Fluorescent analysis of interaction of flavonols with hemoglobin and bovine serum albumin

We have studied the fluorescent properties of flavonols (quercetin, fisetin, morin, rutin) with the aim of studying possible interaction with hemoglobin and bovine serum albumin (BSA). We observed an increase in the intensity of intrinsic fluorescence for all the flavonols except rutin in the presence of BSA. From the changes in the fluorescence spectra, we concluded that tautomeric forms are formed on interaction with hemoglobin. We determined the interconnection between the structure of related flavonols and their fluorescent properties on interaction with proteins, and we determined the binding constants for binding with BSA and hemoglobin. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 74, No. 5, pp. 659–664, September–October, 2007.     

Heterotropic binding of alclofenac and dansylsarcosine to bovine serum albumin

The binding data for the interaction of alclofenac (AF) and dansylsarcosine (DS) to bovine serum albumin (BSA) have respectively yielded nonlinear Scatchard plots. The plots have been subjected to Rosenthal’s method of analysis and thus the ligands have been found to possess two different kinds of sites in BSA. The binding capacities of these sites have been evaluated. The fluorescence competition studies have revealed that the binding of DS to BSA is noncompetitively inhibited by AF. Therefore, the presence of distinct binding sites for AF and DS in BSA could be inferred. The fluorescence quenching studies have also been able to demonstrate this aforesaid fact. The analysis of the quenching data by the modified Stern-Volmer plot has indicated that both the tryptophan (Trp) residues of BSA are accessible to DS for the quenching in absence of AF, but only one of them is accessible in presence of AF. This has led to suggest that the binding site of DS has been in the vicinity of loop 3–4, involving Trp-134 and Trp-212. The binding of AF at a distinct site from that of DS has exerted heterotropic interactions at the DS binding site and thereby inhibited the binding of DS to BSA.     

Multi-Photon Fluorescence Correlation Spectroscopy: a Quantification of Tryptophan Methylester Solutions by Visible Emission

A multi-photon excitation fluorescence correlation system has been developed. The emission from tryptophan methylester solution was observed by this system and analyzed by the intensity correlation function of the visible emission, which originates from the two-photon excitation of photo products generated through a five-photon process. The intensity and the product concentration were proportional to the concentration of tryptophan methylester at a lower concentration range and thus the generation process is a single molecular reaction. The correlation analysis determined the concentration of tryptophan methylester down to 5 μM. The photo product generation from tryptophan solution was enhanced by a potassium iodide addition. These results suggest a new quantification method of tryptophan derivatives.     

Conformational Study of Spectrin in Presence of Submolar Concentrations of Denaturants

The presence of very low concentrations of the commonly used chemical denaturants, guanidinium chloride (GdmCl) and urea brought about conformational changes in the erythrocyte membrane skeletal protein, spectrin. Evidences in support of changes in the quaternary structure of spectrin have been put forward from quenching study of tryptophan fluorescence, by both steady state and time-resolved measurements, using acrylamide as the quencher. It revealed significant differences between the Stern–Volmer quenching constants (K_SV) and the fraction of accessible tryptophans (f_e) observed in absence and presence of GdmCl and urea concentrations below 1 M at which the association of the two subunits remains intact. The steady state anisotropy of both the spectrin tryptophans and the spectrin-bound fluorescence probe, Prodan also indicate changes in the overall flexibility of the spectrin dimer, originating from changes in the quaternary structure of spectrin. Studies on the binding of Prodan, further indicate that conformational changes also occur in spectrin near the Prodan-binding site at the terminal domain of the protein which is reflected in 3–4 fold decrease in the affinity of binding of Prodan to spectrin in the presence of GdmCl and urea compared to that observed in the absence of the denaturants. The dissociation constant (K_d) of Prodan to spectrin is 0.43 M at 25°C.     

Intricate packing in the hydrophobic core of barstar through a CH–π interaction

Identification of specific packing interactions within in the hydrophobic core of proteins is important for understanding the integrity of protein structure. Finding such interactions is challenging because few tools allow monitoring of a specific interaction in the presence of several non‐specific forces that hold proteins together. It is important to understand how and when such interactions develop during protein folding. In this study, we have used the intrinsic tryptophan residue, Trp53, as an ultraviolet resonance Raman probe to elucidate the packing interactions in the hydrophobic core of the protein barstar. Barstar is extensively studied for its folding, unfolding and aggregation properties. The Trp53 residue is known to be completely buried in the hydrophobic core of the protein and is used extensively as an intrinsic probe to monitor the folding and unfolding reactions of barstar. A comparison of the resonance Raman cross sections of some bands of Trp53 with those observed for N‐acetyl‐tryptophanoamide in water suggests that Trp53 in barstar is indeed isolated from water. Intensity ratio of the Fermi doublet suggests that Trp53 is surrounded by several aliphatic amino acid residues in corroboration with the crystal structure of barstar. Importantly, we show that the side chain of Trp53 is involved in a unique CH–π interaction with CH groups of Phe56 as well as a steric interaction with the methyl group of Ile5. Copyright © 2014 John Wiley & Sons, Ltd.     

Investigation of protein FTT1103 electroactivity using carbon and mercury electrodes. Surface-inhibition approach for disulfide oxidoreductases using silver amalgam powder

Recently, it was shown that electrochemical methods can be used for analysis of poorly water-soluble proteins and for study of their structural changes and intermolecular (protein–ligand) interactions. In this study, we focused on complex electrochemical investigation of recombinant protein FTT1103, a disulfide oxidoreductase with structural similarity to well described DsbA proteins. This thioredoxin-like periplasmic lipoprotein plays an important role in virulence of bacteria Francisella tularensis. For electrochemical analyses, adsorptive transfer (ex situ) square-wave voltammetry with pyrolytic graphite electrode, and alternating-current voltammetry and constant-current chronopotentiometric stripping analysis with mercury electrodes, including silver solid amalgam electrode (AgSAE) were used. AgSAE was used in poorly water-soluble protein analysis for the first time. In addition to basic redox, electrocatalytic and adsorption/desorption characterization of FTT1103, electrochemical methods were also used for sensitive determination of the protein at nanomolar level and study of its interaction with surface of AgSA microparticles. Proposed electrochemical protocol and AgSA surface-inhibition approach presented here could be used in future for biochemical studies focused on proteins associated with membranes as well as on those with disulfide oxidoreductase activity.     

Effects of metal ion binding on an oncomodulin mutant containing a novel calcium-binding loop

The Ca²⁺-binding protein oncomodulin was altered by cassette mutagenesis of the CD site (CDOM33) with a sequence that was derived by a consensus method using over 250 known Ca²⁺-binding loop sequences. This mutant was studied using time-resolved and steady-state fluorescence from the Trp residue included at position 7 of the loop (position 57 of the protein sequence). The fluorescence characteristics of this species in the absence and presence of metal ions were compared to those of a tetradecapeptide containing the loop and the single Trp mutant of oncomodulin, Y57W. The fluorescence properties of CDOM33 were quite different from the peptide, both in the apo form and in response to metal binding. The consensus CD loop in CDOM33 exhibited the characteristics of a Ca²⁺/Mg²⁺ site in contrast to the Ca²⁺ specificity of the wild-type CD loop. The Trp analogue, 5-hydroxytryptophan (5HW), was incorporated into both oncomodulin mutants to produce Y75(5HW) and 5HW-CDOM33. Results showed that this intrinsic probe was relatively insensitive to structural changes in the mutants upon metal binding compared to Trp itself.     

Effect of hydrostatic pressure on the fluorescence of indole derivatives

Effects of hydrostatic pressure on the fluorescence emission of L-tryptophan, N-acetyl-L-trytophanamide and indole were investigated. An increase in pressure ranging from 1 bar to 2.4 kbar results in reversible red-shifts of the emission of the three fluorophores. The pressure-induced redshift amounts to about 170 cm^–1 at 2.4 kbar, and appears related to changes in Stokes shift of the fluorophores caused by pressure effects on the dielectric constant and/or refractive index of the medium. As the pressure range investigated here is the range commonly used in studies of protein subunit association and/or folding, these observations raise the need for caution in interpreting pressure-induced spectral shifts. The significance of these observations to pressure studies of proteins is illustrated by investigation of pressure effects on human Cu,Zn Superoxide dismutase (SOD) and azurin fromPseudomonas aeruginosa. A reversible 170 cm^–1 red-shift of the emission of SOD was observed upon pressurization to 2.4 kbar. This might be interpreted as pressure-induced conformational changes of the protein. However, further studies using SOD that had been fully unfolded by guanidine hydrochloride, and fluorescence anisotropy measurements indicated that the observed red-shift was likely due to a direct effect of pressure on the fluorescence of the single tryptophan residue of SOD. Similar pressure-induced red-shifts were also observed for the buried tryptophan residue of azurin or for azurin that had been previously denatured by guanidine hydrochloride. These observations further suggest that the effective dielectric constant of the protein matrix is affected by pressure similarly to water.