Analysis of the interactions of mixtures of two β-agonists steroids with bovine serum albumin: a fluorescence spectroscopy and chemometrics investigation

Abstract

β-Agonists such as ractopamine (RAC) and clenbuterol (CLEN), have similar effects as anabolic steroids i.e. they promote growth of muscular tissue and reduce body fat. They have been used successfully with animals and humans but have also been banned in many countries principally, because of their serious side effects. However, their illegal use persists. Thus, their interaction with biomolecules such as bovine serum albumin (BSA) is of significance, especially the co-operative reaction of mixed ligands with the protein. Fluorescence and UV-vis spectra of complex mixtures of individual ligands, binary and ternary complexes with BSA resulted in significantly overlapping spectral profiles. Qualitative and quantitative information about the various complex ligand-protein species formed, was obtained with the resolution of the excitation-emission fluorescence three-way data matrices by chemometrics methods-MCR-ALS and PARAFAC. Individual spectra of the ligands, their binary complexes with BSA and their ternary complexes were extracted, and quantitative concentration profiles for each species in a particular interaction were constructed. Such analyses made it possible to interpret the role and behaviour of each reaction component. It was found that both ligands, RAC and CLEN, bound co-operatively in site I of the BSA. This was confirmed with the use of site markers such as warfarin (site I) and ibuprofen (site II). However, CLEN formed a 1:1 CLEN-BSA complex, while RAC formed a 2:1 RAC₂-BSA binary species. Interestingly, when CLEN or RAC was added to RAC₂-BSA or CLEN-BSA, respectively, ternary complexes were produced such as RAC₂-BSA-CLEN. Significantly, the presence of the second ligand in such an interaction in excess, appeared to increase the affinity of the added ligand for BSA. This may have consequences on the amount of steroid required to achieve a desired tissue growth effect.