1. Preparation and characterization of two LysB29 specifically labelled fluorescent derivatives of human insulin
Alice Ciencialová, Lenka Záková, Jiri Jirácek, Jana Barthová, Tomislav Barth J Pept Sci. 2004 Jul;10(7):470-8. doi: 10.1002/psc.556.
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 epsilon-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.
2. Assessment of Synthetic Matrix Metalloproteinase Inhibitors by Fluorogenic Substrate Assay
Ty J Lively, Dale B Bosco, Zahraa I Khamis, Qing-Xiang Amy Sang Methods Mol Biol. 2016;1406:161-70. doi: 10.1007/978-1-4939-3444-7_13.
Matrix metalloproteinases (MMPs) are a family of metzincin enzymes that act as the principal regulators and remodelers of the extracellular matrix (ECM). While MMPs are involved in many normal biological processes, unregulated MMP activity has been linked to many detrimental diseases, including cancer, neurodegenerative diseases, stroke, and cardiovascular disease. Developed as tools to investigate MMP function and as potential new therapeutics, matrix metalloproteinase inhibitors (MMPIs) have been designed, synthesized, and tested to regulate MMP activity. This chapter focuses on the use of enzyme kinetics to characterize inhibitors of MMPs. MMP activity is measured via fluorescence spectroscopy using a fluorogenic substrate that contains a 7-methoxycoumarin-4-acetic acid N-succinimidyl ester (Mca) fluorophore and a 2,4-dinitrophenyl (Dpa) quencher separated by a scissile bond. MMP inhibitor (MMPI) potency can be determined from the reduction in fluorescent intensity when compared to the absence of the inhibitor. This chapter describes a technique to characterize a variety of MMPs through enzyme inhibition assays.
