[Chromatographic behavior and purification of synthetic huwentoxin-I]

A comparative study was made to investigate the chromatographic behavior and purification of chemically synthesized and native polypeptide neurotoxin huwentoxin-I (HWTX-I) by means of reversed-phase HPLC and ion-exchange chromatography. The results showed that the synthetic HWTX-I in crude product has a longer retention time than the native HWTX-I, but has the same retention time as the denatured HWTX-I, suggesting it being in a state of complete denaturation which was confirmed by the MALDI-TOF MS analysis. It has also been discovered that, before complete purification, the synthetic HWTX-I, after oxidative treatment, showed wider and more asymmetric peaks than the native HWTX-I that was denatured and then renatured under the same experimental conditions, which suggests that there is even greater conformational unhomogeneity in the synthetic HWTX-I. It was inferred that, in addition to the wrong disulfide bonds formation during oxidative folding, the racemization during chemical synthesis resulted in more stereoisomers of synthetic HWTX-I. It was needed for the renatured synthetic peptides to be purified using different methods alternatively.