Dissociation of bovine seminal ribonuclease into catalytically active monomers by selective reduction and alkylation of the intersubunit disulfide bridges.
The hypothesis previously advanced that interchain disulfide bridges link the two identical subunits of bovine seminal ribonuclease BS-1 has been confirmed. The sedimentation rate and the electrophoretic mobility of the protein are not affected by denaturing agents unless thiol reagents are present in the denaturation mixtures. Reduction under controlled conditions results in the immediate cleavage of only 2 disulfide bonds out of 10 percent in the dimeric protein. Under these conditions, and the results do not change when partial reduction is followed by S-alkylation, 30% of the protein dissociates, while the remaining is found to consist of a dimeric species easily dissociable by denaturing agents without addition of thiol reagents. This indicates that the dimeric structure of seminal ribonuclease is maintained not only by disulfide bridges, but also by noncovalent forces. The protein derivative prepared by selective reduction and alkylation has been identified as monomeric bis-S-carboxymethylcysteine-31,32-ribonuclease BS-1. This is on the basis of the characterization of the 14C-labeled S-carboxymethylated peptides isolated from a thermolytic hydrolysate of the derivative prepared with iodo-2-[14C]acetic acid. Monomeric, selectively alkylated ribonuclease BS-1 is stable and catalytically active. The importance of such a derivative is discussed both in the light of the recent studies on the biological actions of seminal ribonuclease and as the fourth component of an experimental system of ribonucleases consisting of two homologous dimers (bovine seminal ribonuclease BS-1 and dimerized bovine pancreatic ribonuclease A) and two homologous monomers (ribonuclease A and the monomeric derivative of ribonuclease BS-1.
D000478:Alkylation; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002384:Catalysis; D002417:Cattle; D004220:Disulfides; D004355:Drug Stability; D046911:Macromolecular Substances; D008297:Male; D010084:Oxidation-Reduction; D010179:Pancreas / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D011489:Protein Denaturation; D012260:Ribonucleases / Q000032:analysis; D012661:Semen / Q000201:enzymology*
D000596:Amino Acids; D004220:Disulfides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D012260:Ribonucleases