ABSTRACT
The plasmon resonance of gold nanoparticles (GNPs) synthesized on a protein template senses formation of advanced glycosylated end products (AGEs). A graded alteration of plasmon resonance (both the peak and intensity are affected) is observed as the glycation progresses. Transmission electron microscopy shows significant shift of the size distribution of GNPs in presence of glycation. The higher plasmon resonance is thus caused by increased formation of GNPs, which in turn is attributed to a larger number of smaller particles. To study the binding of the protein with the GNP, infrared (IR) spectroscopy and circular dichroism (CD) studies were undertaken. Whereas the CD studies confirmed the emergence of beta-structure and loss of alpha-helix, the IR data indicated glycation-induced alterations in the amide I region. The proposed sensor for formation of AGEs thus apparently operates by direct or indirect conjugation with amino groups. Incidentally, glycation and AGE formation are responsible for a number of diabetes-related clinical conditions, and the present approach could be adopted for use for a simple colorimetric assay for the AGEs.
Subject(s)
Biosensing Techniques/methods , Coated Materials, Biocompatible/chemistry , Glycation End Products, Advanced/analysis , Glycation End Products, Advanced/chemistry , Gold/chemistry , Hemoglobins/chemistry , Nanoparticles/chemistry , Surface Plasmon Resonance/methods , Hemoglobins/ultrastructure , Materials Testing , Nanoparticles/ultrastructure , Nanotechnology/methods , Surface PropertiesABSTRACT
The size of gold nanoparticles is shown here to gradually decrease if it is allowed to grow on a protein template, and the protein is subjected to unfolding by a nonionic denaturant. The correlation between size of the gold nanoparticle formed and the plasmon frequency observed remains linear, except at stages where protein folding intermediates are formed. Higher population of exposed tyrosine residues, number of sulfhydryl groups of the protein, and the overall exposition of the inner hydrophobic core may lead to the generation of smaller particles. The method provides a simple colorimetric sensing of protein conformation and has been tested for both nonheme and heme proteins (hemoglobin and bovine serum albumin). Similarly, protein variants with defects in folding (caused by subunit misassembly or mutation) can also be classified. Possible application of this approach in hemoglobinopathy (e.g., thalassemia carrier detection) is discussed in the text.
Subject(s)
Gold , Hemoglobinopathies/pathology , Hemoglobins/chemistry , Nanoparticles , Serum Albumin, Bovine/chemistry , Animals , Cattle , Fluorescence , Hemoglobins/metabolism , Humans , Particle Size , Protein Conformation , Protein Folding , Serum Albumin, Bovine/metabolism , Surface Plasmon Resonance , Tryptophan , Tyrosine , UreaABSTRACT
Glycation, a local covalent interaction, leads to alterations in secondary and tertiary structures of hemoglobin, the changes produced by fructose being more pronounced than those caused by glucose. The Stokes diameter of hemoglobin increases upon glycation from 7 to 14 nm and a concurrent inter-chain cross-linking and heme loss are also observed, particularly in the later stage of glycation. An initial increase of tryptophan (trp) fluorescence was observed in both glucation and fructation. In case of frucation however there was a decrease in tryptophan fluorescence that was accompanied by an increase in fluorescence of the advanced glycosylation end products (AGEs). This fluorescence behavior is indicative of energy transfer between tryptophan and the AGEs formed during the late stage of glycation. Emergence of an isosbestic point in the fluorescence spectra (taken at different time intervals) implies existence of two distinct glycation stages. The late glycation stage is also marked by an increase of beta structure and random coil at the expense of alpha helix. It is further observed that this compensatory loss of alpha helix (reported for the first time) and increase in beta sheet and random coil elements depend on the number of solvent-accessible glycation sites (rather than total number of such sites) and the subunit assembly of the protein.
Subject(s)
Glucose/chemistry , Proteins/chemistry , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Fluorescence , Glycation End Products, Advanced/chemistry , Heme/chemistry , Protein Structure, SecondaryABSTRACT
The paper describes an experimental construct of the folding route of the heme protein cytochrome-C. The construct highlights a slowing down near the nose of the folding funnel caused by the multiplicity of the energy traps near the native conformation created as a result of complex heme-peptide interaction. Interestingly the hydrodynamic size, the size heterogeneity and peroxidase activity serve as a triple measure of the distance of this near equilibrium departure from native conformation. Accordingly, the folding process is marked with a gradual and reversible reduction of mean hydrodynamic size, size heterogeneity and peroxidase activity (higher in unfolded state). The Dynamic Light Scattering based straightforward illustration of hydrodynamic size variation may serve as a model to slow folding observed in case of heme proteins, the heme itself serving as a natural facilitator for the native peptide conformation.
