Accumulation of the two-UPF 1069 disulfide species occurs in both the selective reduction and the oxidative refolding processes; this suggests that it adopts a highly stable structure and represents a major kinetic trap during MCh-1 folding. The amounts of Ia and IIa increase during the early phase of the oxidative folding process, but decrease during later phases. The native form accumulates along the pathway of oxidative refolding, indicating that the native form is the most stable form. The intermediate IIa was isolated and the folding reaction monitored with HPLC. Four minor two-disulfide scrambled isomers and six minor three-disulfide scrambled isomers were observed along the course of folding of IIa to form the native peptide as shown in Figure 2D. Under the experimental conditions used at room temperature), the disulfide isomers are expected to be freely reversible, allowing the possible disulfide-bonded isomers to accumulate according to their relative stabilities. The native form eluted latest among the three-disulfide scrambled isomers along the oxidative folding pathway, consistent with it being the most stable form. The seeds of Cucurbitaceae species are emerging as a rich source of novel disulfide-rich peptides. In this study we isolated two peptides from the seeds of M. charantia that contain novel sequences and ICK structural motifs. Analysis of the oxidative refolding highlighted a common intermediate present in the folding of a variety of ICK peptides, despite variations in inter-cysteine loop sizes. This two-disulfide intermediate is surprisingly stable and provides new insights into how the cystine knot might have evolved from a simple disulfide framework. A comparison of the sequences of MCh-1 and MCh-2 with peptides isolated from the related species M. cochinchinensis shows that although the peptides all contain six cysteine residues, and loops 3 and 4 all contain the same number of residues, the other inter-cysteine loops are variable. Similarly, the sequences differ from the squash Sodium Danshensu trypsin inhibitors isolated from Momordica species. These sequence differences are reflected in the different retention times observed on RP-HPLC, with the squash trypsin inhibitors being more hydrophilic than MCh-1 and MCh-2. The peptides also differ in activity, as MCh-1 and MCh-2 are not trypsin inhibitors. Given the differences in sequence and activity it is apparent these novel peptides belong to a new subfamily of ICK members. It is interesting to note the differences in retention times of the intermediates during the selective reduction and the stepwise alkylation, or the oxidative refolding and the stepwise alkylation.
