Comparative proton NMR studies on bovine semen and pancreas ribonucleases.

The fine structure of bovine semen RNase was studied with 1H NMR spectroscopy, using the 4-protein system consisting of dimeric bovine semen RNase, its catalytically active monomeric bis-(S-carboxymethyl-31,32) deriv., the naturally monomeric RNase A from the pancreas of the same species, and dimerized RNase A. Only 4 histidine (His) C-2 H resonances were obsd. in the arom. spectrum of bovine semen RNase, which belong to the 4 His residues present in the sequence of bovine semen RNase subunits at positions identical with those of the histidines of RNase A. This indicates identical environments for the individual His residues in both subunits. These resonances were assigned (1) by comparing their titrn. curves with the corresponding curves obtained with RNase A and with monomeric bovine semen RNase and (2) by evaluating the effects on their titrn. curves of nucleotide binding. Very similar NMR parameters were measured for His-105 and for His-119 of seminal and pancreatic RNase, whereas His-12 was found to have different environments in the 2 proteins. The distinctive NMR features of His-48 in bovine semen RNase confirmed the role of the hinge regions of the subunits in maintaining the dimeric structure of the protein. Although monomerization of the semen enzyme reduced the differences between the His C-2 H resonances of RNase A and bovine semen RNase, dimerization of RNase A did not affect the NMR spectrum of this protein, thus indicating that it is unlikely that the quaternary structure of bovine semen RNase resembles that of dimerized RNase A.