Hummel-Dreyer method

Several variants of separation methods based on dialysis, ultrafiltration, and size exclusion chromatography have been developed that work under equilibrium conditions. Size exclusion chromatography especially has become the method of choice for binding measurements. The Hummel-Dreyer method, the vacancy peak method, and frontal analysis are variants that also apply to capillary electrophoresis. In comparison to chromatographic methods, capillary electrophoresis is faster, needs only minimal amounts of substances, and contains no stationary phase that may absorb parts of the equilibrium mixture or must be pre-equilibrated. 

As the other methods, the Hummel-Dreyer method was first developed for chromatography and then adapted to capillary electrophoresis (21). A small sample of the ligand, dissolved in buffer, is injected into the capillary. The capillary as well as source and destination vial are filled with buffer containing the substrate. In the ligand-containing sample, the initial concentration of the free substrate is depleted to the equilibrium concentration (see Fig. 7a). The depletion peak moves with the velocity of the substrate, and its area corresponds to the bound substrate. As demonstrated in Fig. 7b, the... 

Another technique that has been used in CE format for chiral drug-protein interactions is the Hummel-Dreyer method (52). In this technique, the solute is dissolved in the run buffer at varying concentrations, creating a high detector background response. After equilibration of the system, mixtures of the ligand and protein in various ratios are injected into this system as a sample. 

The binding parameters of (RS)-, (R)-, and (S)-carvedilol to human AGT determined by this technique were in good agreement with the parameters obtained by HPLC, which indicates the reliability of the Hummel-Dreyer method in the CE mode for the evaluation of protein-drug interactions. 

Busch et al. used the Hummel-Dreyer method to investigate the interactions between BSA and warfarin (53). For the binding constant calculations these authors used a nonlinear regression of the following equation ... 

The low selectivity and adsorption effect of proteins on capillary wall have been noted as disadvantages of the Hummel-Dreyer method in CE (54). 

Fig. 1 Schematic representation of the experimental setups of the mobility-shift method and the Hummel-Dreyer method (A) the vacancy peak method and the vacancy affinity capillary electrophoresis method (B) the equilibrium-mixture method and the frontal analysis method (C) for drug-protein binding analysis. drug protein gg drug-protein complex Q buffer. (Reprinted with permission from Ref. 38. Copyright 1992 Elsevier Science.)...

In a similar study, the Hummel-Dreyer method was used for the investigation of the affinity of frusemide, ceftriaxone, and ICI 118551 to HSA and AGP, and a focus was also set on a comparison with HPLC results (52). 

Rudnev, A.V., Aleksenko, S.S., Semenova, O., Hartinger, C.G., Timerbaev, A.R., Keppler, B.K. Determination of binding constants and stoichiometries for platinum anticancer drugs and serum transport proteins by capillary electrophoresis using the Hummel-Dreyer method. J. Sep. Sci. 28, 121-127 (2005)... 

The Hummel-Dreyer method is useful for studying competitive interactions of two drugs towards the same protein. The method is mostly applied to drugs that are weakly retained in an aqueous medium on size-exclusion supports. The use of ion exchangers can give better peak resolution. 

Samples to be injected were prepared by dissolving 2.5 mg PDMDAAC per ml in the mobile phase, along with increasing amoimts of BSA. All samples were filtered (0.20 pm Millipore) before injection. Injections were performed in mobile phases of various pH, all below pHcritical- To determine the stoichiometries of polymer-protein complexes, we used the Hummel-Dreyer method (24), as recently applied to dextran sulfate-hemoglobin complexes (25). 

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.

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