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Affinity Capillary Electrophoresis 327
approximate analyte concentration at least 10 times lower than the lowest ligand
concentration.
5. Process peak appearance shift data according to the relations given above. A direct
binding curve of (1/t) as a function of [L] is plotted to estimate the saturability of
the system and to fit the binding isotherm to the experimental data using non-linear
curve fitting methods. This yields the K d from the formula for a one site-binding
hyperbola.
6.2.2. Procedures for CE-FA as Applied to Drug–Plasma Protein
Interactions
The procedures used for studying low-molecular weight drug binding to
human serum albumin (HSA) (93) by CE-FA are given below. The approach
was found to be applicable to a range of ligands with different physicochemical
properties and should be useful for investigating the interactions of other ligands
and proteins with minor modifications.
6.2.2.1. Materials and Instrumentation
1. HSA and drug samples of adequate purity.
2. Sample and electrophoresis buffer solution: 0.067 M sodium phosphate buffer
(pH 7.4).
3. Deionized water, 1 M NaOH and 0.1 M NaOH for capillary conditioning and rinse
procedures.
4. Uncoated fused silica capillary, suitable dimensions may be 57 cm × 50 m ID,
50 cm effective length. Condition capillaries by flushing with 1 M NaOH, water
and electrophoresis buffer for 30 min each.
5. Commercially available CE-instrument with programmable autosampler.
6.2.2.2. Sample Solutions
1. Prepare HAS-stock solution in electrophoresis buffer and drug-stock solutions in
a suitable solvent. Filter HSA and buffer solutions through 0.45 or 0.22 m pore
size filter before use.
2. Prepare a series of samples containing a constant and known concentration of
HSA, e.g. 55 M and varying drug concentrations. Include a sample without drug
added to check for impurities in the protein sample (see Note 9).
3. Prepare a series of standard solutions containing only the drug for construction of
a calibration curve.
6.2.2.3. FA Experiments
The standards and pre-incubated samples are all subjected to the procedure
listed below:
1. Rinse capillary between measurements by flushing for 2 min each with 0.1 M
NaOH and running buffer.