PHYSICOCHEMICAL CHARACTERIZATION OF A OUABAIN ISOMER ISOLATED FROM BOVINE HYPOTHALAMUS

Recent reports have shown the presence of a ouabain-like inhibitor of Na+/K+-ATPase in humans. We have purified a bovine hypothalamic Na+/K-ATPase inhibitory factor (HIF) by using affinity chromatography combi ned with HPLC. This inhibitor has a molecular weight of 584 as determi ned by ion-spray mass spectrometry, making it isobaric with ouabain. G lycosidase treatment or acid hydrolysis of HIF released Only L-rhamnos e, the hexose isomer found in ouabain, as detected by chiral GC/MS. Ad ditionally, enzymatically generated desrhamnosyl HIF was found to have a molecular weight of 438, as does ouabagenin, the aglycone of ouabai n. HIF and its aglycone were indistinguishable from ouabain and ouabag enin, respectively, by reversed-phase HPLC retention times. However, d erivatization with naphthoylimidazole followed by HPLC revealed differ ent retention times for naphthoylation products of HIF and ouabain. Su bsequent CD spectroscopy on isolated naphthoylation products of HIF an d ouabain confirmed that they were different. This study provides chro matographic and spectroscopic evidence that ouabain and HIF are isomer ic cardenolides. The structural difference is presumed to account for the significant differences in biological properties observed for HIF and ouabain.