Time-resolved fluorescence anisotropy as a tool to study guest–cucurbit[n]uril—protein ternary supramolecular interactions

Abstract

Ternary supramolecular complexes involving cucurbit[n]urils and proteins are of potential interest for improving drug transport and delivery. We report here time-resolved fluorescence studies for acridine orange complexes with cucurbit[7]uril and cucurbit[8]uril in the presence of human serum albumin as a model system. A detailed characterization of the fluorescence lifetime and anisotropy properties of the different acridine orange complexes with cucurbit[n]urils and human serum albumin was performed. Of particular importance is the analysis of the stepwise binding for acridine orange-cucurbit[8]uril complexes and the assignment of the fluorescence and anisotropy properties to the 2 : 1 complex. Anisotropy decay measurements were essential to detect protein-bound species and to discriminate between different complexes. Based on the fluorescence evidence, ternary interactions with the protein are suggested for the acridine orange-cucurbit[7]uril complex but not for the cucurbit[8]uril complex. We highlight here the usability and sensitivity of the combined fluorescence analysis.

Photochemical behavior of biosupramolecular assemblies of photosensitizers, cucurbit[n]urils and albumins

“…For this, the molecule has to protrude out of the cavity of CB [7]. 50,52 We observed this behavior for AO + in the presence of CB [7] and HSA, while no interactions were observed for the case of CB [8] probably due to its larger cavity size. 52 The binding of the AO + @CB [7] complex to HSA occurs with lower affinity compared to free AO + ( On the other hand, in the presence of excess CB [8], all AO + is bound to CB [8].…”

“…52 The data indicated mainly a 1:1 interaction, although some minor binding to other sites could not be discarded. 52 A similar interaction was previously reported for AO + with BSA, although with lower affinity (<5×10 3 M -1 ). 69 The same type of binding has been reported for MB + with HSA,…”

“…AO + was irradiated at 492 nm in the absence of CB[n]s and at 485 nm or 465 nm in the presence of CB [7] or CB [8], respectively. 52 The irradiance absorbed by the samples was the same within error and equal to 31 ± 3 W m Laser flash photolysis was used to measure triplet excited state lifetimes for AO + and its complex with CB [7]. The samples were excited using a 266 nm Nd:YAG laser (Quanta-Ray Lab 130-4, Spectra Physics) and the detection of the excited state was done by monitoring the changes in the absorbance of the samples using a Xe-arc lamp from Oriel.…”

“…It must be noted that the formation of 1:1 and 2:1 complexes with CB [8] is stepwise, and their relative abundance depends on the concentration of the dye and the macrocycle. 52 There is likely an interaction between TMPyP 4+ and CB [8], but there are no reported binding constants. 51 CB [8] can also bind two different molecules and form a 1:1:1 ternary complex, and this is particularly interesting when one of the components is a protein, as the one reported for TMPyP 4+ and BSA, where a tryptophan residue in the protein is suggested to be included inside the CB [8] cavity together with one of TMPyP 4+ pyridinium arms.…”

“…We recently reported on the interaction of another well-known photosensitizer acridine orange (AO + ) with CB [7] and human serum albumin (HSA), and found no such interaction with the protein in the presence of CB [8]. 52 The latter is an interesting model system in the first place because AO + is active as a PDT agent in vivo, 53,54 and this molecule forms complexes with either HSA, CB [7] or CB [8], and simultaneously with CB [7] and HSA allowing the possibility to compare the photoactivity of a broad range of AO + species. Therefore, we set out to investigate the photoactivity of the different complexes of AO + and obtain further insights into the mechanism of action and ROS generation in the biosupramolecular complex.…”