Merck
  • Home
  • Search Results
  • Species-dependent binding of tocainide analogues to albumin: affinity chromatography and circular dichroism study.

Species-dependent binding of tocainide analogues to albumin: affinity chromatography and circular dichroism study.

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (2014-01-30)
Marco Pistolozzi, Cecilia Fortugno, Carlo Franchini, Filomena Corbo, Marilena Muraglia, Myriam Roy, Guy Félix, Carlo Bertucci
ABSTRACT

A series of novel tocainide analogues were characterized for their HSA and RSA binding, by using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). In this HPLAC study, HSA and RSA were covalently immobilized to the silica matrix of HPLC columns, with a procedure that maintained unaltered the binding properties of the proteins. The tocainide analogues were ranked for their affinity to HSA and RSA on the basis of their bound fractions measured by the two albumin-based columns. This technique was also applied to characterize the high affinity binding sites of these tocainide analogues to the protein. For this purpose displacement experiments were carried out by means of increasing concentrations in the mobile phase of competitors known to bind selectively to the main binding sites of HSA. The results obtained with the immobilized proteins were confirmed by investigating the same drug-protein systems in solution by circular dichroism. The comparison of the data collected with both methodologies highlighted the dramatic effect of small differences in the amino acidic sequences of the two proteins. In fact, despite their similar primary and secondary structures, a small difference in the amino acidic sequence leads to significant differences in their three-dimensional structure reflecting their different binding capacity and their stereoselectivity. Therefore, this study confirms how it is crucial to consider the significant differences among the animal models when performing pharmacokinetic studies. It is also clear that the knowledge of serum carrier binding parameters at an early stage of drug discovery represents a great advantage that may help to save time and efforts.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ketoprofen, ≥98% (TLC)
Sigma-Aldrich
Ketoprofen, meets USP testing specifications
Sigma-Aldrich
Furosemide
Sigma-Aldrich
Potassium phosphate dibasic, reagent grade, ≥98.0%
Sigma-Aldrich
L-Tryptophan, reagent grade, ≥98% (HPLC)
Sigma-Aldrich
Potassium phosphate dibasic solution, 1.0 M
Sigma-Aldrich
Potassium phosphate dibasic, 99.95% trace metals basis
Sigma-Aldrich
Valproic acid sodium salt, 98%
Sigma-Aldrich
Potassium phosphate monobasic, for molecular biology, ≥98.0%
Sigma-Aldrich
Potassium phosphate monobasic, powder, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.0%
Sigma-Aldrich
Bilirubin, ≥98% (EmM/453 = 60), powder
Sigma-Aldrich
L-Tryptophan, from non-animal source, meets EP, JP, USP testing specifications, suitable for cell culture, 99.0-101.0%
Sigma-Aldrich
Potassium phosphate dibasic, meets USP testing specifications
Sigma-Aldrich
Potassium phosphate monobasic, 99.99% trace metals basis
Sigma-Aldrich
Potassium phosphate dibasic, anhydrous, for luminescence, for molecular biology, BioUltra, ≥99.0% (T)
Sigma-Aldrich
Bilirubin, purum, ≥95.0% (UV)
Sigma-Aldrich
Potassium phosphate monobasic, tested according to Ph. Eur., anhydrous
Sigma-Aldrich
L-Tryptophan, BioUltra, ≥99.5% (NT)
Sigma-Aldrich
Potassium phosphate monobasic, BioUltra, for molecular biology, anhydrous, ≥99.5% (T)
Sigma-Aldrich
Potassium phosphate monobasic, ReagentPlus®
Supelco
Ketoprofen, VETRANAL®, analytical standard
Supelco
Monobasic Potassium Phosphate, Pharmaceutical Secondary Standard; Certified Reference Material
Ketoprofen, European Pharmacopoeia (EP) Reference Standard
USP
Monobasic potassium phosphate, United States Pharmacopeia (USP) Reference Standard
Supelco
L-Tryptophan, certified reference material, TraceCERT®
Furosemide, European Pharmacopoeia (EP) Reference Standard
USP
Furosemide, United States Pharmacopeia (USP) Reference Standard
Supelco
Furosemide, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
Ketoprofen, Pharmaceutical Secondary Standard; Certified Reference Material
USP
Ketoprofen, United States Pharmacopeia (USP) Reference Standard