Spectroscopic characterization of the warfarin drug-binding site of folded and unfolded human serum albumin anchored on gold nanoparticles: effect of bioconjugation on the loading capacity.

Protein-conjugated gold nanoparticles (AuNPs) have recently shown promising applications in medicine, owing to their inertness and biocompatibility. Herein, we studied the spectroscopy of 25 nm diameter AuNPs, coated with human serum albumin (HSA) as a model drug carrier. The morphology and coating of the AuNPs were examined using transmission electron microscopy and dynamic light scattering. Resonance energy transfer from the sole tryptophan of HSA (Trp214) to the AuNPs indicates a single layer of protein coverage. Using fluorescein (FL) to probe the warfarin drug-binding site in HSA revealed an increase in the HSA-FL binding by ∼4.5 times when HSA is anchored on the nanoparticle surface, indicating a rise in the loading capacity. Femtosecond transient absorption measurements of the surface plasmonic resonance band of the AuNPs show three ultrafast dynamics that are involved in the relaxation process. The three decay components were assigned to the electron-electron (∼400 fs), electron-phonon (∼2.0 ps) and phonon-phonon (200-250 ps) interactions. These dynamics were not changed upon coating the AuNPs with HSA which indicates the chemical and physical stability of the AuNPs upon bioconjugation. Chemical unfolding of the warfarin binding site with guanidine hydrochloride (GdnHCl) was studied by measuring the spectral shift in the Trp214 fluorescence and the appearance of the Tyr fluorescence. Unfolding was shown to start at [GdnHCl] ≥ 2.0 M and is complete at [GdnHCl] = 6.0 M. HSA anchored onto the nanoparticle surface shows more resistance to the unfolding effect which is attributed to the stability of the native form of HSA on the nanoparticle surface. On the other hand, upon complete unfolding, a larger red shift in the Trp214 fluorescence was observed for the HSA-AuNP complex. This observation indicates that, upon unfolding, the HSA molecule is still anchored on the AuNP surface in which subdomain IIA is facing the outer water molecules in the bulk solution as well as the hydration shell rather than the core of the nanoparticle. The current study is important for a better understanding of the physical and dynamical properties of protein-coated metal nanoparticles, which is expected to help in optimizing their properties for critical applications in nanomedicine.

Photophysical properties of hydroxyphenyl benzazoles and their applications as fluorescent probes to study local environment in DNA, protein and lipid.

Fluorescence techniques have drawn increasing attention because they provide crucial information about molecular interactions in protein-ligand systems beyond that obtained by other methods. The advantage of fluorescence spectroscopy stems from the fact that the majority of molecules in biological systems do not exhibit fluorescence, making fluorescent probes useful with high sensitivity. Also, the fluorescence emission is highly sensitive to the local environment, providing a valuable tool to investigate the nature of binding sites in macromolecules. In this review, we discuss some of the important applications of a class of molecules that have been used as fluorescent probes in a variety of studies. Hydroxyphenyl benzazoles (HBXs) show distinct spectroscopic features that make them suitable probes for the study of certain biological mechanisms in DNA, protein and lipid. In particular, the complex photophysics of 2-(2'-hydroxyphenyl)benzoxazole (HBO) and the distinguished fluorescence signatures of its different tautomeric forms make this molecule a useful probe in several applications. Among these are probing the DNA local environment, study of the flexibility and specificity of protein-binding sites, and detecting the heterogeneity and ionization ability of the head groups of different lipidic phases. The spectroscopy of HBX molecules and some of their chemically modified structures is also reviewed.

Kinetic studies on the inhibition of GABA-T by gamma-vinyl GABA and taurine.

Gamma-aminobutyric acid transaminase (GABA-T, EC 2.6.1.19) is a pyridoxal phosphate (PLP) dependent enzyme that catalyzes the degradation of gamma-aminobutyric acid. The kinetics of this reaction are studied in vitro, both in the absence, and in the presence of two inhibitors: gamma-vinyl GABA (4-aminohex-5-enoic acid), and a natural product, taurine (ethylamine-2-sulfonic acid). A kinetic model that describes the transamination process is proposed. GABA-T from Pseudomonas fluorescens is inhibited by gamma-vinyl GABA and taurine at concentrations of 51.0 and 78.5 mM. Both inhibitors show competitive inhibition behavior when GABA is the substrate and the inhibition constant (Ki) values for gamma-vinyl GABA and taurine were found to be 26 +/- 3 mM and 68 +/- 7 mM respectively. The transamination process of alpha-ketoglutarate was not affected by the presence of gamma-vinyl GABA, whereas, taurine was a noncompetitive inhibitor of GABA-T when alpha-ketoglutarate was the substrate. The inhibition dissociation constant (Kii) for this system was found to be 96 +/- 10 mM. The Michaelis-Menten constant (Km) in the absence of inhibition, was found to be 0.79 +/- 0.11 mM, and 0.47 +/- 0.10 mM for GABA and alpha-ketoglutarate respectively.