complexes part 11

The equilibrium dialysis procedure was refined for studying the cornplexation between metal ions or small molecules and macromolecules that cannot pass through a semipermeable membrane. According to the equilibrium dialysis method, the serum albumin (or other protein under investigation) is placed in cellulose tubing or similar dialyzing membrane. The tubes are tied securely and suspended in vessels containing the drug in various con¬centrations, and controls and blanks are run to account for the adsorption of the drug and the protein on the membrane.
If binding occurs, the drug concentration in the sac con¬taining the protein is greater at equilibrium than the con¬centration of drug in the vessel outside the sac. Samples are removed and analyzed to obtain the concentrations of free and complexed drug.
Equilibrium dialysis is the classic technique for protein binding and remains the most popular method. Some poten¬tial errors associated with this technique are the possible bind¬ing of drug to the membrane, transfer of substantial amounts of drug from the plasma to the buffer side of the membrane, and osmotic volume shifts of fluid to the plasma side.
Ultrafiltration methods are perhaps more convenient for the routine determination because they are less time con¬suming. The ultrafiltration method is similar to equilibrium dialysis in that macromolecules such as serum albumin are separated from small drug molecules, Hydraulic pressure or centrifugation is used in ultrafiltration to force the solvent and the small molecules, unbound drug, through the membrane while preventing the passage of the drug bound to the pro¬tein. This ultrafiltrate is then analyzed by spectrophotometry or other suitable technique.
The concentration of the drug that is free and unbound, Df, is obtained by use of the Beer s law equation [equation 4-9]
 where A is the spectrophotometric absorbance (dimension-less), ? is the molar absorptivity, determined independently for each drug, c (Df) is the concentration of the free drug in the ultrafiltrate in moles/liter, and b is the optical path length of the spectrophotometer cell, ordinarily 1 cm. The following example outlines the steps involved in calculating the Scatchard r value and the percentage of drug bound.
Example: The binding of sulfamethoxypyridazine to human serum albumin was studied at 25°C, pH 7.4, using the ultrafiltration technique. The concentration of the drug under study, [Dt], is 3.24×10^–5 mole/liter and the human serum albumin concentration, [Pt], is 1.0 ×10^–4 mole/liter. After equilibration the ultrafiltrate has an absorbance, A, of 0.559 at 540 nm in a cell whose optical path length, b, is 1 cm. The molar absorptivity, ?, of the drug is 5.6 ×10⁴ liter/mole.cm. Calculate the Scatchard r value and the percentage of drug bound.
 The concentration of free (unbound) drug, [Df], is given by
 The concentration of bound drug, [Db], is given by
The percentage of bound drug is