Probing the binding mechanism of photoresponsive azobenzene polyamine derivatives with human serum albumin

Abstract

Control over chemical and biochemical processes by agents sensitive either to internal or external stimuli has attracted much attention in recent years. In particular photosensitive polyamines have been recently used to photo-trigger the hybridization/melting of DNA as well as to modify its intrinsic morphology. These results prompted us to synthesize azobenzene-based polyamine derivatives and study their impact towards human serum albumin (HSA), the principal extracellular protein in plasma, which is highly responsible for the proper biological activity exerted by exogenous compounds. It turns out that to assess and understand the binding mechanism of relevant compounds towards the HSA active sites is a critical step for the design of biomolecules-targeted probes. Herein, we show that both the mono-substituted Azo-4N and bi-substituted bis-Azo-4N azobenzene derivatives bind the protein template with moderate affinity and the number of positive charges along the polyamine moiety plays a pivotal role in stabilizing the photochrome–HSA adduct. Changes taking place in the fluorescence intensity of the tryptophan residue enabled us to determine the K_sv and k_q parameters which provide evidence for a quenching driven by a static mechanism. Both ΔH and ΔS of the binding process being negative indicates that the HSA–photochrome association is mainly stabilized by a combination of long-range interactions of ionic nature. The overlap between the donor (Trp-214) and acceptor (photochrome) spectra allowed to calculate the distance (r) and the rate (k_ET) of energy transfer. Investigation of the HSA structural components reveals that the azobenzene derivatives, in both their conformations, slightly affect the overall protein secondary structure and they do not change its native state. Direct comparison of our results achieved by using photosensitive polyamines with those previously reported for biogenic and analogous polyamines bound to HSA reveals that the azo motif does not enhance to a large degree the overall binding affinity of the photoswitches towards the globular protein and contributes only a little to affect its intrinsic morphology.