Absolute on-line molecular mass analysis of basic fibroblast growth factor and its multimers by reversed-phase liquid chromatography with multi-angle laser light scattering detection

The multi-angle laser light scattering (MALLS) detection method was combined with reversed-phase high-performance liquid chromatography to analyze multimerization of basic fibroblast growth factor (bFGF) formed by oxidation of bFGF with air or with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). This analysis provided the absolute molecular mass and the mean square radius for each eluted protein fraction or each slice of the chromatogram. It was shown that depending on the oxidation conditions, bFGF forms different multimeric forms, from dimers to hexamers. It was found that these multimers have varied conformations of the same molecular mass, but different structure. Molecular mass and size analyses provided molecular conformation of the aggregates; the results indicated the formation of rod-like rigid structures. The MALLS analysis confirmed that, during oxidation, each bFGF monomer bound sequentially to form the extended multimer. The proposed scheme of bFGF oxidation with DTNB revealed that the difference in the aggregate structural forms was probably due either to the presence of covalently bound residues of nitrobenzoic acid in the products of oxidation, or to the participation of sulfhydryl groups in disulfide bond formation.