Changes In Tertiary Structure Research Articles

Higher-Order Structure Comparison of a Proposed Biosimilar and the Innovator Biotherapeutic Trastuzumab using Circular Dichroism Coupled with Statistical Analysis

We employ a novel circular dichroism (CD) technology coupled with statistical analysis to assess the higher-order structure (HOS) similarity of commercially available trastuzumab and a proposed biosimilar. This technology shows good potential for enabling the comparison of similarities and differences in secondary and tertiary structures of proteins. Multiple CD spectra in far- and near-UV are obtained reproducibly from a CD spectrometer with fully integrated autosampler and flow cell to eliminate the errors typically associated with sample handling on manual CD spectrometers. The significance of similarities or differences was quantified statistically using three analytical methods (weighted spectral difference, correlation coefficient, and area of overlap). HOS comparison of the secondary structure (far-UV CD spectra) suggested similarity between the innovator drug, trastuzumab (Herceptin®), and biosimilar products. However, the tertiary structure (near-UV CD spectra) suggested statistically significant differences. These differences may be due to changes in tertiary structure or to modifications and degradations during the process of production or storage. The results show that the automated circular dichroism technology coupled with statistical analysis is a robust tool for enabling the comparison of similarities and differences in secondary and tertiary structures of protein products.

Raman spectroscopy analysis of the effect of electrolysis treatment on the structure of soy protein isolate

The effect of electrolysis on soy protein isolate (SPI) was studied by Raman spectroscopy and ultraviolet spectroscopy. The results showed that with the prolongation of electrolysis time, the α-helix content decreased first and then increased. Trp and Tyr residues tended to be exposed from their original embedded state, while electrolysis gradually shifted the SPI disulfide bond from the g–g–g configuration to g–g–t and t–g–t configuration. Therefore, the electrolysis treatment influences the SPI disulfide bond, resulting in intermolecular disulfide bond. Raman spectra showed the secondary and tertiary structure changes of SP and revealed that the structure of SPI would be affected by electrolysis. And the results of UV scanning spectrum confirm the above conclusions.

The interaction of Naphthol Yellow S (NYS) with pepsin: Insights from spectroscopic to molecular dynamics studies

The effects of Naphthol Yellow S (NYS) on the structure and activity of pepsin were carried out using ultraviolet–visible (UV–Vis) spectroscopy, intrinsic fluorescence spectroscopy, circular dichroism (CD), thermal stability, kinetic techniques, as well as molecular docking, and Molecular dynamic simulations (MD) technique. The experimental results from fluorescence spectroscopy showed that the changes in pepsin's tertiary structure were caused by NYS binding. The apparent binding constant Ka, the number of the binding sites, and thermodynamic parameters were computed at three different temperatures. Thermodynamic results revealed that NYS interacts with pepsin spontaneously by hydrogen bond and Van der Waals forces. The result of the circular dichroism spectral suggests the secondary structural changes. An increase in the content of the β-sheet and β-turn structure was shown. Kinetic parameters revealed that NYS inhibited the activity of pepsin by the mixed model. The Molecular dynamic (MD) and docking simulations supported experimental findings. The main interactions between NYS and pepsin are hydrogen bonds and Van der Waals Forces. As a result, NYS could be considered as an inhibitor with adverse effects on pepsin structure and function.

Ultrasonic effects on the headspace volatilome and protein isolate microstructure of duck liver, as well as their potential correlation mechanism

In spite of the high added value and tremendous output from duck processing industries, duck liver (DLv) is underutilized and a major factor is related to its prominent off-flavor perception which hampers the consumption and processing attributes. This work was designed to investigate the impact of low-frequency ultrasound (US) pretreatments on the headspace volatilome evolution of DLv and its isolated protein (DLvP) microstructure, aiming at exploring the potential of US technology to inhibit off-flavor perception and possible mechanisms behind. Results suggested that US pretreatment resulted in decreased lipid oxidation and off-flavor perception, simultaneously without significantly causing physicochemical influence including texture, pH and color. US induced obvious tertiary structural changes of DLvP, as revealed by the intrinsic fluorescence and surface hydrophobicity (H0), whereas the SH, S-S, secondary structure and molecular weight of DLvP remained unaffected, suggesting the presence of molten globule state (MG-state) under ultrasonic conditions. Besides, the headspace contents of flavor compounds, mainly aldehydes and alcohols, were significantly decreased whereas acids were increased. Multivariate analysis suggested an obvious US-induced effect on the volatilome evolution of DLv samples. Discriminant analysis recognized the aroma compounds including aldehydes and alkenals as the major contributors leading to the change of olfactory characteristics of DLv after ultrasonic treatment. Correlation analysis demonstrated the positive relationship between the volatile markers variation and the increased H0 values, a characteristic attribute of MG-state protein. The results obtained in this work suggested that US technology matched with suitable parameters could be a very promising approach to modulate the off-flavor perception of liver products by altering DLvP conformation.

A novel missense mutation of LRP6 identified by whole-exome sequencing in a Chinese family with non-syndromic tooth agenesis.

The aim of this study was to explore the genetic basis of non-syndromic tooth agenesis (TA) in a Chinese family of five individuals using whole-exome sequencing (WES) analysis. Five participants/Family-based study of a non-syndromic TA proband. The proband, proband's mother and grandmother displayed congenital tooth deficiency. Genomic DNA was extracted from the peripheral blood or saliva samples of the proband, her parents and her grandmother, and WES was utilized to identify the causal genetic mutation. The identified mutation was further verified by Sanger sequencing and analysed using bioinformatics tools. A novel missense mutation, c.G711T (p.L237F), was identified in the low-density lipoprotein receptor-related protein 6 (LRP6) gene in all affected individuals. Bioinformatics analysis predicted the mutation to be deleterious, with the mutant LRP6 protein displaying a tertiary structural change that might disturb the Wnt/β-catenin signalling pathway. The identification of the mutation in the LRP6 gene and autosomal dominant inheritance with TA in the generations is consistent with the mutation being responsible for TA in the family, and furthers the association of LRP6 with nonsyndromic TA.

Aminoglycoside antibiotics can inhibit or activate twister ribozyme cleavage

Interactions between aminoglycoside antibiotics and the twister ribozyme were investigated in this study. An initial screen of 17 RNA-binding antibiotics showed that a number of aminoglycosides inhibit the ribozyme, while a subset of aminoglycosides enhances twister cleavage. Initial kinetic analysis of the twister ribozyme showed a sevenfold inhibition of ribozyme cleavage by paromomycin and a fivefold enhancement of cleavage by sisomicin. Direct binding between the twister ribozyme RNA and paromomycin or sisomicin was measured by microscale thermophoresis. Selective 2'-hydroxyl acylation analysed by primer extension shows that both paromomycin and sisomicin induce distinctive tertiary structure changes to the twister ribozyme. Published crystal structures and mechanistic analysis of the twister ribozyme have deduced a nucleobase-mediated general acid-base catalytic mechanism, in which a conserved guanine plays a key role. Here, we show that paromomycin binding induces a structural transition to the twister ribozyme such that a highly conserved guanine in the active site becomes displaced, leading to inhibition of cleavage. In contrast, sisomicin binding appears to change interactions between P3 and L2, inducing allosteric changes to the active site that enhance twister RNA cleavage. Therefore, we show that small-molecule binding can modulate twister ribozyme activity. These results suggest that aminoglycosides may be used as molecular tools to study this widely distributed ribozyme.

Molecular structural modification of duck egg white protein conjugates with monosaccharides for improving emulsifying capacity

Glycosylation is considered to be an effective method to improve the emulsifying property of proteins, and this work investigates and discusses the correlation between the molecular structure of glycosylated egg white protein (EWP) and its emulsifying capacity. Three types of monosaccharides, including glucose, d-galactose and d-xylose, were utilized to conjugate with EWP in a wet glycosylation environment. Molecular structural properties of EWP, involving surface hydrophobicity, secondary and tertiary structures, protein flexibility, and free sulfhydryl group content, were determined. The results showed that the degree of grafting (DG), browning intensity and apparent viscosity increased significantly (P < 0.05) with the monosaccharides proportion improved, and the intrinsic fluorescence intensity and surface hydrophobicity of EWP decreased obviously during glycosylation, and the absolute value of zeta potential, protein flexibility and free sulfhydryl content improved remarkably (P < 0.05). Emulsifying capacity results showed that glycosylation improved emulsifying activity and emulsifying stability of EWP, especially after the addition of d-xylose. The particle size analysis exhibited that the D4,3 and D3,2 declined obviously (P < 0.05) as DG increased. Correlation analysis revealed the improvement of the emulsifying capacity of glycosylated EWP showed in a DG-dependent manner, as remarkably influenced by molecular flexibility, surface hydrophobicity, tertiary structure changes and free sulfhydryl. The work provides a deep insight into the molecular conformation−emulsifiability relationship of EWP−monosaccharides conjugates.

Effects of Maillard reaction on structural modification and potential allergenicity of peanut 7S globulin (Ara h 1).

Ara h 1 is a major food allergen in peanuts. Recently, many studies have revealed that the Maillard reaction (MR) affects the allergenicity of food proteins. To investigate the influence of the MR on the allergenicity of Ara h 1, R-Ara h 1 was processed with glucose in dry heating conditions for different periods. The extent of the MR was assessed by four methods. The changes in secondary and tertiary structures were characterized through spectroscopy assays. Advanced glycation end products (AGE) structures were identified by protein sample dry heating for 60 min, indicating the formation of AGE-Ara h 1. Simulated gastric fluid (SGF) digestion analysis showed that AGE-Ara h 1 has higher resistance to peptic digestion than R-Ara h 1. The BALB/c mouse model was also utilized to explore the effect of the MR on the allergenicity of Ara h 1, and the results showed that the Th2-type cytokines, antibodies, and histamine content increased, and there was a greater degree of degranulation of rat basophilic leukemia (RBL) cells in the AGE-Ara h 1 group compared with the R-Ara h 1 group. During the process of dry heating, proteins participated in the MR with changes in secondary and tertiary structures. The condition applying a temperature of 100 °C for 60 min caused the formation of AGE-Ara h 1. Simulated gastric fluid digestion analysis showed that AGE-Ara h 1 had a greater resistance to peptic digestion than R-Ara h 1. The BALB/c mouse model showed that AGE-Ara h 1 had more allergenicity, indicating that the MR could enhance the allergenicity of Ara h 1. © 2020 Society of Chemical Industry.

Effects of High Intensity Ultrasound on Physiochemical and Structural Properties of Goat Milk β-Lactoglobulin.

This study aimed to compare the effects of high intensity ultrasound (HIU) applied at various amplitudes (20~40%) and for different durations (1~10 min) on the physiochemical and structural properties of goat milk β-lactoglobulin. No significant change was observed in the protein electrophoretic patterns by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Deconvolution and second derivative of the Fourier transform infrared spectra (FTIR) showed that the percentage of β-sheet of goat milk β-lactoglobulin was significantly decreased while those of α-helix and random coils increased after HIU treatment The surface hydrophobicity index and intrinsic fluorescence intensity of samples was enhanced and increased with increasing HIU amplitude or time. Differential scanning calorimetry (DSC) results exhibited that HIU treatments improved the thermal stability of goat milk β-lactoglobulin. Transmission electron microscopy (TEM) of samples showed that the goat milk β-lactoglobulin microstructure had changed and it contained larger aggregates when compared with the untreated goat milk β-lactoglobulin sample. Data suggested that HIU treatments resulted in secondary and tertiary structural changes of goat milk β-lactoglobulin and improved its thermal stability.

Characterization of the apo-form of extracellular hemoglobin of Glossoscolex paulistus (HbGp) and its stability in the presence of urea.

The structural study of small heme-containing proteins, such as myoglobin, in the apo-form lacking heme has been extensively described, but the characterization and stability of the giant Glossoscolex paulistus hemoglobin (HbGp), in the absence of heme groups, has not been studied. Spectroscopic data show efficient extraction of the heme groups from the hemoglobin, with relatively small secondary and tertiary structural changes in apo-HbGp noticed compared to oxy-HbGp. Electrophoresis shows a partial precipitation of the trimer abc (significantly lower intensity of the corresponding band in the gel), due to extraction of heme groups, and the predominance of the intense monomeric d band, as well as of two linker bands. AUC and DLS data agree with SDS-PAGE in showing that the apo-HbGp undergoes dissociation into the d and abc subunits. Subunits d and abc are characterized by sedimentation coefficients and percentage contributions of 2.0 and 3.0S and 76 and 24%, respectively. DLS data suggest that the apo-HbGp is unstable, and two populations are present in solution: one with a diameter around 6.0nm, identified with the dissociated species, and a second one with diameter 100-180nm, due to aggregated protein. Finally, the presence of urea promotes the exposure of the fluorescent probes, extrinsic ANS and intrinsic protein tryptophans to the aqueous solvent due to the unfolding process. An understanding of the effect of heme extraction on the stability of hemoproteins is important for biotechnological approaches such as the introduction of non-native prosthetic groups and development of artificial enzymes with designed properties.

Binding of CML-Modified as Well as Heat-Glycated β-lactoglobulin to Receptors for AGEs Is Determined by Charge and Hydrophobicity.

Intake of dietary advanced glycation end products (AGEs) is associated with inflammation-related health problems. Nε-carboxymethyl lysine (CML) is one of the best characterised AGEs in processed food. AGEs have been described as ligands for receptors present on antigen presenting cells. However, changes in protein secondary and tertiary structure also induce binding to AGE receptors. We aimed to discriminate the role of different protein modifications in binding to AGE receptors. Therefore, β-lactoglobulin was chemically modified with glyoxylic acid to produce CML and compared to β-lactoglobulin glycated with lactose. Secondary structure was monitored with circular dichroism, while hydrophobicity and formation of β-sheet structures was measured with ANS-assay and ThT-assay, respectively. Aggregation was monitored using native-PAGE. Binding to sRAGE, CD36, and galectin-3 was measured using inhibition ELISA. Even though no changes in secondary structure were observed in all tested samples, binding to AGE receptors increased with CML concentration of CML-modified β-lactoglobulin. The negative charge of CML was a crucial determinant for the binding of protein bound CML, while binding of glycated BLG was determined by increasing hydrophobicity. This shows that sRAGE, galectin-3, and CD36 bind to protein bound CML and points out the role of negatively charged AGEs in binding to AGE receptors.

Effect of Antioxidants on Heavy Metals Induced Conformational Alteration of Cytochrome C and Myoglobin.

The exposure to heavy metals due to unrestrained industrialization, pollution and non-degradability imposes a significant risk to human health. Proteins are prime targets of heavy metal stress, however, the underlying mechanisms and its impact on heme proteins is still not entirely clear. To analyze the deleterious effect of heavy metals such as cadmium, chromium and mercury on conformation of two proteins namely, cytochrome c and myoglobin. The protective effect of glycine and ascorbic acid (animal origin), gallic acid and sesamol (plant origin) on heavy metal exposure was studied. Far- and near-UV Circular Dichroism (CD) measurements monitored the changes in secondary and tertiary structure. Absorption Soret spectroscopy study revealed changes in heme-protein interaction. Peroxidase activity has been assayed to measure the absorption of tetraguaiacol. The interaction of heme proteins with different heavy metals was done using docking study. Far- and near-UV CD measurements reveal that heavy metals disrupt the secondary and tertiary structure of heme proteins. Antioxidants counteract the deleterious effect of heavy metals. Absorption spectroscopy revealed changes in the Soret region of these heme proteins. Changes in peroxidase activity was observed on addition of heavy metals and antioxidants. Molecular docking validated interaction of the heavy metals with proteins with a significant binding affinity (-2.3 kcal/- mol). Heavy metals interfered and disrupted both the heme proteins and mercury showed the maximum deleterious effect, further, chromium showed detrimental effect at very small concentration. The antioxidants from animal origin exhibited better protective response than those from plant source.

Interaction mechanism of an antimalarial drug, sulfadoxine with human serum albumin

The main aim of this study was to characterize the molecular interaction between sulfadoxine and the major transport protein in the blood plasma, human serum albumin using fluorescence, absorption, circular dichroism, and voltammetric techniques along with computational methods. Sulfadoxine-induced changes in the fluorescence signal of human serum albumin hinted the complex formation between sulfadoxine and human serum albumin. Both values of the bimolecular quenching rate constant and UV-vis absorption spectral results characterized the quenching of human serum albumin fluorescence as static quenching. Analysis of the quenching results showed a moderate binding affinity of 3.39 × 104 M−1 at 300 K between sulfadoxine and human serum albumin. Thermodynamic data (entropy change = +104.42 J mol−1 K−1, enthalpy change = +5.25 kJ mol−1) suggested the participation of hydrophobic interactions as the main binding force in the complex formation. Secondary and tertiary structural changes along with microenvironmental perturbation around protein fluorophores were also noticed upon sulfadoxine binding. The voltammetric spectral analysis further supported the complex formation between human serum albumin and sulfadoxine. Competitive ligand displacement results, as well as computational analysis, revealed binding of sulfadoxine to Sudlow’s Site I, located in subdomain IIA of human serum albumin.

Cryoprotective effect of egg white proteins and xylooligosaccharides mixture on oxidative and structural changes in myofibrillar proteins of Culter alburnus during frozen storage

The purpose of this research was to investigate the cryoprotective role of EWP-XO in the prevention of oxidative and structural changes in the myofibrillar proteins (MPs). Different concentrations of egg white protein and xylooligosaccharide (EWP-XO) mixture (0, 2, 4 and 6%) were added to the MPs of Culter alburnus fish during frozen storage (−18 °C) of 60 days. During the study, it was observed that EWP-XO significantly (P < .05) reduced the Ca-ATPase activity, which is greatly related with tertiary structural changes. Meanwhile, carbonyl contents of MPs increased in line with frozen storage (control samples). Meanwhile, samples treated with 6% EWP-XO showed less increase in carbonyl content indicating the decreased protein oxidation. The addition of EWP-XO efficiently inhibited the decline in the sulfhydryl contents. Furthermore, through circular dichroism analysis it was confirmed that the addition of EWP-XO increased the secondary structural stability by preventing the reduction in α-helix content. Microstructural analysis confirmed the preservation of a well-structured protein network that reduced the porosity and protein aggregation of MPs gel. It was concluded that 6% EWP-XO was an effective cryoprotectant mixture, which preserved the functional and structural properties of Culter alburnus during 60 days of frozen storage period.

Comprehensive investigations about the binding interaction of acesulfame with human serum albumin.

In this work, the binding interaction of an artificial sweetener, acesulfame (ACS) with human serum albumin (HSA) are investigated at the molecular level by using spectral methods and molecular modeling. ACS has the ability to induce static quenching of the intrinsic fluorescence of HSA by a complex formed between HSA and ACS through weak multi-noncovalent forces including hydrophobic, hydrogen bond and van der Waals forces. ACS enters subdomain IIA of HSA to induce the tertiary structure changes of HSA and decreased the hydrophobicity of protein. In addition, ACS binding does not obviously alter the secondary structure of HSA. This study is hoped to provide some crucial information for further investigations of the biosafety of sweetener.

Enzymatic behavior of bovine liver catalase in aqueous medium of sugar based deep eutectic solvents

Natural deep eutectic solvents are novel green solvent media with outstanding characteristic features which are used in many enzymatic reaction studies in the last decade. There are many possible combinations of natural materials which lead to an individual deep eutectic solvent with unique features. Thereby, they are suitable solvent media to study the functionality of biological macromolecules like enzymes.In current study, the enzymatic efficiency of bovine liver catalase (BLC) was investigated in three sugar based deep eutectic solvents by using various spectroscopic methods of Uv-Visible, fluorescence and circular dichroism (CD). Combination of Glucose, Fructose and Sucrose with Choline chloride as vitamin B4, were used to make three different sugar-based deep eutectic solvents.Kinetic studies was demonstrated that formation of complex hydrogen bond network between Choline chloride and sugar components in Glucose and Sucrose based aqueous deep eutectic solvents of 50% and 60% concentrations keep the catalase activity near to 50%.Fluorescence analysis revealed that catalase fluorophore is fully quenched in 30% concentrated GCH (Glucose: Choline chloride: Water) and SCH (Sucrose: Choline chloride: Water) which is a sign of considerable tertiary structure changes. Also even the CD results which showed that α-Helical content decrease considerably in this concentration but enzyme is 80% active in both Glucose and Sucrose based deep eutectic solvents.From Hofmeister series viewpoint, synergistic relation between Choline chloride component as a well-known chaotropic molecule and sugar components as chaotropes in lower concentrations and kosmotropes in higher concentrations is influential factor on catalase enzyme activity trend in this research study.

Biophysical insight into the interaction of levocabastine with human serum albumin: spectroscopy and molecular docking approach

Interaction of levocabastine with human serum albumin (HSA) is investigated by applying fluorescence spectroscopy, circular dichroism spectroscopy and molecular docking methods. Levocabastine is an important drug in treatment of allergy and currently a target drug for drug repurposing to treat other diseases like vernal keratoconjuctivitis. Fluorescence quenching data revealed that levocabastine bind weakly to protein with binding constant in the order of 103 M−1. Förster resonance energy transfer results indicated the binding distance of 2.28 nm for levocabastine. Synchronous fluorescence result suggest slight blue shift for tryptophan upon levocabastine binding, binding of levocabastine impelled rise in α-helical structure in protein, while there are minimal changes in tertiary structure in protein. Moreover, docking results indicate levocabastine binds to pocket near to the drug site-I in HSA via hydrogen bonding and hydrophobic interactions. Understanding the interaction of levocabastine with HSA is significant for the advancement of therapeutic and diagnostic strategies for optimal treatment results. Communicated by Ramaswamy H. Sarma

Effect of thermosonication pre-treatment on mung bean (Vigna radiata) and white kidney bean (Phaseolus vulgaris) proteins: Enzymatic hydrolysis, cholesterol lowering activity and structural characterization.

Protein hydrolysates have attained great attention due to a good nutritive food ingredient and higher biological activities. In this study, thermosonication, ultrasound and heat were used as a pre-treatment to obtain (<3KDa) hydrolysate from mung bean and white kidney bean to understand the mechanism of cholesterol absorption into micelle and inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) activity. Size exclusion high performance liquid chromatography (SE-HPLC) results of mung bean showed that the concentration of peptides (0.5KDa-1KDa and 1-3KDa) in the hydrolysate were significantly (p<0.05) increased after thermosonication while, the peptides concentration (1-3KDa) in white kidney bean was significantly (p<0.05) decreased. Thermosonication of mung bean hydrolysate exhibited higher inhibition of cholesterol solubilization, hydrophobicity and antioxidant activities. In addition, there was no difference observed in HMG-CoA activity and hydrophobicity between ultrasound alone and ultrasound combined with heat i.e. thermosonication treated hydrolysate of white kidney bean. Changes in secondary and tertiary structures were also analyzed under different processing conditions with maximum change due to thermosonication. Results indicated that mung bean hydrolysate had a great potential for inhibition of cholesterol synthesis and its solubility in the micelle, antioxidant activity and also convinced for its application in food and nutraceutical industries.

Conformational perturbation, allosteric modulation of cellular signaling pathways, and disease in P23H rhodopsin

In this investigation we use THz spectroscopy and MD simulation to study the functional dynamics and conformational stability of P23H rhodopsin. The P23H mutation of rod opsin is the most common cause of human binding autosomal dominant retinitis pigmentosa (ADRP), but the precise mechanism by which this mutation leads to photoreceptor cell degeneration has not yet been elucidated. Our measurements confirm conformational instability in the global modes of the receptor and an active-state that uncouples the torsional dynamics of the retinal with protein functional modes, indicating inefficient signaling in P23H and a drastically altered mechanism of activation when contrasted with the wild-type receptor. Further, our MD simulations indicate that P23H rhodopsin is not functional as a monomer but rather, due to the instability of the mutant receptor, preferentially adopts a specific homodimerization motif. The preferred homodimer configuration induces structural changes in the receptor tertiary structure that reduces the affinity of the receptor for the retinal and significantly modifies the interactions of the Meta-II signaling state. We conjecture that the formation of the specific dimerization motif of P23H rhodopsin represents a cellular-wide signaling perturbation that is directly tied with the mechanism of P23H disease pathogenesis. Our results also support a direct role for rhodopsin P23H dimerization in photoreceptor rod death.

Comprehensive biophysical and functional study of ziv-aflibercept: characterization and forced degradation

Aflibercept (AFL) is an Fc fusion protein used in the treatment of colorectal cancers and different ophthalmological diseases. There are two medicines in which AFL is the active substance: Zaltrap and Eylea, referred as ziv-AFL and AFL respectively. No proper accelerated degradation studies were published on either AFL or ziv-AFL. These studies are essential during research, development and manufacturing stages. Here, we characterized ziv-AFL and submitted it to different stress conditions: light, 60 °C, freeze-thaw cycles, changes in pH, high hypertonic solution and strong denaturing conditions. We used an array of techniques to detect aggregation (SE-HPLC/DAD and DLS), changes in secondary structure (Far-UV circular dichroism), changes in conformation or tertiary structure (Intrinsic tryptophan fluorescence) and alterations in functionality (ELISA). Results indicate that aggregation is common degradation pathway. Two different types of aggregates were detected: dimers and high molecular weight aggregates attributed to β-amyloid-like structures. Secondary structure was maintained in most of the stress tests, while conformation was altered by almost all the tests except for the freeze-thaw cycles. Functionality, evaluated by its immunochemical reaction with VEGF, was found to be stable but with decrease when exposed to light and with likely partial inactivation of the drug when pH was altered.