Characterization of solute-protein interactions using high performance affinity chromatography and affinity capillary electrophoresis.

摘要

High-performance affinity chromatography and affinity capillary electrophoresis is used for the study of interactions between drugs and human serum albumin (HSA). In chapter 2 and 3, frontal analysis was used to determine the association equilibrium constant and binding capacity for carbamazepine on immobilized HSA column at various temperatures. The results indicated that carbamazepine had a single binding site on HSA with an association constant of 5.3 × 103 M−1 at pH 7.4 and 37°C.{09}This was confirmed through the use of zonal elution self-competition studies. The value of ΔG for this reaction was −5.35 kcal/mol at 37°C, with an associated change in enthalpy (ΔH) of −6.45 kcal/mol and a change in entropy (ΔS) of Δ3.56 cal/mol·K. The location of this binding region was examined by competitive zonal elution experiments using probe compounds with known sites on HSA. It was found that carbamazepine had direct competition with L-tryptophan, a probe for the indole-benzodiazepine site of HSA, but allosteric interactions with probes for the warfarin, tamoxifen and digitoxin sites. Changes in the pH, ionic strength, and organic modifier content of the mobile phase were used to identify the predominant forces in the carbamazepine-HSA interaction.; In chapter 4, a method was developed and evaluated for preparing N-hydroxysuccinimide (NHS)-activated silica for use in immobilizing human serum albumin (HSA) in HPLC columns. HSA column prepared in this manner showed excellent chrial separation ability and long term stability on separation of racemic warfarin and tryptophan.; A technique based on affinity capillary electrophoresis (ACE) and chemically-modified proteins was used to screen the binding sites of various drugs on human serum albumin (HSA) in chapter 5. This involved using HSA as a buffer additive, following the site-selective modification of this protein at two residues (tryptophan 214 or tyrosine 411) located in its major binding regions. The results of this method showed good agreement with those of previous reports.; In chapter 6, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and electrospray ionization mass spectrometry (ESI-MS) were used to study the preparation of singly labeled protein from conjugation of myoglobin with fluorescent molecules and the kinetics of this conjugation reaction.