Measurement of Binding Constants

I Nucleus iitsciusitive coiupluxalion Sensitive nucleus Analogous nucleus 

Fluorescence titration measurements are based on the proportion of fluorescence intensity to fluorophore concentration (concentration of fluorescent species in solution this is often a fluorescent guest, G). For a 1 1 complex with host, H, with stability constant = [HG]/[H][G] the fluorescence intensity F is given by  

Combining these two relationships gives Equation (1.15), which provides the basis for most fluori-metric methods for stability constant determination  

This equation is greatly simplified for cases where either the guest or host-guest complex are non-fluorescent (i.e. the fluorescence is turned on by complexation, or in the case of quenching by the host), in which case either kQ or kn become zero. For example, for kQ = kS and ku = 0, we obtain  

A simple plot of FJF against [H] from titration of the quenching host into a guest solution should yield a straight line of slope Kn. Common fluorescent guests such as 8-anilino-l-naphthalenesulfonate (ANS, 1.7) may be used to probe complexation ability of various hosts in this way. 

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Higuchi and Lachman [122] pioneered the approach of improving drug stability by complexation. They showed that aromatic esters can be stabilized in aqueous solutions in the presence of xanthines such as caffeine. Thus, the half-lives of benzocaine, procaine hydrochloride, and tetracaine are increased by approximately two- to fivefold in the presence of 2.5% caffeine. This increase in stability is attributed to the formation of a less reactive complex between caffeine and the aromatic ester. Professor K. A. Connors has written a comprehensive textbook that describes methods for the measurement of binding constants for complex formation in solution—along with discussions of pertinent thermodynamics, modeling statistics,... 

These systems possess the simplest possible variation in that only one parameter, i.e., the fluorescence quantum yield is proton-controlled. The insulation of the fluor and receptor modules in the ground state by the alkyl spacer leads to essentially pH-invariant absorption (position, shape, and intensity) and fluorescence spectra (shape and position only). The same pKa values are obtained from fluorimetry or absorption spectroscopy so that the detection sensitivity of excited state experiments can be used for the measurement of binding constants of the ground state. 

DJ Winzor. Measurement of binding constants by capillary electrophoresis. J Chromatogr A 696 1630-1637, 1995. 

Hattori, T., Hallberg, R., Dubin, P.L. (2000). Roles of electrostatic interaction and polymer structure in the binding of p-lactoglobulin to anionic polyelectroytes measurement of binding constants by frontal analysis continuous capillary electrophoresis. Langmuir, 16, 9738-9743. 

Wieseman, T Williston, S., Brandts, J.F., Lin, L.-N. (1989). Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179, 131-137. 

Numerous studies of the thermodynamics of ligand addition to Fe porphyrins have been reported. Three main methods have been utilized for measurement of binding constants (1) UV-visible spectroscopic techniques based on Beer s (2) electrochemical techniques based... 

The measurement of binding constants (also called stability constants or formation constants) is of interest in many areas of chemistry. Quantitative information concerning the products of acid-base, metal-ligand or enzyme-substrate interactions is invaluable in analytical chemistry, industrial process chemistry, biochemistry, etc. 

Numerous studies of the thermodynamics of ligand addition to Fe porphyrins have been reported. Three main methods have been utilized for measurement of binding constants (1) UV-visible spectroscopic techniques based on Beer s law (2) electrochemical techniques based on the Nernst equation (equations 26-28) " " and (3) NMR techniques based on the relative intensity of peaks that are due to known proton types within the starting material, PFeX, and product, PFe(X)(L) or PFeL2" X" (5ee Stability Constants their Determination). In nonaqueous solvents the reaction of ligands with iron (III) porphyrins is found to proceed as in equations (32) and (33) ... 

As with any spectroscopic probe, the wavelength to be used in CD measurements of binding constants must be chosen carefully. It should be a wavelength at which the difference in CD between the free and complexed analyte is at a maximum, but wavelengths near either the short- or long-range limit of the instrument should be avoided because of deterioration in the SIN ratio. 

Measurement of binding constants (Kj) or 50% inhibition concentrations (150) for Iri vitro events usually provides data of far higher precision than that of Iji vivo studies. When carried out on a series of structurally related compounds, the data set (logl/Kj or pI50) can be analyzed in terms of a linear combination of relevant descriptors to provide a mathematical model of the event. The number of descriptors the model will support depends partly on the size and data span of the set and partly on the strength of statistical mea-2... 

In Table 3. a description of different techniques employed in the measurement of binding constants is presented, where A5( H, N) is the average weighted change in or H-amide chemical shift in two-... 

Wiseman T, WiEiston S, Brandts J, Lin L. Rapid Measurement of Binding. Constants and Heats of Binding Using a New Titration Calorimeter. AnalBiochem 1989 79 131-137. 

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