A study of rose bengal and sulfhydryl reagents on stromal fragments of rabbit and human erythrocytes

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Abstract
Stromal fragments of human and rabbit erythrocytes, prepared by osmotic hemolysis, were used as sources of ATPase in a study of the action of the fluorescein dye, rose bengal, and of sulfhydryl reagents on this membrane component. Only the Na‐K independent ATPase was systematically studied but the dye was observed, at concentrations of 10−3 M, to completely inhibit the Na‐K dependent activity. Over a concentration range of 1.6 × 10−7 to 10−3 M the dye had a graded inhibitory effect on the Na‐K independent ATPase in Tris buffer with little difference in the sensitivity of the enzyme from the stroma of the two species. The activity of the cation independent enzyme was not changed by darkness or anaerobic conditions in the presence of the dye and its action could not be attributed to photosensitization. Repeated washing to remove the dye failed to reduce its effect on rabbit stroma and only partially succeeded with human stromal fragments. The dye was shown not to be a competitive inhibitor of ATP for the stromal ATPase of either species. Attempts to reduce the rose bengal effect by use of the sulfhydryl agents, glutathione or cysteine were unsuccessful although glutathione did protect ATPase against the common sulfhydryl inhibitors. It is concluded that the dye is affecting an essential group on or near the enzyme either by direct bonding or irreversibly changing essential bonds.