Hummel and Dreyer method

STUDY OF THE SIMULTANEOUS BINDING OF ADP AND ATP ON COUPLING FACTOR CFl BY A MODIFICATION OF THE HUMMEL AND DREYER METHOD... 

In the original method, the separation between the free ligand and the complex is based on the difference of size. When there is simultaneous fixation of two ligands of similar molecular weights, separation by gel filtration is difficult or impossible. The respective concentrations of each ligand can be determined if they have different optical characteristics or if one of them is radioactive. In the particular (but frequent) case of an ADP-ATP mixture, the situation is unfavourable same optical spectra, similar sizes. In order to avoid important levels of radioactivity, we have adapted the Hummel and Dreyer method by using anion exchange separation of the protein and of the two nucleotides. 

Study of the Simultaneous Binding of ADP and ATP on Coupling Factor CF by a Modification of the Hummel and Dreyer Method 65... 

Figure 8. Typical elution profiles for A, Hummel and Dreyer method B, "large zone Hu l and Dreyer method" and C, the method of Sebille ( ef 42,43), in which the column is Initially equilibrated with a solution containing both L and M and then a known volume of buffer is injected.

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-... 

Hummel and Dreyer developed a method for determining the stability constant from chromatographic data [48]. A modification of the method was used to calculate the apparent stability constants of steroid-CyD inclusion complexes [49-51]. In this method, varying amounts of CyD are injected on the column equilibrated with the guest compound present in the mobile phase. A negative peak obtained on the chromatogram corresponds to the amount of guest consumed to produce the complex. The area of the positive peak is a measure of the concentration of the complex... 

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... 

An exclusion chromatographic technique has been increasingly used for the studies on metal-ligand binding, which is known as the method of Hummel and Dreyer [ref. 127]. This method allows to determine the stability constants of metal complexes, as well as the metal-ligand binding ratio. The principle of the determination of the stability constants of metal complexes for a simplified model has been intelligibly described by Yoza [ref. 128]. 

The method developed by Hummel and Dreyer is a dynamic equilibration technique based on the fact that complexes, formed between metal ions and the ligand macromolecules, are excluded to a greater or lesser extent from the gel interior whereas the free metal ions completely permeate the gel, thus permitting a separation of the two species. 

An alternative exclusion chromatographic method for the determination of the stability constants of metal complexes was presented by Miyajima et al. [ref. 136, 141]. In contrast to the method of Hummel and Dreyer, this method requires an eluent containing a known concentration of the ligand. The elution volume of metal ion injected, v, was expressed as a function of the ligand concentration in an eluent, [L]q, and the stability constants of the complexes by the following equation... 

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)... 

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... 

A blank control run was performed by injection of the above buffer. Then, at the same eluent composition, vacancy peaks were obtained by injection of the above sample solutions containing only HSA in the buffer. Blank and protein injections were run using the eluents containing different drug concentrations (0.4-50 pM) in an automated sequence, controlled by the workstation. The experimental data obtained by the modified Hummel-Dreyer method were analyzed using Scatchard plots. Binding affinity was calculated from a Langmuir-type equation. 

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.

The Hummel-Dreyer method [25] of size-exclusion chromatography (SEC) has been used for the determination of the stoichiometries of protein-polyelectrolyte complexes. Samsonov [26] in 1969 showed that the strength of the complexes formed by ribonuclease (RNAse) and copolymers of sodium vinyl pyrrolidone and vinyl sulfonate diminishes with reduction in the content of sodium vinyl... 

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