ABSTRACT
The formation of folding intermediates blocked by iodoacetate from the main toxin of Naja naja samarensis was monitored by FPLC and the populations were identified by amino acid analyses. We have determined conditions allowing for the highest yield in the populations with two blocked cysteines. The free cysteines remaining under these conditions were labeled with iodo[14C]acetate and localized by peptide mapping in one of the products isolated by ion-exchange chromatography (NT3III). We have also investigated the effects on the native-like characteristics of such molecules of an incubation at equilibrium with a mixture of cysteine and cystine. We find that many different molecular populations are present during the folding process and that disulfide exchanges allow for the reconstitution of native-like products having open disulfides even under strongly denaturing conditions.
Subject(s)
Cysteine , Elapid Venoms/metabolism , Amino Acid Sequence , Animals , Carbon Radioisotopes , Disulfides/analysis , Iodoacetates/metabolism , Iodoacetates/pharmacology , Iodoacetic Acid , Kinetics , Peptide Fragments/analysis , Protein Binding , Protein Conformation , TrypsinABSTRACT
The main toxin of Naja naja samarensis is a very small and rigid protein (Mr 6850, 8 cysteines). When fully reduced, it regains its native conformation by a mechanism involving a rapid cysteine oxidation and a slower, less temperature-dependent disulfide exchange. In a native-like form of the protein we observed a population whose cysteines were incompletely reoxidized. When intermediates with blocked cysteines were incubated with oxidized and reduced glutathione, the percentage of native-like molecules increased. These findings suggest a multiple folding pathway.
