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

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.