Giovanna Navarra

Thermal aggregation of glycated bovine serum albumin

Aggregation and glycation processes in proteins have a particular interest in medicine fields and in food technology. Serum albumins are model proteins which are able to self-assembly in aggregates and also sensitive to a non-enzymatic glycation in cases of diabetes. In this work, we firstly reported a study on the glycation and oxidation effects on the structure of bovine serum albumin (BSA). The experimental approach is based on the study of conformational changes of BSA at secondary and tertiary structures by FTIR absorption and fluorescence spectroscopy, respectively. Secondly, we analysed the thermal aggregation process on BSA glycated with different glucose concentrations. Additional information on the aggregation kinetics are obtained by light scattering measurements. The results show that glycation process affects the native structure of BSA. Then, the partial unfolding of the tertiary structure which accompanies the aggregation process is similar both in native and glycated BSA. In particular, the formation of aggregates is progressively inhibited with growing concentration of glucose incubated with BSA. These results bring new insights on how aggregation process is affected by modification of BSA induced by glycation.

Influence of metal ions on thermal aggregation of bovine serum albumin: Aggregation kinetics and structural changes

Metal ions are implicated in protein aggregation processes of several neurodegenerative pathologies. In this work the effects of Cu(II) and Zn(II) ions on heat-induced structural modifications of bovine serum albumin (BSA) were studied, with the aim of delineating the role of these ions in the early stages of proteins aggregation kinetics. A joint application of different techniques was used. The aggregate growth was followed by dynamic light scattering measurements, whereas the conformational changes occurring in the protein structure were monitored by Raman and IR spectroscopy. Both in absence and in presence of metal ions, heating treatment gave rise to beta-structures to the detriment of alpha-helix conformation of BSA. The temperature of protein unfolding was not sensitively affected by the presence of Zn(II) or Cu(II) ions; on the contrary, only Zn(II) ions slightly promoted the heat-induced aggregation of the protein, since bigger aggregates were formed in their presence. The different efficacy of the Cu(II) and Zn(II) ions in promoting the BSA aggregation were highlighted by Raman measurements, assessing the role of His residues in metal binding. A distinct polypeptide folding of the two metal-BSA systems takes place, since the predominant mode of metal binding depends on metal. In particular, in Zn-BSA the metal coordination involves the imidazole N(tau) atom of His which can promote inter-molecular cross-linking.

Metal ions modulate thermal aggregation of beta-lactoglobulin: A joint chemical and physical characterization

Molecular basis of the role played by Cu(2+) and Zn(2+) ions during the thermal aggregation processes of beta-lactoglobulin (BLG) was studied by using a joint application of different techniques. In particular, Raman spectroscopy was very useful in identifying the different effects caused by the two metals at molecular level (i.e. changes in His protonation state, disulfides bridge conformation, and micro-environment of aromatic residues), evidencing the primary importance of the protein charge distribution during the aggregation process. Both metal ions are able to act on this factor and favor the protein aggregation, but Zn(2+) is able to alter the natural conformational state of BLG, causing a slight unfolding, whereas Cu(2+) ions play a role only during the thermal treatment. Thus, Zn(2+) ions favor the formation of bigger aggregates and branched fibril-like structures, whereas for Cu(2+) ions a greater number of cross-beta structures during thermal incubation and finally, fibrillar structures. The aggregation process occurs in two phases, as suggested by the measurements on the time evolution of the BLG aggregates: the first one is characterized by a partial unfolding of the protein and aggregate growth, forming oligomers and protofibrils, whereas the second one is characterized by further supramolecular assembly, leading to the formation of fibrils.

Deciphering metal-induced oxidative damages on glycated albumin structure and function

BACKGROUND Metal ions such as copper or zinc are involved in the development of neurodegenerative pathologies and metabolic diseases such as diabetes mellitus. Albumin structure and functions are impaired following metal- and glucose-mediated oxidative alterations. The aim of this study was to elucidate effects of Cu(II) and Zn(II) ions on glucose-induced modifications in albumin by focusing on glycation, aggregation, oxidation and functional aspects. METHODS Aggregation and conformational changes in albumin were monitored by spectroscopy, fluorescence and microscopy techniques. Biochemical assays such as carbonyl, thiol groups, albumin-bound Cu, fructosamine and amine group measurements were used. Cellular assays were used to gain functional information concerning antioxidant activity of oxidized albumins. RESULTS Both metals promoted inhibition of albumin glycation associated with an enhanced aggregation and oxidation process. Metal ions gave rise to the formation of β-amyloid type aggregates in albumin exhibiting impaired antioxidant properties and toxic activity to murine microglia cells (BV2). The differential efficiency of both metal ions to inhibit albumin glycation, to promote aggregation and to affect cellular physiology is compared. CONCLUSIONS AND GENERAL SIGNIFICANCE Considering the key role of oxidized protein in pathology complications, glycation-mediated and metal ion-induced impairment of albumin properties might be important parameters to be followed and fought.

Characterization of the nucleation process of lysozyme at physiological pH: Primary but not sole process

We report on a kinetic study of the heat-induced aggregation process of lysozyme at physiological pH. The time evolution of the aggregation extent and the conformational changes of the protein were followed by dynamic light scattering (DLS) and FTIR spectroscopy, respectively, whereas the morphology of the aggregates was observed by Atomic Force Microscopy (AFM). The conformational changes of the secondary and tertiary structures were simultaneous and distinct in time with respect to the formation of aggregates. Oligomer formation occurred through at least two different aggregation processes: a nucleation process and a homogeneous non-nucleative diffusion-controlled process. FTIR measurements showed that supramolecular aggregation proceeded without the formation of β-aggregated structures and AFM images revealed the presence of oligomers and amorphous aggregates; no fibrillar structures were observed.

Simultaneous Determination of Caffeine and Chlorogenic Acids in Green Coffee by UV/Vis Spectroscopy

A simple method for the simultaneous determination of caffeine and chlorogenic acids content in green coffee was reported. The method was based on the use of UV/Vis absorption. It is relevant that the quantification of both caffeine and chlorogenic acids was performed without their preliminary chemical separation despite their spectral overlap in the range 250–350 nm. Green coffee was extracted with 70% ethanol aqueous solution; then the solution was analyzed by spectroscopy. Quantitative determination was obtained analytically through deconvolution of the absorption spectrum and by applying the Lambert-Beer law. The bands used for the deconvolution were the absorption bands of both caffeine and chlorogenic acids standards. The molar extinction coefficients for caffeine and chlorogenic acid in ethanol solution at 70% were calculated by using the chemical standards; the estimated values were  M−1 cm−1 for caffeine and  M−1 cm−1 for chlorogenic acids molecules, respectively. The estimate of concentration values was in agreement with the one obtained by High Performance Liquid Chromatography quantification. The method is fast and simple and allows us to realize routine controls during the coffee production. In addition, it could be applied on roasted coffee and espresso coffee.

Chemical and physical characterization of thermal aggregation of model proteins modulated by zinc(II) and copper(II) ions

BACKGROUND: Metal ions are implicated in protein aggregation processes of several neurodegenerative pathologies, where the protein deposition occurs, and in the biotechnology field like the food technology where many processes in food manufacturing are based on thermal treatments. OBJECTIVE: The influence of Cu 2+ or Zn 2+ ions on the thermal aggregation process of Bovine β-lactoglobulin (BLG) and Bovine Serum Albumin (BSA), two protein models, was studied with the aim of delineating the role of these ions in the protein aggregation kinetics and to clarify the related molecular mechanisms. METHODS: The protein structure changes were monitored by Raman spectroscopy, whereas the aggregate growth was followed by Dynamic Light Scattering measurements. RESULTS: Both metal ions are able to favour the BLG aggregation, whereas only Zn 2+ ions have a promoter effect on the thermal aggregation of BSA. The reason of this different behaviour is that the BLG aggregation evolution is manly affected by the redistribution of charges, whereas that of BSA by the metal coordination binding which depends on metal. CONCLUSIONS: Raman spectroscopy, combined with dynamic light scattering experiments, was very useful in identifying the role played by Cu 2+ and Zn 2+ on the aggregation pathways of BLG and BSA. The results provide evidence for the role of histidine residues both in the redistribution of charges and in the two modes of metal binding that take place in BLG- and BSA-containing systems, respectively.

Data concerning the proteolytic resistance and oxidative stress in LAN5 cells after treatment with BSA hydrogels

Proteolytic resistance is a relevant aspect to be tested in the formulation of new nanoscale biomaterials. The action of proteolytic enzymes is a very fast process occurring in the range of few minutes. Here, we report data concerning the proteolytic resistance of a heat-set BSA hydrogel obtained after 20-hour incubation at 60 °C prepared at the pH value of 3.9, pH at which the hydrogel presents the highest elastic character with respect to gel formed at pH 5.9 and 7.4 “Heat-and pH-induced BSA conformational changes, hydrogel formation and application as 3D cell scaffold” (G. Navarra, C. Peres, M. Contardi, P. Picone, P.L. San Biagio, M. Di Carlo, D. Giacomazza, V. Militello, 2016) [1]. We show that the BSA hydrogel produced by heating treatment is protected by the action of proteinase K enzyme. Moreover, we show that LAN5 cells cultured in presence of BSA hydrogels formed at pH 3.9, 5.9 and 7.4 did not exhibit any oxidative stress, one of the first and crucial events causing cell death “Are oxidative stress and mitochondrial dysfunction the key players in the neurodegenerative diseases?” (M. Di Carlo, D. Giacomazza, P. Picone, D. Nuzzo, P.L. San Biagio, 2012) [2] “Effect of zinc oxide nanomaterials induced oxidative stress on the p53 pathway” (M.I. Setyawati, C.Y. Tay, D.T. Leaong, 2013) [3].