Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry

作者：Gardner Qurra tul Ann Afza^*; Younas Hooria; Akhtar Muhammad

来源：Biochimica et Biophysica Acta-Proteins and Proteomics, 2013, 1834(1): 182-190.

DOI：10.1016/j.bbapap.2012.09.004

• Comparative kinetics by mass spectrometry and HPLC
• Proinsulin kinetic parameter
• Conversion of proinsulin to insulin
• Human di-arginyl insulin
• Des-(B30)-insulin
• Bovine/buffalo proinsulin

Human M-proinsulin was cleaved by trypsin at the (RR32)-R-31-E-33 and (KR65)-R-64-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 (KR60)-R-59-G(61) bond but at the B/C junction cleavage occurred at the (RR32)-R-31-E-33 as well as the R-31-(RE33)-E-32 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 k(cat)/K-m values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02 +/- 0.08 x 10(5) s(-1) M-1) and the cleavage of the K-29-T-30 bond of M-insulin-RR (1.3 +/- 0.07 x 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 (k(cat)/K-m values around 1000 s(-1) M-1). Hence, as the biosynthetic path follows the sequence; proinsulin -%26gt; insulin-RR -%26gt; insulin, the K-29-T-30 bond becomes shielded, exposed then shielded again respectively.

• 出版日期2013-1