Bovine seminal ribonuclease (
BS-RNase) is a homologue of bovine
pancreatic ribonuclease (
RNase A). Unlike
RNase A,
BS-RNase has notable toxicity for human
tumor cells. Wild-type
BS-RNase is a homodimer linked by two intermolecular
disulfide bonds. This quaternary structure endows
BS-RNase with resistance to inhibition by the
cytosolic ribonuclease inhibitor protein (RI), which binds tightly to
RNase A and monomeric
BS-RNase. Here, we report on the creation and analysis of monomeric variants of
BS-RNase that evade RI but retain full enzymatic activity. The cytotoxic activity of these monomeric variants exceeds that of the wild-type dimer by up to 30-fold, indicating that the dimeric structure of
BS-RNase is not required for cytotoxicity. Dimers of these monomeric variants are more cytotoxic than wild-type
BS-RNase, suggesting that the cytotoxicity of the wild-type
enzyme is limited by RI inhibition following dissociation of the dimer in the reducing environment of the cytosol. Finally, the cytotoxic activity of these dimers is less than that of the constituent monomers, indicating that their quaternary structure is a liability. These data provide new insight into structure-function relationships of
BS-RNase. Moreover,
BS-RNase monomers described herein are more toxic to human
tumor cells than is any known variant or homologue of
RNase A including
Onconase, an amphibian homologue in phase III clinical trials for the treatment of unresectable
malignant mesothelioma.