Table 5. Relative rates of accumulation of peaks generated by the NEP and NEP mutant dependent hydrolysis of insulin B chain.

Single Cleavage
	Peak	NEP	S546E	F563L
Cleavage Site		Δarea/min/ng	Δarea/min/ng	Δarea/min/ng
H5-L6	1–5	59	12	83
H10-L11	1–10	163	87	135
L11-V12	1–11	88	30	16
A14-L15	1–14	167	145	266
	15–30	135	116	282
Y16-L17	1–16	31	17	25
	17–30	171	81	112
G23-F24	24–30	268	142	290

Time course assays were carried out by incubation of NEP with insulin B chain using conditions as described in Table 2 . At 0, 30, 60, 90, 120, and 180 min., aliquots of 100 µL were removed followed by the addition of 10 µL of 5% TFA to stop further hydrolysis. Each reaction mixture was subjected to HPLC analysis as in Table 4 and peak areas measured. The rate of accumulation for each peak was calculated from the linear phase of the reaction.