Published in:
Biochemical Journal, 358 (2001) 3, Seite 681-692
Language:
English
DOI:
10.1042/bj3580681
ISSN:
0264-6021;
1470-8728
Origination:
Footnote:
Description:
Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K+ channel subtypes. MTX adopts a disulphide bridge organization of the type C1–C5, C2–C6, C3–C4 and C7–C8, and folds according to the common α/β scaffold reported for other known scorpion toxins. Here we have investigated the process and kinetics of the in vitro oxidation/folding of reduced synthetic L-MTX (L-sMTX, where L-MTX contains only l-amino acid residues). During the oxidation/folding of reduced L-sMTX, the oxidation intermediates were blocked by iodoacetamide alkylation of free cysteine residues, and analysed by MS. The L-sMTX intermediates appeared sequentially over time from the least (intermediates with one disulphide bridge) to the most oxidized species (native-like, four-disulphide-bridged L-sMTX). The mathematical formulation of the diffusion-collision model being inadequate to accurately describe the kinetics of oxidation/folding of L-sMTX, we have formulated a derived mathematical description that better fits the experimental data. Using this mathematical description, we have compared for the first time the oxidation/folding of L-sMTX with that of D-sMTX, its stereoisomer that contains only d-amino acid residues. Several experimental parameters, likely to affect the oxidation/folding process, were studied further; these included temperature, pH, ionic strength, redox potential and concentration of reduced toxin. We also assessed the effects of some cellular enzymes, peptidylprolyl cis–trans isomerase (PPIase) and protein disulphide isomerase (PDI), on the folding pathways of reduced L-sMTX and D-sMTX. All the parameters tested affect the oxidative folding of sMTX, and the kinetics of this process were indistinguishable for L-sMTX and D-sMTX, except when stereospecific enzymes were used. The most efficient conditions were found to be: 50mM Tris/HCl/1.4mM EDTA, pH7.5, supplemented by 0.5mM PPIase and 50units/ml PDI for 0.1mM reduced compound. These data represent the first report of potent stereoselective effects of cellular enzymes on the oxidation/folding of a scorpion toxin.