyte properties. This counterion binding equilibrium may have been observed in ultrasonic studies of sodium decyl sulphate. Attempts by the former authors to modify the Aniansson and Wall theory... 

Although this experiment is written as a dry-lab, it can be adapted to the laboratory. Details are given for the determination of the equilibrium constant for the binding of the Lewis base 1-methylimidazole to the Lewis acid cobalt(II)4-trifluoromethyl-o-phenylene-4,6-methoxysalicylideniminate in toluene. The equilibrium constant is found by a linear regression analysis of the absorbance data to a theoretical equilibrium model. 

For determining the adsorption isotherm, the equilibrium concentrations of bound and free template must be reliably measured within a large concentration interval. Since the binding sites are part of a solid, this experiment is relatively simple and can be carried out in a batch equilibrium rebinding experiment or by frontal analysis. 

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