Ion-exchange chromatographic assay of peptidases acting on the C-terminal hexapeptide sequence of substance P

A rapid and sensitive assay for peptidases acting on the C-terminal hexapeptide sequence of the neuropeptide substance P is described. The radiolabelled substrate, N^α-[¹²⁵I]desaminotyrosyl-substance P (6-11) is easily prepared by coupling commercially available radioiodinated Bolton-Hunter reagent with substance P (6-11). Peptidase activity is determined by quantitative separation of the degradation products from the intact substrate on small QAE-Sephadex columns. The assay has been used to measure degradation of the substrate by rat parotid and diencephalon slices. The peptidase activity in the latter system was inhibited by substance P and substance P fragments and was sensitive to metal chelators and thiol reagents.