In order to gain understanding of the hemolytic action of DIG in plasma membrane, a ^2H NMR study was attempted using CHOL-d_6 and SA-d_3 probes together with DIG and its analogs in the multibilayers of egg yolk PC. It was found that DIG changed the ^2H NMR spectra of the former probe more than that of the latter. Thus, CHOL is proved as the target molecule for DIG. As ^2H NMR spectra of CHOL-d_6 in the equimolecular complex gave the quadrupole splitting close to that of solid CHOL-d_6, the complex in the multibilayers of egg yolk PC is characterized by the rigid structure as in the "digitonides" as occurring as precipitates in aqueous ethanol. It was also found that the excess CHOL not directly complexed with DIG (in lower DIG: CHOL ratio) gave rise to ^2H NMR spectra with reduced quadrupole splitting and larger linewidths compared with those of the absence of DIG. Therefore, the alteration of the bilayer structure is not always restricted to the vicinity of CHOL complexed with DIG. It may be concluded that the hemolytic activity of DIG can be explained by the presence of such disordered CHOL and lipids in the bilayers. Furthermore, prosapogenins with reduced number of sugar moiety exhibited quite different behavior. Therefore, configuration of the sugar moiety in DIG plays a crucial role in the interaction with CHOL.
