Azobis 2-amidinopropane Dihydrochloride Research Articles

Influence of peroxyl radical-induced oxidation on structural characteristics, emulsifying, and foaming properties of α-lactalbumin

In this research, 2, 2′-azobis (2-amidinopropane) dihydrochloride (AAPH) was chosen to evaluate the influences of peroxyl radical-induced oxidation on structural characteristics, emulsifying, and foaming properties of α-lactalbumin. Oxidative modification of α-lactalbumin resulted in the gradual increase of protein carbonyl groups from 1.7 to 5.5 nmol/mg and dityrosine concentration from 33.5 to 225 but the gradual decrease of free sulfhydryl groups from 8.2 to 2.0 nmol/mg. Moderate oxidation led to slight decreases of surface hydrophobicity of α-lactalbumin, whereas excessive oxidation resulted in the increase of surface hydrophobicity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated the formation of α-lactalbumin aggregates partly through the disulfide bond, consistent with the increase of particle size after oxidative modifications. Moderate oxidation was beneficial to interfacial properties, whereas exorbitant oxidation (AAPH>1 mmol/L) could lead to the reduction. Peroxyl radical-induced oxidative modification exhibited great potential to ameliorate the emulsifying, and foaming properties of α-lactalbumin.

The Link Between 15-F2t-Isoprostane Activity and Acute Bovine Endothelial Inflammation Remains Elusive.

Modern dairy cattle suffer from increased incidence and severity of mastitis during major physiological transitions of the lactation cycle. Oxidative stress, a condition resulting from inadequate antioxidant defense against reactive oxygen and nitrogen species, is a major underlying component of mastitis pathophysiology. Isoprostanes (IsoP) are molecules derived from cellular lipid membranes upon non-enzymatic interaction with reactive species during inflammation, and are regarded as highly sensitive and specific biomarkers of oxidative stress. Changes in IsoP concentrations have been noted during major physiological transitions and diseases such as coliform mastitis in dairy cattle. However, the biological role of IsoP during oxidative stress in dairy cows has not been well-elucidated. Therefore, this study aimed to characterize the impacts of IsoP on oxidative stress outcomes in a bovine model of acute endothelial inflammation. Bovine aortic endothelial cells (BAEC; n = 4) were stimulated with 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) or lipopolysaccharide (LPS) with or without 15-F2t-IsoP to determine how IsoP influence oxidative stress outcomes. Our endothelial inflammation model showed relatively decreased reactive metabolites and increased barrier integrity in cells treated with both the agonist and IsoP compared to agonist treatment alone. However, IsoP do not appear to affect oxidative stress outcomes during acute inflammation. Understanding the effect of IsoP on BAEC is an early step in elucidating how IsoP impact dairy cows during times of oxidative stress in the context of acute clinical mastitis. Future studies should define the optimal dosing and treatment timing of IsoP to maximize their cytoprotective potential during acute inflammation.

Antihemolytic and antioxidant activities of aerial parts extracts of Zygophyllum album L. and Globularia alypum L. from Algeria

The main intrest of this study was to evaluate the antihemolytic and the antioxidant activities of the aqueous extracts of Globularia alypum and Zygophyllum album prepared by three different methods: decoction, infusion and maceration. Phytochemical tests, total phenolic, flavonoid and tannins contents of the different aqueous extracts were determined. DPPH radical scavenging assay and Ferric Reducing Antioxidant Power (FRAP) were employed for the evaluation of the antioxidant activity. Whereas, the antihemolytic activity was carried out by the evaluation of the protective effect of the various prepared extracts on red blood cells against the radical attack induced by 2,2′-azobis(2-amidinopropane) di-hydrochloride (AAPH). The aqueous extract of Globularia alypum prepared by decoction showed the highest level of total phenolic contents (196.52± 10.76 µg GAE/mg Extract). These findings confirmed the remarkably antioxidant properties of this plant with DPPH scavenging activity and FRAP test. A concentration of 160 µg/ml of this extract showed a protective effect against hemolysis induced by AAPH radical. Hence, Zygophyllum album extracts demonstrated lower level of total phenolic contents (30.41 ± 2.01 µg GAE/mg Extract) and lower antioxidant and antihemolytic activities. Globularia alypum extracts presented a powerful antioxidant and antihemolytic activities especially for the decoction extract whereas Zygophyllum album revealed a weaker protective effect except its maceration extract.

Peroxyl radical induced membrane instability of giant unilamellar vesicles and anti-lipooxidation protection

The present work is intended to investigate the morphological instability of lipid membrane induced by peroxyl radical (ROO•) and the underlying mechanism. To this end, the giant unilamellar vesicle (GUV) made from phosphatidylcholine was employed as a membrane model, and the azo compounds 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN) and 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) were used as the precursors of ROO•. Upon mild pyrolysis, the GUV immobilized in agarose gel was followed by conventional optical microscopy in real time, and the morphological variation was quantified by the image heterogeneity, perimeter and area all as a function of time for up to an hour. Lipid oxidation initiated from lipid phase with AMVN and from aqueous phase with AAPH led to different types of morphological changes, i.e. membrane coarsening and vesicle deformation/budding, respectively. Based on the compositional analysis of lipid oxidation products, we propose that ROO• as the primary radical initiator is responsible for the morphological changes of the GUV-AMVN while both ROO• and RO• are responsible for the morphological changes of the GUV-AAPH system. Lipophilic β-carotene and amphipathic plant phenols as antioxidants are found to be able to stabilize the membrane integrity effectively, in corroboration with the proposed mechanisms for membrane destruction.

Efficacy of Propyl Selenoethers Against Peroxyl Radical Induced Protein Damage: Effect of Functional Group Substitution

Background: Proteins are the critical bio-molecules for cellular function and are also considered to be highly susceptible to reactive oxygen species (ROS)-induced oxidative damages. Methods: The present study aimed to evaluate some simple water-soluble aliphatic organoselenium compounds bearing different functional groups but of similar carbon chain (propyl) length for a protective effect against 2,2’-azobis-(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative damage using mitochondria as an in vitro model. Results: The results indicated that APPH (20 mM) treatment of mitochondrial fraction induced protein carbonylation, leading to inactivation of redox enzymes and electron transport chain (ETC) and, ultimately the mitochondrial dysfunction. On the other hand, treatment with 0.5 mM of monoselenobutyric acid (SeBA) and monoselenopropyl alcohol (SePOH) significantly prevented APPH-induced protein carbonylation in mitochondrial fraction and restored activities of mitochondrial proteins. On the contrary, monoselenopropyl amine (SePAm) treatment at identical concentrations did not show significant protection to mitochondrial activity from AAPH-induced oxidative damages. The above results concur with the reported peroxyl radical scavenging activities of the above molecules. Conclusion: In conclusion, SeBA and SePOH are potential candidate molecules to protect proteins from oxidative damage and, therefore, can be useful for managing oxidative stress in cellular models.

Oxidation of specific tryptophan residues inhibits high-affinity binding of cocaine and its metabolites to a humanized anticocaine mAb

Cocaine addiction remains a serious problem lacking an effective pharmacological treatment. Thus, we have developed a high-affinity anti-cocaine monoclonal antibody (mAb), h2E2, for the treatment of cocaine use disorders. We show that selective tryptophan (Trp) oxidation by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) resulted in a loss of high-affinity binding of cocaine to this mAb. The newly developed use of excess methionine (Met) to protect mAb met residues from AAPH oxidation did not substantially attenuate the effects of oxidation on cocaine binding but greatly decreased the modification of met residues in the mAb. Similar large decreases in ligand affinity (5000–10,000-fold) upon oxidation were observed using cocaine and two cocaine metabolites, cocaethylene and benzoylecgonine, which also bind with nanomolar affinity to this h2E2 mAb. The decrease in binding affinity was accompanied by a decrease of approximately 50% in Trp fluorescence, and increases in mAb 310 to 370 nm absorbance were consistent with the presence of oxidized forms of Trp. Finally, mass spectral analysis of peptides derived from control and AAPH-oxidized mAb indicated that excess free met did effectively protect mAb met residues from oxidation, and that AAPH-oxidized mAb heavy-chain Trp33 and light-chain Trp91 residues are important for cocaine binding, consistent with a recently derived h2E2 Fab fragment crystal structure containing bound benzoylecgonine. Thus, protection of the anti-cocaine h2E2 mAb from Trp oxidation prior to its clinical administration is critical for its proposed therapeutic use in the treatment of cocaine use disorders.

A Newly Synthesized Flavone from Luteolin Escapes from COMT-Catalyzed Methylation and Inhibits Lipopolysaccharide-Induced Inflammation in RAW264.7 Macrophages via JNK, p38 and NF-κB Signaling Pathways.

Luteolin is a common dietary flavone possessing potent anti-inflammatory activities. However, when administrated in vivo, luteolin becomes methylated by catechol-O-methyltransferases (COMT) owing to the catechol ring in the chemical structure, which largely diminishes its anti-inflammatory effect. In this study, we made a modification on luteolin, named LUA, which was generated by the chemical reaction between luteolin and 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Without a catechol ring in the chemical structure, this new flavone could escape from the COMT-catalyzed methylation, thus affording the potential to exert its functions in the original form when administrated in the organism. Moreover, an LPS-stimulated RAW cell model was applied to detect the anti-inflammatory properties. LUA showed much more superior inhibitory effect on LPS-induced production of NO than diosmetin (a major methylated form of luteolin) and significantly suppressed upregulation of iNOS and COX-2 in macrophages. LUA treatment dramatically reduced LPS-stimulated reactive oxygen species (ROS) and mRNA levels of pro-inflammatory mediators such as IL-1β, IL-6, IL-8 and IFN-β. Furthermore, LUA significantly reduced the phosphorylation of JNK and p38 without affecting that of ERK. LUA also inhibited the activation of NF-κB through suppression of p65 phosphorylation and nuclear translocation.

Effect of Peroxyl Radical-Induced Oxidation on Functional and Structural Characteristics of Walnut Protein Isolates Revealed by High-Resolution Mass Spectrometry.

The present study aims to investigate the structural and functional properties of oxidated walnut protein isolates (WPI) by 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH). The oxidation degree, changes in structural characteristics, processing properties, and protein modifications of WPI were measured. The results showed that oxidation significantly induced structural changes, mainly reflected by the increasing carbonyl content, and decreasing sulfhydryl and free amino groups. Moreover, the secondary structure of WPI was altered in response to oxidation, and large aggregates formed through disulfide cross-linking and hydrophobic interactions. Almost all the property indicators were significantly decreased by oxidation except the foaming property and water/oil holding capacity. Mass spectrometry analysis showed that 16 different modifications occurred in amino acid side chains, and most of the protein groups with higher numbers of modifications were found to be associated with allergies, which was further confirmed by the reduction in antigenicity of the major allergen (Jug r 1) in WPI. Meanwhile, we used oxidation-related proteins for gene ontology (GO) enrichment analyses, and the results indicated that 115, 204 and 59 GO terms were enriched in terms of biological process, molecular function, and cellular component, respectively. In conclusion, oxidation altered the groups and conformation of WPI, which in turn caused modification in the functional properties correspondingly. These findings might provide a reference for processing and storage of walnut protein foods.

Destruction of polyunsaturated alkyl/acyl and alkenyl/acyl glycerophosphocholine of plasma lipoproteins during incubation with group V and X secretory phospholipase A2s

Plasma lipoproteins are carriers of various glycerophospholipids including diacyl, alkenyl/acyl, and alkyl/acyl glycerophosphocholines (GPCs), which become distributed among cells and tissues during metabolism. For metabolic function, these phospholipids require hydrolysis by phospholipases, but the responsible enzymes have not been identified. We had previously shown that after complete digestion of lipoprotein diacyl- and oxo-diacyl-GPCs, degradation of residual alkyl/acyl and alkenyl/acyl GPCs continues, despite the fact that ether lipids are resistant to hydrolysis by Ca2+ -activated secretory PLA2 s and require the presence of the Ca2+ -independent PLA2 . In the course of further investigation, we came across a report by Khaselev and Murphy in which the autoxidative degradation of plasmalogens in the presence of 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) proceeded beyond the formation of dihydroperoxides, hydroxides and epoxides, and led to an attack on the enyl bond of the plasmalogen, resulting in formation of 1-OH/2-20:4-GPC and 1-formyl/2-20:4-GPC. Our preliminary investigation indicated that lipoprotein 16:0p/20:4ω6-GPC yielded the same autoxidation products as those reported for synthetic 16:0p/20:4ω6-GPC in the presence of AAPH. Such autoxidative degradation of lipoprotein plasmalogens had not been previously reported with or without AAPH. Subsequent study led to the conclusion that this reaction was not limited to arachidonates, but extended to other polyunsaturated eicosanoids, docosanoids, and tetracosanoids, as well as oligounsaturated octadecanoids. These observations led to a hypothesis that the autoxidative cleavage of the lipoprotein plasmalogens proceeded under the influence of apo-protein-derived free radicals as intermediates of oxidative processes.

Changes in the content and antioxidative activity of β‐carotene and its metabolite vitamin A during gastrointestinal digestion and absorption and optimisation of HPLC‐based detection

SummaryThe β‐Carotene (BC), an important precursor of vitamin A (VA), possesses antioxidant activity but is fat‐soluble and has low bioavailability. In previous in‐vitro assays evaluating antioxidant and 2,2′‐azobis(2‐amidinopropane) dihydrochloride (AAPH) free radical scavenging, both BC and VA showed a strong ability to scavenge radicals and protected cells from oxidative stress. Here, we used artificially simulated gastrointestinal digestion and Caco‐2 cell absorption models to evaluate the bioavailability of the BC during gastrointestinal digestion and absorption using high‐performance liquid chromatography (HPLC) analysis. We observed high absorptive and transfer rates of BC and detected retinol metabolites (Vitamin A). Therefore, BC can be detected in the acidic gastrointestinal environment using HPLC. Optimised method provided better separation of BC and VA in the column, improving the accuracy of the test results.

Development of hypoallergenic ovalbumin with improving functional properties by AAPH and acrolein treatment

• OVA showed lower allergenicity in vitro and in vivo after oxidation. • Oxidation increased digestibility and functional properties of OVA. • Oxidation provide a new method to process hypoallergenic OVA. Ovalbumin (OVA) is one of the major allergenic proteins in egg that contain numerous functional properties. Here, oxidation-induced changes to the structure, functional properties, and allergenicity of OVA were evaluated after 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) and acrolein treatment. Oxidation complexes were prepared by the lipid peroxidation, which caused structural changes, and increased digestibility, foaming, and emulsifying properties. The IgE-binding capacity of OVA decreased after oxidation. KU812 cell assay showed that the histamine and IL-4 levels decreased upon oxidation with AAPH and acrolein. A mouse model study showed that oxidation caused a reduction in the allergenicity by reducing IgE and IgG1 levels and the production of Th2 cytokines after acrolein (1 mmol/L) and AAPH (5 mmol/L) treatment. Thus, OVA could cause changes to structural and functional properties, modulating Th1/Th2 immunobalance, and reducing potential allergenicity after oxidation by lipid peroxidation, which might provide an effective and potential method to process hypoallergenic OVA.

Targeting multiple mediators of sepsis using multifunctional tannic acid-Zn2+-gentamicin nanoparticles

Targeting multiple mediators of sepsis using multifunctional tannic acid-Zn2+-gentamicin nanoparticles

Determination of antioxidant capacity in some foods by oxygen radical absorbance capacity (ORAC)

Oxygen radical absorbance capacity (ORAC) assay has been applied to determine the Hydrophilic – ORAC index in food. The method measures antioxidant scavenging activity against peroxyl radical induced by 2,2′-azobis(2-amidino-propane) dihydrochloride (AAPH) at 37ºC, used fluororescein as the fluorescent probe. The antioxidant capacity is measured by assemssing the fluorescence decay curve (AUC) of the sample as compared to that of the blank in which no antioxidant is present. Results expressed in ORAC units, equivalent to micromole Trolox per 100 grams sample (Trolox equivalent). The method was shown with high selectivity, a wide linear range, from 5 to 50µM Trolox with linear coefficient R2 = 0.9987. The recovery from 93.2% to 104.4% and repeatability (RSD) was less than 6.60%. The limits of detection and quantitation were 5 and 10µM Trolox, respectively. The method has been applied to determine of H-ORAC index in 65 samples including vegetables, fruits, vegetable products and health supplements with content ranging from 720 µM TE/100g to 310878 µM TE/100g.

Lipid oxidation induced egg white protein foaming properties enhancement: The mechanism study revealed by high resolution mass spectrometry

Lipid oxidation often occurs during egg white protein (EWP) storage and processing periods. Here, 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) was performed to simulate lipid oxidation to probe the oxidation effects on foaming and structural properties of EWP. Results indicated that EWP structure became unfolding and flexible after oxidation, resulting in more hydrophobic groups and negative charge exposed and soluble aggregates formed, which revealed by the results of DLS and AFM. Additionally, high resolution mass spectrometry results evidenced that ovotransferrin and lysozyme trended to be new oxidation targets with the AAPH concentration increasing, and the oxidation sites inside lysozyme proved that EWP unfolding and exposure of internal hydrophobic amino acids, which were related to the enhancement of EWP foaming properties. Overall, our study provided a further analysis of the lipid induced oxidation of EWP, which may contribute to provide a more accurate strategy for enhancing protein foaming properties in food industry.

Anti-Diabetic and Antioxidant Activities of Red Wine Concentrate Enriched with Polyphenol Compounds under Experimental Diabetes in Rats.

We obtained red wine concentrate, which was enriched with natural polyphenolic compounds (PC concentrate). The main purpose was to study the hypoglycemic and antioxidant effects of the red wine concentrate, and its impact on key hematological parameters of rats with experimental diabetes mellitus. While administrating the red wine concentrate to rats with diabetes, partial recovering of glucose tolerance was promoted, as well as normalization of glycated hemoglobin level, an increase in the quantity of erythrocytes and hemoglobin concentration. PC concentrate had anti-radical effect, which was determined using 2,2-diphenyl-1-picrylhydrazylradical (DPPH) method and effectively inhibited oxidation of phosphatidylcholine liposomes, induced by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) as a free radical generator. It was also confirmed that PC concentrate had antioxidant properties in vivo. The contents of lipid peroxidation and protein oxidation products, the activity of catalase, and glutathione peroxidase (GPx) were increased in the plasma of rats with diabetes mellitus. At the same time, the activity of superoxide dismutase (SOD) was decreased. The concentrate of red wine had a corrective effect on investigated indicators and caused their normalization in plasma of diabetic animals.

Hair growth-promotion effects at the cellular level and antioxidant activity of the plant-based extract Phyllotex™

Hair loss and predominantly female hair loss is a common dermatologic condition with serious psychosocial consequences. Effective treatments remain scarce mainly due to the multifactorial elements involved in the onset of this chronic condition. The approved drugs available are based on molecules designed towards a single pharmacological target and do not interact with the various biochemical mechanisms involved in alopecia. Phytochemical compounds and their derivatives represent a plethora of biologically active agents, which act in synergism and simultaneously activate different biochemical pathways. Here we present an herbal formulation composed of herbs, vitamins, and minerals acting on hair regrowth and hair micro vascularization.This study aimed at evaluating the potential of Phyllotex™ to treat multifactorial androgenetic alopecia (AGA) in males and females, as well as delving into its molecular mechanisms of action. In vitro studies showed that the herbal formula stimulates cell proliferation of both dermal papilla and HaCaT cells and increases the phosphorylated form of the extracellular signal-regulated kinase 1 and 2 (ERK1/2), a well-known marker for cell proliferation. Surprisingly, expression of TGF-β1 was significantly suppressed without blocking DHT production. Additionally, the formula was able to rescue cells from the oxidative stress conditions generated by 2,2′-Azobis (2-amidinopropane) dihydrochloride (AAPH), a high oxidative agent. This data supports the potential use of this formulation as a hair growth-promoting agent for the treatment of both male and female AGA due to its multifactorial composition, which grants it the ability to cope with the different mechanisms involved in alopecia.

Protective Effect of Various Extracts of Allium hirtifolium and Satureja khuzestanica Plants on AAPH-Induced Oxidative Hemolysis

Background: Allium hirtifolium and Satureja khuzestanica are the Iranian endemic plants and proper candidates for antioxidant studies. This study investigated the antioxidant, anti-hemolytic properties, and phytochemical composition in different extracts of A. hirtifolium and S. khuzestanica. Methods: Hydroalcoholic, methanolic, and n-hexane extracts of A. hirtifolium and S. khuzestanica plants were prepared using soaking and ultrasonic methods. Different plant extracts were evaluated for the presence of secondary metabolites using standard methods based on colorimetric analysis. The antioxidant properties of the compounds were measured by the ferric reducing ability of power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods. Finally, the anti-hemolytic effects of the extracts were investigated using the 2,2’-Azobis(2-amidinopropane) dihydrochloride (AAPH) methods. Results: Phytochemical analysis indicated the presence of flavonoids, phenolic acids, glycosides, steroids, and terpenoids in S. khuzestanica, as well as amino acid compounds, flavonoids, saponins, glycosides, and phenols in A. hirtifolium. FRAP assay showed that the methanolic extract of S. khuzestanica and the n-hexane extract of A. hirtifolium have the highest total antioxidant activity. The results of the DPPH assay also demonstrated that the minimum IC50 was related to the hydroalcoholic extract of S. khuzestanica (18.58 µg/mL) and the n-hexane extract of A. hirtifolium (87.95 µg/mL). In general, the extracts of both plants could reduce the percentage of AAPH-induced hemolysis while being significant only in some concentrations (P<0.001). Conclusion: Overall, A. hirtifolium and S. khuzestanica can be used as a herbal supplement in the human diet due to their anti-hemolytic effects.

Effect of ultrasonic on the structure and quality characteristics of quinoa protein oxidation aggregates

Protein oxidation leads to covalent modification of structure and deterioration of functional properties of quinoa protein. The objective of this study was to investigate the effects of ultrasonic treatment on the functional and physicochemical properties of quinoa protein oxidation aggregates. In this concern, 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) was selected as oxidative modification of quinoa protein. The microstructure of quinoa protein displayed by scanning electron microscope (SEM) indicated that oxidation induced extensive aggregation, leading to carbonylation and degradation of sulfhydryl groups. Aggregation induced by oxidation had a negative effect on the solubility, turbidity, emulsifying stability. However, according to the analysis of physicochemical properties, ultrasonic significantly improved the water solubility of quinoa protein. The quinoa protein treated by ultrasonic for 30 min exhibited the best dispersion stability in water, which corresponded to the highest ζ-potential, smallest particle size and most uniform distribution. Based on the FT-IR, SDS-PAGE and surface hydrophobicity analysis, the increase of α-helix, β-turn and surface hydrophobicity caused by cavitation effect appeared to be the main mechanism of quinoa protein solubilization. In addition, the hydrophobic region of the protein was re-buried by excessive ultrasonic treatment, and the protein molecules were reaggregated by disulfide bonds. Microstructural observations further confirmed that ultrasonic treatment effectively inhibited protein aggregation and improved the functional properties of quinoa protein.

Synthesis of molecularly imprinted polymers using an amidine-functionalized initiator for carboxylic acid recognition

In traditional molecular imprinting reactions, the initial radicals generated by thermo- or photo-decomposition are randomly distributed in the reaction mixture. Because a noticeable portion of the initial radicals is able to cause self-polymerization of the crosslinking monomer, a significant part of the polymer product does not contain successfully imprinted molecular recognition sites. To solve this problem, we designed a molecular imprinting method using functionalized radical initiator to replace the conventional combination of initiator and functional monomer. Since the active radicals in the reaction mixture carry a template-binding moiety, the actual radical polymerization becomes more likely to take place nearby the molecular template. As a result, the efficiency of molecular imprinting can be improved. In this work, we report the use of amidine-functionalized initiator to synthesize molecularly imprinted polymers (MIPs) for selective recognition of methotrexate, a cytostatic drug used for cancer therapy. As glutamic acid represents a substructure of methotrexate, we select to use N-terminal protected glutamic acid as template to synthesize the MIPs. An initiator, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) is used to provide strong interaction with the molecular template. Two MIPs synthesized using glutamic acid derivatives as templates display specific binding to the fluorescent amino acid derivative Fmoc-Glu. When the molecular binding is tested against methotrexate, the MIP particles also exhibit specific binding for the cytostatic drug. Using cationic functional initiator to target carboxyl epitope of molecular target, this work provides an additional example of molecular imprinting based on functionalized radical initiators.

Reducing the Allergenicity of α-Lactalbumin after Lipid Peroxidation.

This study analyzed the effect of lipid peroxidation using 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) and acrolein on the in vitro and in vivo allergenicity of α-lactalbumin (α-La). The structure of oxidized α-La was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, fluorescence spectroscopy, and circular dichroism, whereas the changes in the allergenic properties were evaluated. Lipid peroxidation induced changes to the structural properties that might destroy and/or mask α-La epitopes. In comparison to native α-La, oxidation complexes caused a decrease in the immunoglobulin E (IgE) binding capacity, as observed via immunoblotting. Moreover, the capacity to release mediators and cytokines from KU812 cells was also greatly reduced. In vivo, oxidation with AAPH and acrolein caused a significant reduction in IgE, IgG, IgG1, mast cell protease 1, and plasma histamine, along with the reduction of mast surface c-Kit+ and FcεRI+ expression. Therefore, these results indicate that oxidation via AAPH and acrolein can potentially reduce the allergenicity of α-La, which can help with the better understanding of the changes in allergenicity of milk allergen by lipid peroxidation.