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Preparation and characterization of two LysB29 specifically labelled fluorescent derivatives of human insulin
Author(s) -
Ciencialová Alice,
Žáková Lenka,
Jiráček Jiří,
Barthová Jana,
Barth Tomislav
Publication year - 2004
Publication title -
journal of peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 66
eISSN - 1099-1387
pISSN - 1075-2617
DOI - 10.1002/psc.556
Subject(s) - fluorescence , chemistry , peptide , lysine , human insulin , trypsin , amino acid , semisynthesis , combinatorial chemistry , insulin , peptide synthesis , biochemistry , enzyme , biology , physics , quantum mechanics , endocrinology
The preparation and characterization of two novel LysB29 selectively labelled fluorescent derivatives of human insulin are described. Two probes were chosen: 4‐chloro‐7‐nitrobenz‐2‐oxa‐1,3‐diazole (NBD) and 7‐methoxycoumarin‐4‐acetic acid (MCA), which have a relatively small, compact structure and are able to react with amino groups to form highly fluorescent derivatives. The combination of solid phase peptide synthesis and enzymatic semisynthesis was chosen for preparation of these fluorescent derivatives. Using two different protocols of solid‐phase peptide synthesis, two fluorescent octapeptides were prepared corresponding to the position B23–B30 of human insulin, each with a different fluorescent label, NBD or MCA, on the ε‐amino group of lysine. Then, the fluorescent octapeptides were coupled to desoctapeptide‐(B23–B30)‐insulin by a trypsin catalysed reaction. The receptor binding affinities of two novel fluorescent derivatives of human insulin with NBD and MCA (HI‐NBD and HI‐MCA) were determined on rat adipose tissue plasma membranes. Both fluorescent insulins, HI‐NBD and HI‐MCA, had only slightly reduced binding affinity and will be used for studying the interaction of insulin with its receptor. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.