An investigation of the different molecular forms of IGFBP-1 using immobilised metal-, immuno- and lectin-affinity chromatography

The insulin-like growth factor-binding protein I (IGFBP-1) is a member of a family of six homologous proteins that regulate the action of the insulin-like growth factors. IGFBP-1 is a 25 kDa protein that, in addition to its native form, may exist in several phosphoforms (30 kDa), which are predominant in the circulation of humans. Phosphorylation of IGFBP-1 is a post-translational modification that has a great influence on the action of IGF-I. IGFBP-1 forms multimers and complexes with alpha 2-macroglobulin (alpha 2M). Polymerisation of IGFBP-I was also reported. In order to analyse and separate these IGFBP-1 molecular species, affinity chromatography methods were used in this study. The results demonstrated that most of the IGFBP-1 circulates in complexes with alpha 2M, which can be isolated by affinity chromatography using immobilised anti-alpha 2M antibodies. IGFBP-1/alpha 2M complexes may be differentiated from IGFBP-1 dimer and multimers using lectin-affinity chromatography, since the latter do not interact with lectins. It seems that the complexes contain not only monomeric IGFBP-1, but also its multimers. The dimer and multimers are stable under reducing conditions, suggesting a covalent linkage between the units. Free IGFBP-1 monomer can be separated from multimers using Con A-affinity chromatography. The concentration of free IGFBP-1 is relatively low in the circulation.