Parameters affecting in vitro oxidation/folding of maurotoxin, a four-disulphide-bridged scorpion toxin

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 ac cording to the common alpha/beta 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-sMT X, the oxidation intermediates were blocked by iodoacetamide alkylation of free cysteine residues, and analysed by MS. The L-sMTX intermediates appear ed 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 bei ng inadequate to accurately describe the kinetics of oxidation/folding of L -sMTX, we have formulated a derived mathematical description that better fi ts the experimental data. Using this mathematical description, we have comp ared 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 experi mental parameters, likely to affect the oxidation/folding process, were stu died further; these included temperature, pH, ionic strength, redox potenti al 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: 50 mM Tris/HCl/1.4 mM EDTA, pH 7.5, supplemented by 0.5 mM PPIase and 50 units/ml PDI for 0.1 mM reduced compound. These data repre sent the first report of potent stereoselective effects of cellular enzymes on the oxidation/folding of a scorpion toxin.