Dissociation and Reconstitution of Bovine Seminal RNAase: Construction of a Hyperactive Hybrid Dimer

The quaternary structure of bovine seminal ribonuclease is mantained both by noncovalent forces and by two interchain disulfides. The available monomeric derivatives of the enzyme may not be reassembled into dimers. They are catalitically active, but do not retain certain properties of the dimeric enzyme, such as (i) the ability to respond cooperatively to increasing substrate concentrations in the rate-limiting reaction step, and (ii) the antitumor and immunosuppressive actions. In this report we describe the preparation of stable monomers of seminal ribonuclease which can be reassociated into covalent dimers, indistinguishable from the native protein. With this procedure a hubrid dimer was constructed, made up of native subunits associated to a subunit catalitically inactivated by selective alkylation of the active site His 119. This dimer was found to have enzymic properties typical of monomeric ribonucleases, such as a hyperbolic saturation curve in the hydrolytic rate-limiting step of the reaction. However, the hybrid dimer was order-of-magnitude more active than the dimeric enzyme.