Tuning the mechanical and physicochemical properties of cross-linked protein films.

Films derived from natural sources such as proteins provide an advantage over synthetic films due to their noncytotoxicity, biodegradability, and vast functionality. A new protein source gained from the cataractous eye protein isolate (CEPI) obtained after surgery has been investigated for this purpose. Glycerol has been employed as the plasticizer and glutaraldehyde (GD) as a cross-linker. Fourier transform infrared spectroscopy was employed to characterize the films. Nanoindentation and thermogravimetric analyses reveal improved mechanical and thermal properties of the cross-linked films. The films with 20% (w/w) GD exhibited properties such as the highest modulus and low water solubility. It is possible to tune the properties based on the extent of cross-linking. All the films were completely degraded by the enzyme trypsin. The similarity of these films was checked by using the prepared films as a delivery vehicle for a model compound, ampicillin sodium. The encapsulation efficiency was found to be 74%, and in vitro release studies showed significant amounts of drug release at physiological pH. This study will help us understand how the properties of protein films can be tuned to obtain the desired physicochemical properties. These biodegradable protein films could find use in pharmaceutical industries as delivery carriers.

Probing the inhibitory potency of epigallocatechin gallate against human γB-crystallin aggregation: Spectroscopic, microscopic and simulation studies.

Aggregation of human ocular lens proteins, the crystallins is believed to be one of the key reasons for age-onset cataract. Previous studies have shown that human γD-crystallin forms amyloid like fibres under conditions of low pH and elevated temperature. In this article, we have investigated the aggregation propensity of human γB-crystallin in absence and presence of epigallocatechin gallate (EGCG), in vitro, when exposed to stressful conditions. We have used different spectroscopic and microscopic techniques to elucidate the inhibitory effect of EGCG towards aggregation. The experimental results have been substantiated by molecular dynamics simulation studies. We have shown that EGCG possesses inhibitory potency against the aggregation of human γB-crystallin at low pH and elevated temperature.

ß-cyclodextrin encapsulated polyphenols as effective antioxidants.

Formation of dityrosine (DT) cross-linkages in proteins is one of the most widely used markers of oxidative stress. Ribonuclease A (RNase A) has 6 Tyr residues and shows a characteristic DT fluorescence peak upon oxidation in addition to major changes in its secondary structure. DT formation can be prevented by using polyphenols (GA, ECG, and EGCG) which are known to have strong antioxidant activity. However, it has been observed that ECG and EGCG initiate protein oligomerization due to protein-polyphenol cross-linkages. To prevent the formation of such cross-linkages we have used ß-cyclodextrin (ß-CD) to encapsulate the polyphenols and studied its antioxidant properties along with that of free polyphenols. The polyphenol/ß-cyclodextrin (ß-CD) inclusion complexes not only prevent DT formation but also reduce protein oligomerization. This may be attributed to the fact that the quinone forming rings of ECG and EGCG become encapsulated in the cavity of ß-CD and are no longer available for protein cross-linking.

Green tea flavanols protect human γB-crystallin from oxidative photodamage.

Age related cataract is a major cause of visual loss worldwide that is a result of opacification of the eye lens proteins. One of the major reasons behind this deterioration is UV induced oxidative damage. The study reported here is focused on an investigation of the oxidative stress induced damage to γB-crystallin under UV exposure. Human γB-crystallin has been expressed and purified from E. coli. We have found that epicatechin gallate (ECG) has a higher affinity towards the protein compared to epigallocatechin (EGC). The in vitro study of UV irradiation under oxidative damage to the protein in the presence of increasing concentrations of GTPs is indicative of their effective role as potent inhibitors of oxidative damage. Docking analyses show that the GTPs bind to the cleft between the domains of human γB-crystallin that may be associated with the protection of the protein from oxidative damage.

Glycation of human γB-crystallin: A biophysical investigation.

Glycation of ocular lens proteins plays a vital role in the development of diabetic cataract. In order to investigate the role of glycation in cataractogenesis, the extent of glycation of human γB-crystallin was determined by an in vitro glycation study in a solution of high glucose content for upto 28days. The glycated protein has been purified and the formation of advanced glycation end products (AGEs) has been monitored spectroscopically. Size exclusion chromatographic studies showed that the covalent intermolecular crosslinking in the dimer formed was not due to disulfide bond formation. MALDI-TOF spectroscopy was employed to determine the number of glucose moieties attached to the protein due to glycation.

Protection of human γB-crystallin from UV-induced damage by epigallocatechin gallate: spectroscopic and docking studies.

The transparency of the human eye lens depends on the solubility and stability of the structural proteins of the eye lens, the crystallins. Although the mechanism of cataract formation is still unclear, it is believed to involve protein misfolding and/or aggregation of proteins due to the influence of several external factors such as ultraviolet (UV) radiation, low pH, temperature and exposure to chemical agents. In this article, we report the study of UV induced photo-damage (under oxidative stress) of recombinant human γB-crystallin in vitro in the presence of the major green tea polyphenol, (-)-epigallocatechin gallate (EGCG). We have shown that EGCG has the ability to protect human γB-crystallin from oxidative stress-induced photo-damage.

Hydrophobic tail length plays a pivotal role in amyloid beta (25-35) fibril-surfactant interactions.

The amyloid ß-peptide fragment comprising residues 25-35 (Aß25-35 ) is known to be the most toxic fragment of the full length Aß peptide which undergoes fibrillation very rapidly. In the present work, we have investigated the effects of the micellar environment (cationic, anionic, and nonionic) on preformed Aß25-35 fibrils. The amyloid fibrils have been prepared and characterized by several biophysical and microscopic techniques. Effects of cationic dodecyl trimethyl ammonium bromide (DTAB), cetyl trimethylammonium bromide (CTAB), anionic sodium dodecyl sulfate (SDS), and nonionic polyoxyethyleneoctyl phenyl ether (Triton X-100 or TX) on fibrils have been studied by Thioflavin T fluorescence, UV-vis spectroscopy based turbidity assay and microscopic analyses. Interestingly, DTAB and SDS micelles were observed to disintegrate prepared fibrils to some extent irrespective of their charges. CTAB micelles were found to break down the fibrillar assembly to a greater extent. On the other hand, the nonionic surfactant TX was found to trigger the fibrillation process. The presence of a longer hydrophobic tail in case of CTAB is assumed to be a reason for its higher fibril disaggregating efficacy, the premise of their formation being largely attributed to hydrophobic interactions. Proteins 2016; 84:1213-1223. © 2016 Wiley Periodicals, Inc.

Preparation of albumin based nanoparticles for delivery of fisetin and evaluation of its cytotoxic activity.

Fisetin is a well known flavonoid that shows several properties such as antioxidant, antiviral and anticancer activities. Its use in the pharmaceutical field is limited due to its poor aqueous solubility which results in poor bioavailability and poor permeability. The aim of our present study is to prepare fisetin loaded human serum albumin nanoparticles to improve its bioavailability. The nanoparticles were prepared by a desolvation method and characterized by spectroscopic and microscopic techniques. The particles were smooth and spherical in nature with an average size of 220 ± 8 nm. The encapsulation efficiency was found to be 84%. The in vitro release profile showed a biphasic pattern and the release rate increases with increase in ionic strength of solution. We have also confirmed the antioxidant activity of the prepared nanoparticles by a DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. Further its anticancer activity was evaluated using MCF-7 breast cancer cell lines. Our findings suggest that fisetin loaded HSA nanoparticles could be used to transfer fisetin to target areas under specific conditions and thus may find use as a delivery vehicle for the flavonoid.

EGCG prevents tryptophan oxidation of cataractous ocular lens human γ-crystallin in presence of H2O2.

Disruption of the short range order of proteins present in the ocular lens leads to cataract resulting in a loss of transparency. Human γ-crystallin (HGC), a water soluble protein present in the lens is known to aggregate with aging. A modified form of HGC (HGC(c)) was isolated from cataractous human ocular lens extract and the number of Trp residues that undergo oxidation was determined. The extent of oxidized Trp (N-formyl kynurenine) in HGC due to cataract formation was determined, primarily using fluorescence spectroscopy. The ability of (-)-epigallocatechin gallate (EGCG) to retain its antioxidant effect even in the presence of H2O2 was investigated. This was monitored by its ability to prevent the modification of intact Trp residues in HGC(c) isolated from cataractous human eye lens. Significant Trp fluorescence quenching occurs on interaction of the green tea component, EGCG with HGC(c) accompanied by a red shift. Docking studies were employed to substantiate the experimental results. As eye lens proteins are prone to oxidative stress it is essential that a clear understanding of the effects of the components generated in vivo vis-à-vis the antioxidant effects of natural polyphenols be obtained.

Distribution of protein Ramachandran psi (ψ) angle using non-resonance visible raman scattering measurements.

Knowing the distribution of Ramachandran angles helps in understanding peptide and protein backbone conformation. Empirical relations are proposed to correlate the spectral profile of the amide III3 band, obtained from ultraviolet resonance Raman measurements (UVRR), with the Ramachandran dihedral psi angle distribution in small peptide and protein molecules, in different environmental conditions (Mikhonin et al. J. Phys. Chem. B 2006, 110, 1928-1943). It has also been used for more complicated structures, like large globular proteins and protein fibrils. In our work here, we use visible Raman spectra and available empirical relations to obtain similar correlations for human serum albumin, hen egg white lysozyme, and human gamma crystallin. We also report the dihedral angle distribution in fibrils and a denatured protein in an ethanol environment using the same spectroscopic technique.