The NTNHAs of M-TCs produced by serotype A [15], C [16], and D [16], [17] and [18] strains have always been found to be nicked at unique sites. This leads to the appearance of two bands, 15 and 115 kDa, on SDS–PAGE, while the NTNHAs in the L-TCs remained unprocessed single-chain polypeptide. Previously we found that AZD-4547 a unique strain, D-4947, produces both intact M- and L-TC without any nicking [16]. In addition, we inadvertently found that both isolated NTNHA and NTNHA in the M-TC spontaneously converted to the nicked forms [16]. In the present study, we investigated spontaneous nicking of rNTNHA. As shown in Fig. 2A and B, when the rNTNHA was incubated for long periods at 25 °C without addition of protease, SDS–PAGE analysis showed that disappearance of the 140 kDa band was accompanied by the appearance 18 and 115 kDa bands, and the latter bands increased with longer incubation times. The N-terminal amino acid sequences of the 18 and 115 kDa bands were determined to be MRGSHHH and KTPKSN, corresponding to the N-terminal amino acid sequence of rNTNHA and residues K127 to N132 of NTNHA, respectively (Fig. 2A).