Figure 3.

Time dependence of the deamidation of Asn A21 in insulin fibrils. (A) Insulin fibrils formed by seeding (top) and then reincubated at 65°C (pH 2.0) for 3 or 13 d (middle and bottom) were analyzed by HPLC under reducing conditions. The A-chains and B-chains are clearly resolvable (A,B) and the deamidation that occurs can be detected (A′, B′). After 13 d of incubation, the relative amount of A-chains that are deamidated at Asn A21 (A′) is very close to 50%. (B) Time course for deamidation of Asn A21 in the fibrils. The percent of deamidated Asn A21 was determined from HPLC analysis (e.g., Fig. 3A ▶); squares and diamonds represent two different batches of insulin. The maximum amount of deamidated Asn A21 in the fibrils is 52 ± 5%.