Inactivation of ribonuclease inhibitor by thiol-disulfide exchange.
Porcine ribonuclease inhibitor (RI) contains 30 1/2-cystinyl residues, all of which occur in the reduced form. Reaction of the native protein with 5,5'-dithiobis (2-nitrobenzoic acid) resulted in the release of 30 mol of the product 5-mercapto-2-nitrobenzoate, and the loss of the RNase inhibitory activity. A linear relationship between the degree of modification and inactivation was observed. The rate of modification was greatly increased in the presence of 6 M guanidinium HCl. Reaction with substoichiometric amounts of 5,5'-dithiobis(2-nitrobenzoic acid) was found to yield a mixture of fully reduced active molecules, and fully oxidized inactive ones, but no partially oxidized forms were detected. This suggests that an "all-or-none" type of modification and inactivation took place. All 1/2-cystinyl residues in the inactive, monomeric inhibitor had formed disulfide bridges, judged by the absence of either free thiol groups or mixed disulfides with 5-mercapto-2-nitrobenzoate. This fully disulfide-cross-linked molecule had an open conformation compared to the native one, as shown by gel filtration and limited proteolysis. Reaction of phenylarsinoxide with vicinal dithiols yields products that are much more stable than those with monothiols. Titration of RI with this reagent yielded complete inactivation at a reagent/thiol ratio of 0.5. Taken together, these observations suggest that the thiol groups in RI have a diminished reactivity due to three-dimensional constraints. After the initial modification of a small number of thiol groups, a conformational change occurs which causes an increase in reactivity of the remaining thiols. The thiol groups are situated close enough together to permit the formation of 15 disulfide bridges in the inactive molecule.
Fominaya
J M
JM
Friedrich Miescher-Institut, Basel, Switzerland.
Hofsteenge
J
J
D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D002417:Cattle; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D011487:Protein Conformation; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013552:Swine
D001152:Arsenicals; D003432:Cross-Linking Reagents; D004220:Disulfides; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D011994:Recombinant Proteins; C029341:oxophenylarsine; C066953:ribonuclease inhibitor, porcine; D004228:Dithionitrobenzoic Acid; D012259:Ribonuclease, Pancreatic