Conjugation of a Dipicolyl Chelate to Polypeptide Conjugates Increases Binding Affinities for Human Serum Albumin and Survival Times in Human Serum

Abstract

The affinity for human serum albumin (HSA) of a series of 2-5 kDa peptides covalently linked to 3,5-bis[[bis(2-pyridylmethyl)amino]methyl]benzoic acid, a dipicolyl chelator with micromolar affinity for Zn²⁺ , was found by surface plasmon resonance to increase in the presence of 1 μm ZnCl₂ at physiological pH. The dependence on polypeptide hydrophobicity was found to be minor, thus suggesting that the conjugates bound to the metal-binding site and not to the fatty-acid-binding site. The affinity of the conjugates increased strongly with the positive charge of the polypeptides, thus implicating the negatively charged protein surface surrounding the metal-binding site. The survival times of the peptides in human serum were extended as a consequence of stronger binding to HSA, thus suggesting that Zn²⁺ -chelating agents might provide a general route to increased survival time of peptides in serum in therapeutic and diagnostic applications without significantly increasing their molecular weights.

Keywords: affinity; biosensor; human serum albumin; peptides; zinc-binding site.