Scatchard Technique

The arch for target(s) in vivo is practically insuperable. Thus, the only possible approach is to investi te in vitro eventual interactions of the drug with biological targets such as DNA, RNA, proteins and others. The first step of such in vitro studies consists in observing interactions with DNA, owing to the fact that this nucleic acid is generally considered as the most deeply involved in the intimate processes of life and death. This explains why we focused firstly our attention and efforts on the study of MYKO 63-DNA eventual interactions by two suitable techniques for this purpose, namely the Scatchard technique and Raman spectroscopy... 

The scatchard technique consists of studying the difficulties encountered by a fluorescent dye, ethidium bromide (Fig. 19), with respect to its intercalation between the base pairs of DNA when the secondary structure of the macromolecule is modified by binding (or intercalation) of a drug. 

Nevertheless there exist strong covalent interactions in vitro between MYKO 63 and its relatives and DNA. However, the Scatchard technique does not give the actual sites the drug will graft on. In other words, we had to look for a more local technique which would allow to reach the location in DNA of these sites. Raman spectroscopy was chosen for this purpose because it had previously proved successful for studying in this way interactions of many biological systems with nucleosides or DNA... 

During the study of N3P3AZ6-DNA complexes by the Scatchard technique it was shown that an incubation time of at least 5 days is necessary after mixing the MYKO 63 with DNA on Day D before complete interaction is observed. These results are clearly confirmed by the present work as no fundamental discrepancy was observed between the spectra of the free and bound MYKO 63 when the latter was deduced from the recorded spectrum of the MYKO 63-DNA complex on Day (D -h 1). 

Anyhow, a combination of the Scatchard technique and Raman spectroscopy shows (i) that SOAz actually interacts with DNA at the level of ribose backbones and (ii) that this kind of interaction does not drastically modify the DNA secondary structure, ethidium bromide encountering no more difficulty to intercalate between DNA plates SOAz being grafted or not on the nucleic acid. Thus, the behaviour of MYKO 63 and of SOAz appears quite different with respect to their mode of interaction with DNA despite their close chemical and molecular structure. This surprising observation may be of interest for understanding why SOAz does not induce any cumulative toxicity in vivo in contrast with MYKO 63. 

There are many ways in which hormone-receptor interactions may be studied, the classic method being the Scatchard technique, named in honor of George Scatchard. This technique is applicable to any protein-small molecule interaction, and it provides a means of determining the heterogeneity of binding sites, dissociation constants, and the number of binding sites per receptor unit. The last may be a protein molecule, a cell, a cell membrane fragment, or a unit volume of cytosol with a known protein content. 

A historically important graphical technique to describe binding data (see O Figure 10-2) is the Scatchard plot (1949). This is simply a linearization of the function ... 

The cohesive energy density of a pure compound can be estimated by different techniques (Scatchard, 1949 Hoy, 1970 Fedors, 1974). One method (Hildebrand et al., 1970) relates this parameter to the molar enthalpy of vaporizaMiflv, of the same compound ... 

Bmax A direct measure of receptor concentration or density determined using radioligand-binding techniques, commonly in units such as pmoles/mg membrane protein. It is the asymptotic maximum of a binding isotherm, but is usually determined by calculation from the form of the isotherm (or less satisfactorily from the Scatchard plot). It is the measured... 

There are instances where data are compared to models that predict linear relationships between ordinates and abscissae. Before the widespread availability of computer programs allowing nonlinear fitting techniques, linearizing data was a common practice because it yielded simple algebraic functions and calculations. However, as noted in discussions of Scatchard analysis (Chapter 4) and double reciprocal analysis (Chapter 5), such procedures produce compression of data points, abnormal emphasis on certain data points, and other unwanted aberrations of data. For these reasons, nonlinear curve fitting is... 

A novel new technique employing fluorescence correlation spectroscopy has confirmed specific interactions between a tetramethyirhodamine-labeled kavain derivative and human cortical neurons. Human cortical neurons were incubated with 1 nM of the dye-labeled kavain derivative. A total binding of 0.55 nM was found after an incubation period of 60 minutes. Fifty percent of the total binding was specifically displaced in the presence of 1 XM non-labeled (+)-kavain. Evidence for these specific interactions was verified by a saturation experiment. Both the non-linear Scatchard plot and the n value of 1.58 0.07 in the sigmoid Hill plot indicated binding sites with different binding affinities (Boonen etal., 2000). 

The concentration dependence of ICD has widely been used to investigate intermo-lecular interactions in solution [5, 7, 18]. Several techniques for the calculation of association constants (K ss) have been applied, for instance Benesi-HUdebrand [19], Scatchard plots, [20, 21] and, with the growing capacities of computer calculations, nonlinear curve-fitting procedures [22, 23]. As already mentioned, CyD complexes of type A seem especially well suited for determination of complex stabilities by ICD. Of course, the reliability of the calculated K ss largely depends... 

Gaw and Swinton eliminated the external manometer by using a differential technique. The difference between the vapour pressure of the mixture and the vapour pressure of one of the pure components was measured directly. Adsorption problems were reduced by using mercury cut-off devices in place of taps. With the differential method the error due to temperature differences among a set of supposedly isothermal measurements is much reduced. Scatchard,... 

The above conclusions do not imply a general preference for cooling curves since the static technique of Giauque and Kunzler (1952), which was pioneered by Scatchard et al. (1932) and Prentiss and Scatchard (1933), is certainly the ultimate in precision and accuracy. 

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