Modified Hummel-Dreyer method

The experimental data obtained by the modified Hummel-Dreyer method were analyzed using Scatchard plots. Binding affinity values were calculated from a Langmuir-type equation. When a compound binds to only one binding site on a protein, the model ean be represented by the following equation ... 

Figure 9.1 Chromatograms obtained using the modified Hummel-Dreyer method. Reproduced by permission of John Wiley Sons, ref. 55.

Fig ure 9.2 Scatchard plots obtained using the modified Hummel-Dreyer method with HSA containing various percentages of glycosylated human serum albumin (GHSA). The GHSA contents of DHSA-II, DHSA-IV, DHSA-VI, DHSA-VII, and HSA II are 33.6, 3.23, 36.9, 63.0, and 59.9%, respectively. 

The k values of acidic and basic drugs measured using the above systems were correlated to their binding affinity log nAT values. The log tiK" values of acidic drugs have been measured previously, and those of basic drugs were measured by the modified Hummel-Dreyer method. These log nK" values are listed in Table 18 of the Appendix (p. 315). The calculated results are shown... 

Table 9.3 Molecular properties of some basic compounds, n i values were measured using a modified Hummel-Dreyer method, values were measured by a two-column method, 11X3 values were measured by a one-column method, log k values were measured by a one-column method, MIPS values were calculated at pH 7.5. Reproduced by permission of Elsevier.

A modified Hummel-Dreyer method was carried out using an inner hydro-phobic bonded silica gel, Wako WS GP-N6 from Wako Chemical (Osaka,... 

Table 18 Human serum albumin-drug binding affinity and drug properties. rrSTi represents log k measured using an immobilized-HSA phase. nKa represents predicted log hsa-represents log k of acidic compounds at pH 7.4. k represents log k of basic compounds at pH 7.4. MIFa and MIFb represents the molecular interaction energy values of acidic and basic compounds, resepectively. nKs represents log nST measured using a modified Hummel-Dreyer method. nJQ, nKs, nSTg and nKj represent values. PB and PB2 represent the binding %. Log Pc are predicted log P values, and log Pm are measured values. 7.4 represents pH 7.4. Reproduced by permission of Bentham Science, ref. 20.

An alternative to dialysis methods is the gel filtration technique of Hummel and Dreyer (57), as modified by Price (58). A column of Sephadex G-25 is equilibrated with a buffer containing a desired concentration of calcium and 45CaCl2, and is used for gel filtration of the binding protein. As the process of gel filtration proceeds, the protein migrates in the excluded volume of the column, removing calcium ions from the column buffer until equilibrium is reached. The protein peak, in the void volume of the column, contains above-base line amounts of calcium. The amount of calcium bound to the protein can be found by dividing the molar concentration of calcium above the base line value by the molar protein concentration. A separate gel filtration run is used to determine each point on the binding plot. This is at once time-consum-... 

In this work, we have used the chromatographic method of Hummel and Dreyer (5) and modified it in order to study the simultaneous binding of ADP and ATP on coupling factor CFl. In the original method, a known quantity q of macromolecule is injected on a gel filtration column which is preequilibrated with a fixed concentration (A) of ligand. 

In addition to endogenous ADP, nucleotide binding to CFl is measured according to Hummel and Dreyer, with a gel filtration TSK SW 2000 (30 cm x 0.75 cm) column, and in the modified method, with an anion exchange TSK DEAE 2SW (25 cm x 0.46 cm) colximn. Eluents... 

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