Human M-proinsulin was cleaved by trypsin at the R
31R
32–E
33 and K
64R
65–G
66 bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K
59R
60–G
61 bond but at the B/C junction cleavage occurred at the R
31R
32–E
33 as well as the R
31–R
32E
33 bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the
kcat./
Km values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02 ± 0.08 × 10
5 s
− 1 M
− 1) and the cleavage of the K
29–T
30 bond of M-insulin-RR (1.3 ± 0.07 × 10
5 s
− 1 M
− 1). However, the K
29–T
30 bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (
kcat./
Km values around 1000 s
− 1 M
− 1). Hence, as the biosynthetic path follows the sequence; proinsulin → insulin-RR → insulin, the K
29–T
30 bond becomes shielded, exposed then shielded again respectively.
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