Refolding kinetics of partially reduced and S-carboxymethylated trypsin-subtilisin inhibitor from marine turtle eggwhite.
Three accessible disulphide bonds of basic trypsin-subtilisin inhibitor from marine turtle eggwhite have been reduced with 0.1M NaBH4 at 0 degree C under nitrogen atmosphere at pH9.8 and then S-carboxymethylated. The partially reduced inhibitor retains 80% of the native inhibitory activity towards trypsin and subtilisin. The S-carboxymethylated inhibitor undergoes slower refolding than the native inhibitor from its fully denatured and reduced state at pH 8.5 in the presence of oxidised and reduced glutathione. The refolding process was characterised by the attainment of the inhibitory activity towards trypsin and subtilisin. The values of the second order rate constant for the refolding reactions of the modified protein are 0.02 x 10(2)M-1sec1 and 0.033 x 10(2)M-1sec-1 for its trypsin and subtilisin inhibiting domains and their energies of activation are 20.1 Kcal/mole and 24.6 Kcal/mole. The partially modified inhibitor does not regain complete inhibitory activity even after long incubation in the oxido-shuffling buffer. From the above findings it can be concluded that the three disulphide bonds of the native inhibitor are not essential for the inhibitory activity of the trypsin-subtilisin inhibitor but they help in the correct refolding of the inhibitor by forming transient disulphide bonds with the external disulphide reagents as well as with the internal sulphydryl groups.
Chaudhuri
T K
TK
Department of Chemistry, Bose Institute, Calcutta, India.
D000818:Animals; D004527:Egg Proteins / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D007700:Kinetics; D008745:Methylation; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000037:antagonists & inhibitors*; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000378:metabolism; D014426:Turtles
D004527:Egg Proteins; D014361:Trypsin Inhibitors; D013381:Subtilisins