Characterization of bovine serum albumin/chlorogenic acid solution mixtures by analytical ultracentrifugation

摘要

The aim of this work was to investigate a possible interaction between bovine serum albumin (BSA) and chlorogenic acid (CGA) in KH2PO4/K2HPO4 buffer (pH 7.0; ionic strength 0.1 mol/l) depending on the BSA/CGA molar ratio by analytical ultracentrifugation (sedimentation velocity) using an OPTIMA XL-A AUC equipped with a UV–VIS absorption optical system. The protein concentration of all the solution mixtures with BSA-to-CGA molar (mass) ratios of 1:19 (10:1) up to 1:95 (2:1) was 0.50 g/l. The investigations were carried out under experimental conditions where the possibility of derivatization of the BSA (covalent bonding) is minimized (pH, temperature, investigation of the solutions immediately after mixing). At 280 nm both sedimenting BSA and BSA with non-covalent-bound CGA can be detected. In the region of 320 nm BSA shows no absorption, and thus only BSA with bound CGA (absorption maximum at 325 nm) can be monitored. The results indicate weak interaction between BSA and CGA. At a scan wavelength of 280 nm sample solutions with a BSA-to-CGA molar ratio of 1:19 revealed a major component of 4.2 S similar to the major component of the BSA control sample. However a further component with a lower Sc,20 value (2.3–2.5 S) appears and this is not present in the BSA control sample. At 320 nm the sedimentation of a major component with an Sc,20 value of 1.8–2.0 S followed by a 3.1 S component could be demonstrated. This indicates that the binding of CGA to BSA results in considerably lower Sc,20 value components. The cause could be a more asymmetric particle, a swollen structure and/or ligand-induced partial unfolding of the BSA molecule. Similar results have been obtained with BSA/CGA solutions up to a molar ratio of (1:95). UV–VIS absorption measurements and studies of intrinsic tryptophan fluorescence of BSA/CGA solution mixtures at various CGA concentrations also indicate such interactions between BSA and CGA. Some possible physiological consequences of the BSA/CGA interaction are considered.