ONe-step isolation of human chopionic ganadotropin in milligram amounts, using selective steady-state stacking on...

Human chorionic gonadotropin (hCG) has been isolated in a single step from a crude, commercially available urinary preparation. The isolated hormone, and highly purified standard hormone preparations, each exhibit on PAGE (pH 5.5), as on electrofocusing, 3 to 5 major "change isomeric" species within preparations. In addition, PAGE (pH 5.5) detects differences in molecular size between charge isomeric species of identical net charge occurring in the various preparations. For the purposes of isolation by Steady-State Stacking, each of these charge isomeric families of hCG components was collectively separated from faster migrating proteins in crude, commercial hCG by first allowing them to unstack while the more highly mobile contaminating proteins in the preparation remained stacked and were eluted as a stack from an elution-PAGE apparatus. After some time of electrophoresis the unstacked hCG components were then combined ("restacked") by sweeping them into a second moving boundary and eluted from a preparative elution-PAGE apparatus in a single zone. The product exhibits on PAGE re-run molecular species related to hCG standard as either "size-" or "charge-isomers". After dissociation into subunits, all preparations yield a protein indistinguishable in size or net charge from a standard preparation of the beta-chain of hCG. The alpha- and beta-chains derived from the isolated preparations exhibited dose-response curves parallel to those of standard hCG alpha and hCG beta respectively. The biological activity of the product was 8-12.000 IU/mg, as compared to 3,000 IU/mg for the starting material. The total yield was 20% by dry weight of the crude starting material.