Degradation Of Bovine Serum Albumin Research Articles

Near UV and Visible Light-Induced Degradation of Bovine Serum Albumin and a Monoclonal Antibody Mediated by Citrate Buffer and Fe(III): Reduction vs Oxidation Pathways.

Light exposure during manufacturing, storage, and administration can lead to the photodegradation of therapeutic proteins. This photodegradation can be promoted by pharmaceutical buffers or impurities. Our laboratory has previously demonstrated that citrate-Fe(III) complexes generate the •CO2- radical anion when photoirradiated under near UV (λ = 320-400 nm) and visible light (λ = 400-800 nm) [Subelzu, N.; Schöneich, C. Mol. Pharmaceutics 2020, 17 (11), 4163-4179; Zhang, Y. Mol. Pharmaceutics 2022, 19 (11), 4026-4042]. Here, we evaluated the impact of citrate-Fe(III) on the photostability and degradation mechanisms of disulfide-containing proteins (bovine serum albumin (BSA) and NISTmAb) under pharmaceutically relevant conditions. We monitored and localized competitive disulfide reduction and protein oxidation by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis depending on the reaction conditions. These competitive pathways were affected by multiple factors, including light dose, Fe(III) concentration, protein concentration, the presence of oxygen, and light intensity.

Multiphase reactions of proteins in the air: Oligomerization, nitration and degradation of bovine serum albumin upon ambient exposure

Proteins in atmospheric aerosol can react with atmospheric pollutants such as ozone (O3) and nitrogen dioxide (NO2) in the atmosphere via the reactions of oxidation, nitration, and cross-linking etc. Currently, the reactions have been more thoroughly studied in the laboratory but rarely investigated in the ambient environment. In this study, we used bovine serum albumin (BSA) as the model protein to conduct the exposure experiment in the ambient environment in southern China, an area with increasing oxidative capacity, to investigate the reactions of proteins in the atmosphere. We observed the occurrence of oligomerization, nitration and degradation of BSA upon exposure. The mass fraction of BSA monomer decreased by 5.86 ± 1.61% after exposure and those of dimers, trimers and higher oligomers increased by 1.04 ± 0.49%, 1.37 ± 0.74% and 3.40 ± 1.06%, respectively. Simultaneously, the nitration degrees of monomers, dimers, trimers and higher oligomers increased by 0.42 ± 0.15%, 0.53 ± 0.15%, 0.55 ± 0.28% and 2.15 ± 1.01%, respectively. The results show that oligomerization was significantly affected by O3 and temperature and nitration was jointly affected by O3, temperature and relative humidity, indicating the important role of atmospheric oxidants in the atmospheric reactions of protein. Atmospheric degradation of BSA was observed with the release of free amino acids (FAAs) such as glycine, alanine, serine and methionine. Glycine was the dominant FAA with a molar yield ranging from ∼8% to 33% for BSA. The estimated stoichiometric coefficient (α) of glycine is 10−7–10−6 for the degradation of BSA upon O3. Our observation suggests the occurrence of protein reactions in the oxidative ambient environment, leading to the production of nitrated products, oligomers and low molecular weight products such as peptides and FAAs. This study may deepen the current understanding of the atmospheric reaction mechanisms and reveal the influence of environmental factors in the atmosphere.

Chemical composition, anti-inflammatory and anticholinesterase activities of the essential oils of Elionurus platypus (Trin.) Hack. from Congo

This study reports the chemical composition, anti-inflammatory and anticholinesterase activities of the essential oils of Elionurus platypus (Trin.) Hack. The plant organs studied were the flowers (EOF), leaves (EOL), rhizomes (EORh) and roots (EOR). The essential oils were obtained by steam distillation. The chemical composition was determined using GC-MS and GC-FID. The anti-inflammatory activity was evaluated by lipoxygenase inhibition method and bovine serum albumin degradation inhibition method. The anticholinesterase activity was determined by Ellman’s method. Twenty nine compounds were identified in the essential oils of different organs, with the main one being antsorenone. The content of this compound varied from one organ to another: leaves (58.51%), rhizomes (54.98%), roots (39.90%) and flowers (32.62%). Other compounds were also identified such as myrcene (28.97%) and prezizaene (5.35%) in the EOF; camphene (4.11%) and -acorenone (4.31%) in the EOR; prezizaene (5.10%) and -acorenone (4.24%) in the EORh; epiprezizaene (4.03%) and acorenone B (2.95%) in the EOL. The anti-inflammatory activity of the essential oils of the flowers (IC50 =15.46 ± 0.54 -16.40 ± 0.47 µg/mL) and leaves (IC50 = 15.78 ± 0.57- 16.41 ± 0.46µg/mL) was high. The essential oil anticholinesterase activity of the root with an IC50 = 18.06 ± 1.85µg/mL exhibited the best inhibition. Keywords: Elionurus platypus (Trin.) Hack, essential oil, antsorenone, antiinflammatory and anticholinesterase activities.

Effect of heat treatment on the physical stability, interfacial composition and protein-lipid co-oxidation of whey protein isolate-stabilised O/W emulsions

This work aimed to investigate the effects of heat treatments at different temperatures (60, 70 and 90 °C, expressed as HT-60, HT-70 and HT-90) on interfacial composition and protein-lipid co-oxidation in whey protein isolate (WPI)-stabilised O/W emulsions during storage. Compared with control group, all heated emulsions exhibited weaker physical stability over 10 days of storage, which verified by the increased droplet size, as well as decreased adsorbed protein levels and absolute ζ-potential values. Moreover, proteins recovered from the HT-90 emulsion showed the highest fluorescence intensity and red-shift of the maximum emission wavelength, indicating partial unfolding of the protein structure. Meanwhile, severe changes in protein structure were also observed in the HT-70 and HT-90 emulsions, which clearly verified by the degradation of bovine serum albumin, α-lactalbumin and β-lactoglobulin. Furthermore, HT-70 and HT-90 emulsions showed lower levels of lipid hydroperoxides and thiobarbituric acid reactive substances. In contrast, the recovered proteins were subject to severe oxidative stress as indicated by carbonyl and N’-formyl-L-kynurenine. Hierarchical cluster and correlation analysis implied that the process of protein-lipid co-oxidation is inevitable, but it can be retarded by heat treatment. Our results clearly revealed the relevance among heat treatment, interfacial adsorption property, and the protein-lipid co-oxidation of O/W emulsions.

New insights into the cleaning mechanisms of conductive membrane by NaClO and electric field in terms of protein and polysaccharide degradation

Membrane fouling can be effectively alleviated by using conductive membrane and external electric field in membrane bioreactor. However, the transformation and removal mechanism of foulants during the electrochemical process needs to be further explored. The present work investigated the degradation mechanisms and cleaning efficiency of organic foulants by electric field and sodium hypochlorite (NaClO) on the composite conductive microfiltration membranes. The results show that the electrochemical oxidation obtained a great degradation efficiency of bovine serum albumin (BSA) at current densities of 40 and 80 A/m2, which was equivalent to that of high-dose NaClO (160 mg/L). However, the degradation efficiency of sodium alginate (SA) was much lower than that of BSA under the same cleaning strategy, due to its strong antioxidant activity. It was also found that NaClO oxidation can decompose BSA macromolecules into small molecules instead of mineralization, while electrochemical oxidation can convert dissolved BSA into foams and flocs by electrocoagulation, thus reducing the BSA concentration in the solution. During the electrochemical cleaning, indirect electrochemical oxidation through the formation of strong oxidants played an important role in the degradation of BSA and SA, and the role of OH· was more significant than that of O2·-. The combination of electric fields and NaClO showed great membrane cleaning efficiency. Moreover, the combination of electrochemical cleaning could not only prevent a large number of precursors from being converted to halogenated by-products, but also shorten the chlorination time and reduce the chlorine concentration, which can alleviate the secondary pollution of cleaning solution.

Peracetic acid integrated catalytic ceramic membrane filtration for enhanced membrane fouling control: Performance evaluation and mechanism analysis

Endowing ceramic membrane (CM) catalytic reactivity can enhance membrane fouling control in the aid of in situ oxidation process. Peracetic acid (PAA) oxidant holds great prospect to integrate with CM for membrane fouling control, owing to the prominent advantages of high oxidation efficacy and easy activation. Herein, this study, for the first time, presented a PAA/CM catalytic filtration system achieving highly-efficient protein fouling alleviation. A FeOCl functionalized CM (FeOCl-CM) was synthesized, possessing high hydrophilicity, low surface roughness, and highly-efficient activation towards PAA oxidation. Using bovine serum albumin (BSA) as the model protein foulant, the PAA/FeOCl-CM catalytic filtration notably alleviated fouling occurring in both membrane pores and surface, and halved the flux reduction degree as compared with the conventional CM filtration. The PAA/FeOCl-CM catalytic oxidation allows quick and complete disintegration of BSA particles, via the breakage of the amide I and II bands and the ring opening of the aromatic amino acids (e.g., Tryptophan, Tyrosine). In-depth investigation revealed that the in situ generated •OH and 1O2 were the key reactive species towards BSA degradation during catalytic filtration, while the organic radical oxidation and the direct electron transfer pathway from BSA to PAA via FeOCl-CM played minor roles. Overall, our findings highlight a new PAA/CM catalytic filtration strategy for achieving highly-efficient membrane fouling control and provide an understanding of the integrated PAA catalytic oxidation - membrane filtration behaviors.

RETRACTED: Stoichiometry of Anaerobic Protein Fermentation

Abstract The stoichiometric reactions that characterize protein fermentation during anaerobic wastewater treatment were investigated using bovine serum albumin (BSA) as the model protein. A methodology was developed to describe anaerobic protein degradation to volatile fatty acids (VFAs) using a single reaction step based on the dominant amino acid fermentation pathways. The importance of this work stems from the burgeoning need for resource recovery by production of VFAs from waste activated sludge at municipal wastewater treatment plants. The optimum initial fermentation conditions for BSA degradation to organic acids were first established at a pH of 8 and fermentation time of 3 days. The results from two batch experiments of BSA at concentrations of 2 g/L and 4 g/L showed that the stoichiometric coefficients for acetic, butyric, and valeric acids generally compared very well with the theoretical except for propionic acid which resulted from an alternative fermentation pathway for arginine and proline without propionic acid production. The organic acids produced at each BSA concentration varied respectively by ± 15 % and ± 12 % from the theoretical and the amino acid fermentation pathways remained the same irrespective of concentration differences. The experimental stoichiometric coefficients for iso-butyric and iso-valeric acids were within 10%–15% of the theoretical values, thus validating the use of a single stoichiometry for the overall catabolic reaction of anaerobic protein fermentation to organic acids.

Inhibitory Effect of Crude Walnut Leaf Polysaccharides on Oxidative Damage of Proteins and Proliferation of SGC-7901 Cells

In this paper, we studied the inhibitory effect of crude Walnut Leaf Polysaccharides (WLP) on free radical-induced oxidative damage of proteins and anti-proliferation activity on human Gastric Cancer cells (SGC-7901). The oxidative degradation of Bovine Serum Albumin (BSA), mouse brain proteins (MBP) and Mouse Liver Proteins (MLP) induced by hydroxyl radical and alcoxyl radical was analyzed by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). In addition, MTT method was used to analyze the effect of WLP on cell viabilities of SGC-7901 cells. The results indicated that WLP significantly inhibited the oxidative damage of BSA induced by hydroxyl radical within the concentration range of 0.5~20 mg/mL. Similar protective effects were found in hydroxyl radical-induced oxidative damage on mouse tissue proteins. WLP inhibited the oxidative damage of three protein models induced by alcoxyl radical in a dose-dependent manner. Furthermore, WLP inhibited the growth of SGC-7901 cells in time- and concentration-dependent manner. In conclusion, WLP show significant inhibitory effect on protein oxidative damage induced by hydroxyl radical and alcoxyl radical as well as anti-proliferation capacity on SGC-7901 cells.

Influence of melanoidins on acidogenic fermentation of food waste to produce volatility fatty acids

Few studies on hydrothermal treatment (HT) of food waste (FW) considered the impact of melanoidins formation due to Maillard reaction on acidogenic fermentation. Here, the effects of different melanoidins doses on volatile fatty acid (VFA) production were investigated. Results showed that the solubilization and degradation of proteins can be inhibited by the presence of melanoidins. At the high-dose melanoidins, VFA production from FW was reduced by 12%. Besides, the bovine serum albumin degradation rate declined 22% with the high-dose melanoidins effectively identified their inhibition effect. However, the unaffected carbohydrates utilization led to insignificant VFA disparity at lower doses of melanoidins, because carbohydrates contributed the major VFA yield. The consumption of substrates due to melanoidins formation mainly caused VFA reduction, which contributed to 82% of substantial VFA loss. Therefore, controlling the formation of melanoidins may help the application of HT and enhance the resource recovery from FW.

A Multiparticulate Delivery System for Potential Colonic Targeting Using Bovine Serum Albumin as a Model Protein : Theme: Formulation and Manufacturing of Solid Dosage Forms Guest Editors: Tony Zhou and Tonglei Li.

There are many important diseases whose treatment could be improved by delivering a therapeutic protein to the colon, for example, Clostridium difficile infection, ulcerative colitis and Crohn's Disease. The goal of this project was to investigate the feasibility of colonic delivery of proteins using multiparticulate beads. In this work, bovine serum albumin (BSA) was adopted as a model protein. BSA was spray layered onto beads, followed by coating of an enteric polymer EUDRAGIT® FS 30 D to develop a colonic delivery system. The secondary and tertiary structure change and aggregation of BSA during spray layering process was examined. The BSA layered beads were then challenged in an accelerated stability study using International Council for Harmonization (ICH) conditions. The in vitro release of BSA from enteric coated beads was examined using United States Pharmacopeia (USP) dissolution apparatus 1. No significant changes in the secondary and tertiary structure or aggregation profile of BSA were observed after the spray layering process. Degradation of BSA to different extents was detected after storing at 25°C and 40°C for 38days. Enteric coated BSA beads were intact in acidic media while released BSA in pH7.4 phosphate buffer. We showed the feasibility of delivering proteins to colon in vitro using multiparticulate system.

Proteolytically-induced changes of secondary structural protein conformation of bovine serum albumin monitored by Fourier transform infrared (FT-IR) and UV-circular dichroism spectroscopy

Enzymatically-induced degradation of bovine serum albumin (BSA) by serine proteases (trypsin and α-chymotrypsin) in various concentrations was monitored by means of Fourier transform infrared (FT-IR) and ultraviolet circular dichroism (UV-CD) spectroscopy. In this study, the applicability of both spectroscopies to monitor the proteolysis process in real time has been proven, by tracking the spectral changes together with secondary structure analysis of BSA as proteolysis proceeds. On the basis of the FTIR spectra and the changes in the amide I band region, we suggest the progression of proteolysis process via conversion of α-helices (1654cm−1) into unordered structures and an increase in the concentration of free carboxylates (absorption of 1593 and 1402cm−1). For the first time, the correlation between the degree of hydrolysis and the concentration of carboxylic groups measured by FTIR spectroscopy was revealed as well. The far UV-CD spectra together with their secondary structure analysis suggest that the α-helical content decreases concomitant with an increase in the unordered structure. Both spectroscopic techniques also demonstrate that there are similar but less spectral changes of BSA for the trypsin attack than for α-chymotrypsin although the substrate/enzyme ratio is taken the same.

Thermal degradation of high molar mass hyaluronan in solution and in powder; comparison with BSA

The aim of the present work was to compare the thermal degradation of bovine serum albumin and hyaluronic acid of different molar masses by determining the loss in molecular weight by means of SEC-MALLS (size exclusion chromatography – multi angle laser light scattering). For all measured samples, the results obtained by this method were compared with the results for stability determined by electrophoretic light scattering. The degradation study was performed in solution and in powder.Bovine serum albumin (also known as BSA) is a protein derived from cows, which has many biochemical applications. Hyaluronic acid (hyaluronan or HA) is an anionic nonsulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues.The powder and solutions of BSA and HA were heated at different temperatures ranging from 37 °C to 120 °C for certain periods (the highest temperature was used only for the powder). The observed degradation increased with the duration of heating and with temperature for all hyaluronic acid samples analyzed in this work, in accord with Arrhenius law.At 37 °C and 60 °C, only moderate degradation was observed for hyaluronic acid solutions. For BSA at 37 °C no degradation was observed and at 60 °C significant aggregation occurred.

Differential Proteinase Patterns among Candida albicans Strains Isolated from Root Canal and Lingual Dorsum: Possible Roles in Periapical Disease

IntroductionProteinases play pivotal roles in Candida albicans infections. Although the yeast can colonize the pulpal environment, there is no information about the enzymatic profile of this organism. This in vitro study aimed to determine the proteolysis levels and to investigate differences in the expression of aspartyl proteinase genes (Sap 1, Sap 2, and Sap 4) among various root canal strains and clinical isolates from the lingual dorsum. MethodsThe extracellular proteinase activity of 104 C. albicans samples isolated from the lingual dorsum and from necrotic root canals was measured with respect to bovine serum albumin degradation after 5 days of incubation at 37°C. We used reverse-transcription polymerase chain reaction, a highly sensitive method, to detect messenger RNA transcripts of aspartyl proteinase genes (Sap 1, Sap 2, and Sap 4). The C. albicans strain SC 5314 was used as a positive control for both experiments because it is recognized as being highly proteolytic. All tests were performed in triplicate. ResultsRegardless of the isolation site, all C. albicans strains produced an opaque precipitation halo around the colonies, indicating some proteinase activity. However, the production of proteinase on the plates was significantly greater (P < .05) by the endodontic samples. Sap 2 was the most commonly expressed gene in all samples. Among the root canal samples, the detection of Sap 1 transcripts was always associated with the expression of Sap 2 and Sap 4. Sap 4 gene expression was detected in all root canal samples. The simultaneous expression of the 3 investigated Sap genes (Sap 1, Sap 2, and Sap 4) was more common in strains isolated from the lingual dorsum (50%) than in those isolated from root canals (29.4%). ConclusionsThe increased proteolytic activity as well as the distinct pattern of Sap expression observed among the root canal samples may suggest a pathogenic role for C. albicans in endodontic infections.

Antioxidant properties of xanthones from Calophyllum brasiliense: prevention of oxidative damage induced by FeSO4

BackgroundReactive oxygen species (ROS) are important mediators in a number of degenerative diseases. Oxidative stress refers to the imbalance between the production of ROS and the ability to scavenge these species through endogenous antioxidant systems. Since antioxidants can inhibit oxidative processes, it becomes relevant to describe natural compounds with antioxidant properties which may be designed as therapies to decrease oxidative damage and stimulate endogenous cytoprotective systems. The present study tested the protective effect of two xanthones isolated from the heartwood of Calophyllum brasilienses against FeSO4-induced toxicity.MethodsThrough combinatory chemistry assays, we evaluated the superoxide (O2●—), hydroxyl radical (OH●), hydrogen peroxide (H2O2) and peroxynitrite (ONOO—) scavenging capacity of jacareubin (xanthone III) and 2-(3,3-dimethylallyl)-1,3,5,6-tetrahydroxyxanthone (xanthone V). The effect of these xanthones on murine DNA and bovine serum albumin degradation induced by an OH• generator system was also evaluated. Additionally, we investigated the effect of these xanthones on ROS production, lipid peroxidation and glutathione reductase (GR) activity in FeSO4-exposed brain, liver and lung rat homogenates.ResultsXanthone V exhibited a better scavenging capacity for O2●—, ONOO- and OH● than xanthone III, although both xanthones were unable to trap H2O2. Additionally, xanthones III and V prevented the albumin and DNA degradation induced by the OH● generator system. Lipid peroxidation and ROS production evoked by FeSO4 were decreased by both xanthones in all tissues tested. Xanthones III and V also prevented the GR activity depletion induced by pro-oxidant activity only in the brain.ConclusionsAltogether, the collected evidence suggests that xanthones can play a role as potential agents to attenuate the oxidative damage produced by different pro-oxidants.

Characterisation of protein stability in rod-insert vaginal rings

A major goal in vaccine development is elimination of the ‘cold chain’, the transport and storage system for maintenance and distribution of the vaccine product. This is particularly pertinent to liquid formulation of vaccines. We have previously described the rod-insert vaginal ring (RiR) device, comprising an elastomeric body into which are inserted lyophilised, rod-shaped, solid drug dosage forms, and having potential for sustained mucosal delivery of biomacromolecules, such as HIV envelope protein-based vaccine candidates. Given the solid, lyophilised nature of these insert dosage forms, we hypothesised that antigen stability may be significantly increased compared with more conventional solubilised vaginal gel format. In this study, we prepared and tested vaginal ring devices fitted with lyophilised rod inserts containing the model antigen bovine serum albumin (BSA). Both the RiRs and the gels that were freeze-dried to prepare the inserts were evaluated for BSA stability using PAGE, turbidimetry, microbial load, MALDI-TOF and qualitative precipitate solubility measurements. When stored at 4°C, but not when stored at 40°C/75% RH, the RiR formulation offered protection against structural and conformational changes to BSA. The insert also retained matrix integrity and release characteristics. The results demonstrate that lypophilised gels can provide relative protection against degradation at lower temperatures compared to semi-solid gels. The major mechanism of degradation at 40°C/75% RH was shown to be protein aggregation. Finally, in a preliminary study, we found that addition of trehalose to the formulation significantly reduces the rate of BSA degradation compared to the original formulation when stored at 40°C/75% RH. Establishing the mechanism of degradation, and finding that degradation is decelerated in the presence of trehalose, will help inform further development of RiRs specifically and polymer based freeze-dried systems in general.

Imaging lysosomal enzyme activity in live cells using self-quenched substrates

Endocytosis, the internalization and transport of extracellular cargo, is an essential cellular process. The ultimate step in endocytosis is the intracellular degradation of extracellular cargo for use by the cell. While live cell imaging and single particle tracking have been well-utilized to study the internalization and transport of cargo, the final degradation step has required separate biochemical assays. We describe the use of self-quenched endocytic cargo to image the intracellular transport and degradation of endocytic cargo directly in live cells. We first outline the fluorescent labeling and quantification of two common endocytic cargos: a protein, bovine serum albumin, and a lipid nanoparticle, low-density lipoprotein. In vitro measurements confirm that self-quenching is a function of the number of fluorophores bound to the protein or particle and that recovery of the fluorescent signal occurs in response to enzymatic degradation. We then use confocal fluorescence microscopy and flow cytometry to demonstrate the use of self-quenched bovine serum albumin with standard fluorescence techniques. Using live cell imaging and single particle tracking, we find that the degradation of bovine serum albumin occurs in an endo-lysosomal vesicle that is positive for LAMP1.

Sonocatalytic damage of bovine serum albumin (BSA) under ultrasonic irradiation with TiO2/tooth composite

BACKGROUND: In order to effectively degrade bovine serum albumin (BSA) under ultrasonic irradiation, biological mineral material (tooth powder) was adopted to mix with nano-sized TiO2 powder. A TiO2/tooth composite with high sonocatalytic activity and remarkable selectivity was prepared. RESULTS: TiO2/tooth composite with tooth content of 30% (w/w) heat-treated at 500 °C for 40 min was used as sonocatalyst and the catalytic degradation of BSA under ultrasonic irradiation was examined. Some influencing factors, such as ultrasonic irradiation time, TiO2/tooth catalyst amount, solution acidity and NaCl concentration, were studied by UV-vis and fluorescence spectroscopic analysis. Furthermore, the BSA attack site for the TiO2/tooth composite was identifies by synchronous fluorescence spectra. CONCLUSION: The results indicated that, under ultrasonic irradiation, the TiO2/tooth composite can promote the degradation of BSA more effectively than pure nano-sized TiO2 powder. The attack site is identified as tyrosine (Tyr) residue. These results are of great significance for the use of a sonocatalytic method to treat tumours in clinical applications. Copyright © 2011 Society of Chemical Industry

Photocatalytic inactivation of bacteriophages by TiO2-coated glass plates under low-intensity, long-wavelength UV irradiation

The International Organization for Standardization (ISO) was used to evaluate antibacterial activity by titanium dioxide (TiO(2)) photocatalysis since 2006. We evaluated photocatalytic inactivation of Qβ and T4 bacteriophages induced by low-intensity, long-wavelength ultraviolet A (UVA; 0.1 mW cm(-2) and 0.001 mW cm(-2)) irradiation on a TiO(2)-coated glass plate using the ISO methodology. The results indicated that both bacteriophages were inactivated at 0.001 mW cm(-2) UVA. The intensity of UV light, including long-wavelength light (UVA), is very low in an actual indoor environment. Thus, TiO(2) photocatalysis can be beneficial for inactivating viruses in an indoor environment. Experiments using qPCR and bovine serum albumin degradation assume that viral inactivation is caused by outer viral protein disorder and not by viral RNA reduction by reactive oxygen species produced during TiO(2) photocatalysis. Furthermore, we showed that the ISO methodology for standard testing of antibacterial activity by TiO(2) photocatalysis can be applied to assess antiviral activity.

Characterization of the protease activity of detergents laboratory practicals for studying the protease profile and activity of various commercial detergents

Detergent enzymes account for about 30% of the total worldwide production of enzymes and are one of the largest and most successful applications of modern industrial biotechnology. Proteases can improve the wash performance of household, industrial, and institutional laundry detergents used to remove protein-based stains such as blood, grass, body fluids, and food soils. This article describes two easy and cheap laboratory exercises to study the presence, profile, and basic enzymology of detergent proteases. These laboratory practicals are based on the determination of the detergent protease activity of various commercial detergents using the N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine p-nitroanilide method and the bovine serum albumin degradation capacity. Students are also required to elucidate the enzymatic subtype of detergent proteases by studying the inhibitory potential of several types of protease inhibitors revealed by the same experimental methodology. Additionally, the results of the exercises can be used to provide additional insights on elementary enzymology by studying the influence of several important parameters on protease activity such as temperature (in this article) and the influence of pH and effects of surfactants and oxidizers (proposed). Students also develop laboratory skills, problem-solving capacities, and the ability to write a laboratory report. The exercises are mainly designed for an advanced undergraduate project in the biochemistry and biotechnology sciences. Globally, these laboratory practicals show students the biotechnological applications of proteases in the detergent industry and also reinforce important enzymology concepts.

Inhibition of proteolysis in luminal extracts from the intestine of the brushtail possum.

The proteolytic activity of luminal extracts from five regions (duodenum, jejunum, ileum, caecum and colon) of the brushtail possum intestine towards bovine serum albumin (BSA) and human luteinizing hormone releasing hormone (LHRH) was investigated. There were no significant differences in degradation rates between fresh and previously frozen extracts from any region of the possum intestine. The inhibition of degradation of BSA by luminal extracts from two regions (jejunum and ileum) and of LHRH from four regions (jejunum, ileum, caecum and colon) was evaluated. Soybean trypsin-chymotrypsin inhibitor (SBTI), sodium deoxycholate, Carbopol 934P, bacitracin and bestatin significantly inhibited the degradation of both LHRH and BSA (P < 0.05). SBTI almost totally inhibited the proteolysis of BSA and the peptidolysis of LHRH in extracts from the small intestine. This finding suggests that serine proteases such as chymotrypsin are responsible for the protein and peptide degradation in luminal extracts. It is concluded that including serine protease inhibitors in a formulation may enhance oral delivery of bioactive peptides and proteins to possums.