Hierarchy of N-acylation Sites in Human Insulin Studied by RP-HPLC and Mass Spectrometry

作者：Zain Hina; Ahmad Munir; Gardner Qurra tul Ann Afza; Akhtar Muhammad^*

来源：Journal of the Chemical Society of Pakistan, 2015, 37(6): 1262-1268.

• Mass spectrometry
• Regiospecificity of acylation
• Mono-acetyl insulin
• Mono-dodecanoyl (lauryl) insulin

Acylation of human insulin was studied using, C-2 and C-12 transfer reagents, N-succinimidyl acetate and N-succinimidyl dodecanoate (lauryl) respectively. In general, the acylation reaction was more specific at high pH values (12.00) for the modification of the e-amino group of K-29B. At pH around 8.5, multiple modifications occurred at other sites, in particular the N-terminal glycine of A-chain (G(1A)). WithN-succinimidyl dodecanoate, about 88 % of the acylation was established to be at the epsilon-amino of K-29B, as shown using tryptic digestion and identification of the products by mass spectrometry. While with N-succinimidyl acetate, singly acetylated product with acetyl group at the epsilon-amino of K-29B, as well as doubly modified species containing acetyl group in the epsilon-amino group and the amino-terminal of G(1A)were produced in equal amounts. The high regiospecificity of modification at the epsilon-amino group, by the C-12 reagent, is attributed to steric factors. Moreover, the above investigation also highlights the purification regime of singly and doubly acetylated products on RP-HPLC using a shallow gradient, though the difference between two species, in the case of acetyl group, is of 43 Da. The overall results allow us to establish the hierarchy of accessibility of the three amino group; epsilon-amino group K-29B is the least hindered and accessible to C-2 as well as C-12 reagents, then is the amino group of G(1A) which is accessible to the C-2 but not the C-12 reagent, finally that of F-1B is most hindered and accessible to neither.

• 出版日期2015-12