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Purification of an Endogenous Inhibitor of the Sodium Pump from the Toad Venom Chan′su

An endogenous inhibitor of the sodium pump from the toad venom Chan′su was purified. The dry substance Chan′su was extracted with methanol. The dry residue was passed subsequently through membrane filters with the exclusion size of 10 000, 3 000 and 1 000 at 1 psi in a Filtron Pro Vario-3-System and applied to thin layer chromatography made of Silica 60 F 254+366 separated in CHCl 3∶MeOH∶H 2O. The R f 0.55 fractions inhibiting the sodium pump were purified on a HPLC-RP C18 using a linear H\-2O/methanol gradient at 220 nm and 250 nm on DAD detector. The bio-activity was measured by a specific enzyme-linked immunosorbent assay (ELISA) and 86 Rb-uptake into human red blood cells. In Chan′su there exists an inhibitor of the sodium pump which interacts with antibodies against ouabain and proscillaridin A, and inhibits the sodium pump activity in the red blood cells. The results show that a low molecular weight compound has been isolated from the toad venom Chan′su. It inhibits the sodium pump, interacts preferentially with antibodies against ouabain. It is water soluble and shows a maximum of absorbance at 250 nm. Several milligrams of the compounds from one kilogram were obtained in pure form. The inhibitor differs considerably in its chemical structure from ouabain, because ouabain shows an UV absorbance maximum at 220 nm while the new inhibitor at 250 nm.