Oligomeric hepatoproliferin disaggregates into an active monomer that was purified and forcefully dissociated into two different ionic species

Abstract

Hepatoproliferin (HPF), a liver regeneration factor, was isolated initially as an aggregated molecule (big-HPF) and was purified into two homogeneous, bioactive species of 14 kDa and 18.5 kDa. These two big-HPFs were disaggregated to completion into two monomeric forms (small-HPFs) when incubated for 10 days in 0.15 M ammonium bicarbonate at 25 degrees C. Both monomeric forms were purified to homogeneity as active entities, one with a molecular mass of 944 Da and one with a molecular mass of 1066 Da. Each of the two (35)S-labelled small-HPFs was found, by enzymic analysis, to contain a charged sulfonated saccharide, which was neutralized by a specific amine. Monomeric HPF is therefore a stable ionic complex formed between these two ionic species. So strong was the electrostatic association that small-HPF remained intact in solution and no amine was displaced by the ammonium ions of the buffer. Small-HPF remained unimpaired during purification, since all activity was retained despite alternating acidic and basic conditions. However, when small-HPF was brought into contact with either a cationic or an anionic resin, it was dissociated to completion when mixed continuously with the resin for 4 days. The ionic entity that was released had no bio-activity and was either a pure radioactively labeled saccharide or a non-labeled amine, depending on the kind of resin used. When incubated together, the separated counterions combine to regain full activity after 2 days of reassociation. However, with incubation for longer, this reassociated small-HPF formed different oligomeric HPFs by aggregation. Small-HPF is therefore a new kind of growth enhancer, consisting of an acidic sulfonated saccharide and a basic amine assembled into a stable active ionic complex that has a tendency to aggregate.