Translational and Rotational Motions of Albumin Sensed by a Non-Covalent Associated Porphyrin Under Physiological and Acidic Conditions: A Fluorescence Correlation Spectroscopy and Time Resolved Anisotropy Study

Abstract

The interaction between a free-base, anionic watersoluble porphyrin, TSPP, and the drug carrier protein, bovine serum albumin (BSA) has been studied by time-resolved fluorescence anisotropy (TRFA) and fluorescence correlation spectroscopy (FCS) at two different pH-values. Both rotational correlation times and translational diffusion times of the fluorescent species indicate that TSPP binding to albumin induces very little conformational changes in the protein under physiological conditions. By contrast, at low pH, a biexponential decay is obtained where a short rotational correlation time (7 int =1.2 ns) is obtained, which is likely associated to wobbling movement of the porphyrin in the protein binding site. These physical changes are corroborated by circular dichroism (CD) data which show a 37% loss in the protein helicity upon acidification of the medium. In the presence of excess porphyrin formation of porphyrin Jaggregates is induced, which can be detected by timeresolved fluorescence with short characteristic times. This is also reflected in FCS data by an increase in molecular brightness together with a decrease in the number of fluorescent molecules passing through the detection volume of the sample.

Ordered Self‐assembly of Protonated Porphyrin Induced by the Aqueous Environment of Biomimetic Systems

“…However, a dependence of D with the solution pH is detected: At pH = 7.4, D = (0.65 ± 0.05) × 10 −10 m 2 s −1 , in agreement with literature data obtained with mesoporphyrin IX dihydrochloride by FCS, 38 whereas at pH = 2.5, D = (0.90 ± 0.08) × 10 −10 m 2 s −1 ( 4). A similar dependence with pH had already been found by time‐resolved fluorescence anisotropy, in which rotational correlation times obtained pointed to smaller rotating entities at low pH 28 . These findings are consistent with circular dichroism data that show the loss of the protein tertiary structure together with a decrease in the percentage of α‐helix content upon acidification of the medium, which was partially recovered upon TSPP association 28 …”

“…A similar dependence with pH had already been found by time‐resolved fluorescence anisotropy, in which rotational correlation times obtained pointed to smaller rotating entities at low pH 28 . These findings are consistent with circular dichroism data that show the loss of the protein tertiary structure together with a decrease in the percentage of α‐helix content upon acidification of the medium, which was partially recovered upon TSPP association 28 …”

“…This short lifetime may safely be attributed to the J-aggregate's fluorescence resulting from the interaction of TSPP with the DMPC vesicles. 21 This short component could be detected only in aqueous solution upon excitation at such wavelengths as 480 nm, 28 where an average value of 106 ps has been reported. 29 The longer lifetime is similar to that obtained in the absence of DMPC and ascribed to the monomer adsorbed.…”

A green method for the preparation of fluorescent hybrid structures of gold and corrole

“…Fluorescence was collected by the same microscope objective, passed through the dichroic mirror and appropriate band-pass filter, and focused through a pinhole (30 mm), to reject out-offocus light, onto a single-photon counting avalanche photodiode SPAD (Perkin-Elmer) whose signal was processed by TimeHarp 200 TC-SPC PC-board (PicoQuant) working in the special time-tagged time-resolved mode. (Andrade et al 2008) Fluorescence lifetimes were obtained in the same equipment with a time-correlated single-photon counting (TC-SPC) technique. The goodness of the fit was evaluated by the usual statistical criteria and by visual inspection of the weighted residual distribution and the autocorrelation function.…”

Mobility of Green Fluorescent Protein in Hydrogel‐Based Drug‐Delivery Systems Studied by Anisotropy and Fluorescence Recovery After Photobleaching

“…In addition, the size of GFP (26 kDa) is in the same range of order as that of antibody fragments. Furthermore, the chromophore of GFP has a clear‐defined binding situation and environment and is tightly bound inside the β‐barrel 26, 27. Local motions and a strong influence of the solvent are severe problems associated with fluorophore‐labeled proteins 28, 29.…”