Detergent-assisted refolding of guanidinium chloride-denatured rhodanese. The effect of lauryl maltoside.
For the first time, the enzyme rhodanese (thiosulfate:cyanide sulfurtransferase; EC 2.8.1.1) has been renatured from 6 M guanidinium chloride (GdmCl) by direct dilution of the denaturant at relatively high protein concentrations. This has been made possible by using the nonionic detergent dodecyl-beta-D-maltoside (lauryl maltoside). Lauryl maltoside concentration dependence of the renaturation and reactivation time courses were studied using 50 micrograms/ml rhodanese. There was no renaturation at lauryl maltoside (less than 0.1 mg/ml), and the renaturability increased, apparently cooperatively, up to 5 mg/ml detergent. This may reflect weak binding of lauryl maltoside to intermediate rhodanese conformers. The renaturability began to decrease above 5 mg/ml lauryl maltoside and was significantly reduced at 20 mg/ml. Individual progress curves of product formation, for rhodanese diluted into lauryl maltoside 90 min before assay, showed induction phases as long as 7 min before an apparently linear steady state. The induction phase increased with lauryl maltoside concentration and could even be observed in native controls above 1 mg/ml detergent. These results are consistent with suggestions that refolding of GdmCl-denatured rhodanese involves an intermediate with exposed hydrophobic surfaces that can partition into active and inactive species. Further, lauryl maltoside can stabilize those surfaces and prevent aggregation and other hydrophobic interaction-dependent events that reduce the yield of active protein. The rhodanese-lauryl maltoside complex could also form with native enzyme, thus explaining the induction phase with this species. Finally, it is suggested that renaturation of many proteins might be assisted by lauryl maltoside or other "nondenaturing" detergents.
Tandon
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Horowitz
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D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004793:Enzyme Reactivators; D005960:Glucosides / Q000494:pharmacology*; D006027:Glycosides / Q000494:pharmacology*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase
D004793:Enzyme Reactivators; D005960:Glucosides; D006027:Glycosides; D006146:Guanidines; C040358:dodecyl maltoside; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; D019791:Guanidine