The inside of living cells is crowded by extremely high concentrations of biomolecules, and thus globular proteins should have been developed to increase their solubility under such crowding conditions during organic evolution. The O2-storage protein myoglobin (Mb) is known to be expressed in myocytes of diving mammals in much larger quantities than those of land mammals. We have previously resurrected ancient whale and pinniped Mbs and experimentally demonstrated that the diving animal Mbs have evolved to maintain high solubility under the crowding conditions or to increase their tolerance against macromolecular precipitants, rather than solubility in a dilute buffer solution. However, the detail of chemical mechanisms of the precipitant tolerance remains unclear. Here, we investigated pH dependence of the precipitant tolerance (β, slope of the solubility against precipitant concentration) of extant Mbs and plotted the β values, as well as those of ancestral Mbs, against their surface net charges (ZMb). The results demonstrated that the precipitant tolerance was approximated by the square of ZMb, that is, β = aZMb2 + b, in which a and b are constants. This effect of ZMb against the precipitation is not predicted by a classical excluded volume theory that gives constant β for Mbs but can be explained by electrostatic repulsion between Mb molecules. The present study elucidates how Mb molecules have evolved to increase their in vivo solubility and shows the physiological significance of either neutral or basic isoelectric points (pI) of the natural Mbs, rather than acidic pI.
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