MW Protein Research Articles

Composition, characterisation and emulsifying properties of natural nanoparticles in chickpea aquafaba for the formation of chilli oleoresin-in-water Pickering emulsions

This study aims to assess the compositional characteristics of British Kabuli chickpea aquafaba (AF) by conducting a comprehensive analysis from raw chickpeas to centrifugation fractions of AF and its potential use in forming and stabilising capsaicin-loaded O/W Pickering systems by exploiting the presence of self-assembled nanoparticles and proteins, which act as natural-coating agent. To this end, chickpeas were soaked (16 h, 4 °C, 1:4 chickpea: water) and pressure-cooked (20 min, 113 °C, 10 psig, 1:5 chickpea: water). The dry weight-based (DWB) compositions included total carbohydrate (76.33 ± 4.20%), protein (16.29 ± 0.43%), total phenolics (7.05–8.77 mg/g) and saponins (39.95 ± 0.89 mg/g), thus confirming the leaching of these components from seeds to AF. SDS-PAGE electrophoresis analysis revealed the presence of low MW proteins (≤∼16 kDa). The monosaccharides comprised d-glucose, d-galactose, l-arabinose, d-xylose and d-fructose. AF's particle size distribution revealed the occurrence of a bimodal population of nanoparticles (Dh ≤∼1000 nm and Dh ≤∼100 nm), further characterised by SAXS and TEM imaging. O/W emulsions were prepared with three chilli oleoresin types (capsicum, chilli birds' eye, and chilli ancho) by high-pressure homogenisation. The emulsion with the highest capsaicin content (capsicum oleoresin) was the most stable while the emulsion with the lowest capsaicin content (chilli ancho oleoresin) was the least stable. The presence of incidental nanoparticles and denatured proteins in AF was reasoned to account for the formation and stabilisation of chilli oleoresin-in-water Pickering emulsions, a newly offered explanation for its interfacial properties that will be pursued further in future studies.

Integrating multi-index determination coupled with hierarchical cluster analysis to evaluate the quality consistency of PVE30, an anti-HSV "glycoprotein" macromolecule of Prunellae Spica.

Prunellae Spica (PS), derived from the dried fruit spikes of Prunella vulgaris L., is a traditional Chinese medicinal herb. Our previous studies found that PVE30, a water-extracting ethanol-precipitating "glycoprotein" macromolecule of PS, was a potential anti-herpes simplex virus (HSV) candidate. However, due to the complex structure and diverse bioactivity of the "glycoprotein", ensuring its quality consistency across different batches of PVE30 becomes particularly challenging. This poses a significant hurdle for new drug development based on PVE30. Our study aimed to integrate multi-index determination coupled with hierarchical cluster analysis (HCA) to holistically profile the quality consistency of "glycoprotein" in PVE30. High-performance gel permeation chromatography with refractive index detector (HPGPC-RID) was used to characterise the molecular weight (Mw) distribution, HPLC-PDA was used to quantitatively analyse the composed monosaccharides and amino acids, and UV-VIS was used to quantify the contents of polysaccharides and proteins. Qualitative and quantitative consistency was analysed for each single index in 16 batches of PVE30, and a 16 × 38 data matrix, coupled with HCA, was used to evaluate the holistic quality consistency of PVE30. The newly developed and validated methods were exclusive, linear, precise, accurate, and stable enough to quantify multi-indexes in PVE30. Single-index analysis revealed that 16 batches of PVE30 were qualitatively consistent in Mw distribution, polysaccharides and proteins, and the composition of composed monosaccharides and amino acids but quantitatively inconsistent in the relative contents of some "glycoprotein" macromolecules, as well as the composed monosaccharides/amino acids. HCA showed that the holistic quality of PVE30 was inconsistent, the inconsistency was uncorrelated with the regions where PS was commercially collected, and the contents of 17 amino acids and 2 monosaccharides contributed most to the holistic quality inconsistency. Multi-index determination coupled with HCA was successful in evaluating the quality consistency of PVE30, and the significant difference in quantitative indices was not caused by the origin of PS. The cultivating basis should be confirmed for PVE30-based new drug development.

Protein Fractions from Flaxseed: The Effect of Subsequent Extractions on Composition and Antioxidant Capacity.

Flaxseed proteins exhibit functionalities interesting for the food industry, including antioxidant capacity. Antioxidant activity depends on the protein composition and the presence of phenolic compounds extracted with them from the matrix. The research focused on the effect of subsequent protein extractions (water, salt and alkaline) of flaxseed meals (of three cultivars) on the protein fraction composition and its relations to antioxidant capacity. The protein and phenolic profiles and antioxidant functionalities (in antiradical ORAC and emulsion assays) were analysed. Spectroscopic characteristics of the fractions (fluorometric and FT-IR analysis) were also included. Our study has shown the effect of fractionation on the share of proteins at MW from 56-38 kDa (globulin-like) and <15 kDa (albumin-like) in the protein profiles. The highest globulin share was in the alkaline-extracted fractions (AEF) and albumin in the salt-extracted (SEF) ones. SDG (secoisolariciresinol diglucosides) and phenolic acids (p-coumaric and ferulic) were extracted with flaxseed proteins. Their contents were fraction-dependent and the highest in AEF. The concentration of phenolics in AEF corresponded with the highest antiradical capacity (ORAC) compared with the other fractions. However, the SEF showed a higher ability to inhibit oxidation in emulsions than AEF, which could be associated with the higher content of the low MW proteins.

Genetic variation and structural diversity in major seed proteins among and within Camelina species

Main conclusionGenetic variation in seed protein composition, seed protein gene expression and predictions of seed protein physiochemical properties were documented in C. sativa and other Camelina species.Seed protein diversity was examined in six Camelina species (C. hispida, C. laxa, C. microcarpa, C. neglecta, C. rumelica and C. sativa). Differences were observed in seed protein electrophoretic profiles, total seed protein content and amino acid composition between the species. Genes encoding major seed proteins (cruciferins, napins, oleosins and vicilins) were catalogued for C. sativa and RNA-Seq analysis established the expression patterns of these and other genes in developing seed from anthesis through to maturation. Examination of 187 C. sativa accessions revealed limited variation in seed protein electrophoretic profiles, though sufficient to group the majority into classes based on high MW protein profiles corresponding to the cruciferin region. C. sativa possessed four distinct types of cruciferins, named CsCRA, CsCRB, CsCRC and CsCRD, which corresponded to orthologues in Arabidopsis thaliana with members of each type encoded by homeologous genes on the three C. sativa sub-genomes. Total protein content and amino acid composition varied only slightly; however, RNA-Seq analysis revealed that CsCRA and CsCRB genes contributed > 95% of the cruciferin transcripts in most lines, whereas CsCRC genes were the most highly expressed cruciferin genes in others, including the type cultivar DH55. This was confirmed by proteomics analyses. Cruciferin is the most abundant seed protein and contributes the most to functionality. Modelling of the C. sativa cruciferins indicated that each type possesses different physiochemical attributes that were predicted to impart unique functional properties. As such, opportunities exist to create C. sativa cultivars with seed protein profiles tailored to specific technical applications.

Cross Protectivity Analysis of 49.8 kDa Pili Subunits of S. flexneri against Vibrio cholerae Infection.

Background Although the AMV and AMS vaccine candidates have similar characteristics as hemagglutinin and adhesive molecules, there are differences in molecular weight. Objective The research aims to determine the immunological cross-reaction between AMS and AMV. Method Antihemagglutination test used the anti-adhesion molecular antibody AMS. Next, we examined the immune response that has to be linked with protectivity. The model of the research uses MLIL. The sample separated the mice into four groups, and each group had five mice. The first group was the negative control group. The second group was given AMV and infected with Shigella flexneri. The third group was immunized with AMV before being exposed to Shigella flexneri. The last group was infected with Vibrio cholerae. The immune response results were evaluated by calculating the weight of MLIL and counting the colony of bacteria. We also examined other AMS immune responses, namely, β-defensin and s-IgA levels. To get the data, we measured the number of Th17 immune effector cells, T-reg, and proinflammatory cytokine IL-17A. Data analysis was performed using ANOVA, independent t-test, Kruskal–Wallis, and Mann–Whitney tests. Results An antihemagglutination cross immune response, intestinal weight, the number of bacterial colonies, and other findings were found to be significant (p < 0.05) for the levels of β-defensin, s-IgA, Th17, T-reg, and IL-17A. Conclusion The 49.8 kDa·MW protein subunit of the Shigella flexneri adhesion molecule could act as a candidate vaccine homologous for shigellosis and cholera in the future.

Crc of Pseudomonas syringae Lz4W utilizes the dominant RNA binding site for mutually exclusive interactions with Hfq:mRNA and CrcY/Z RNA

Crc is a global modulator involved in Carbon Catabolite Repression (CCR) in Pseudomonas, manipulating the uptake and assimilation of preferred carbon substrates. It is postulated that the free Crc is regulated by a complex and yet unknown process involving small non-coding regulatory RNAs, CrcY and CrcZ. While Crc's interaction with AANAANAA rich target mRNA was proposed initially, but the conclusive proof was never obtained. Recent cryoEM structural studies derived a ternary complex formed by the hexameric RNA chaperon Hfq, Crc, and an mRNA fragment to highlight the assembly of these major elements in P. aeruginosa CCR. Although Crc was shown to kinetically favor and stabilize the association between Hfq with RNA, the interaction between Crc and substrate RNA remains enigmatic. Here, we present the solution structure, dynamics, and function of Crc (∼ 31 kDa, PsCrc) from a psychrotrophic Antarctic bacterium P. syringae Lz4W exclusively using NMR spectrometer operating at 600 MHz 1H frequency. Further, by utilizing several in vitro biophysical and in vivo functional assays to study the interaction between PsCrc and a representative small RNA as well as endogenous CrcY and CrcZ RNA, we demonstrate the significance of residues D100 and K101 in the activity of PsCrc. Our studies suggest that PsCrc has divergently evolved from Apurinic/apyrimidinic (AP) endonuclease by gaining a non-canonical RNA binding region. Our study proposes a dynamic and complex association of PsCrc:CrcY/Z RNA and Hfq:Crc:mRNA to regulate inactive and active CCR. Moreover, the combination of existing solution NMR approaches used in this study paves a path for meaningfully studying high MW proteins with the help of relatively lower field-strength spectrometers.

Protein profile of scorpion venom from Hottentotta rugiscutis and its immunogenic potential in inducing long term memory response

The scorpions of the Buthidae family exhibit diverse toxins with proven pharmacological activities and yet underexplored. The Hottentotta rugiscutis is a commonly found south-Indian buthid scorpion, whose venom proteomic profile is unknown. In this study, the venom was biochemically and immunologically characterized by SDS-PAGE, MALDI-TOF MS, Western blot and ELISA. The regional and seasonal variation in the venom composition from the same species was also assessed at the molecular mass level. The venom was further studied in albino mice to understand its impact on various blood parameters. The venom has varied MW proteins from 6 to 275 kDa, four of them were found to be major immunodominant proteins. The mass spectra have revealed that some proteins are predominantly present in the venom of 3–4.5 kDa or 6.5–8.0 kDa, which could be the K+ or Na+ channel blockers respectively whose ratio varied by season. The obtained venom-mass spectra could also be used as H. rugiscutis specific finger-print in identifying the region-specific species. The venom was found to elicit a stress-induced innate immune response in mice, giving rise to a strong Th2 mediated humoral immune response. Overall, this study has provided a glimpse of the venom composition and its immunogenicity.

Downregulation of protein and mRNA levels of vimentin in periodontitis – A potential biomarker candidate for periodontal severity?

ObjectiveTo identify a differentially expressed structural protein in moderate and severe periodontitis and to quantify its corresponding mRNA levels. The secondary objective is to find correlation, if any, between Porphyromonas gingivalis load and mRNA levels of the protein. Materials and methodsTotal protein was extracted from 30 human gingival tissues (10 each from healthy, moderate and severe periodontitis) and subjected to 2D-gel electrophoresis. Protein identification was done by LC-MS/MS. The mRNA levels of protein and load of Porphyromonas gingivalis were further quantified by qRT-PCR. Results2D-gel Image Master 2D Platinum 7.0 analysis revealed a significant decrease (p ≤ 0.02) in the protein of MW 53.6 kDa in severe periodontitis compared to moderate periodontitis and healthy controls. LC-MS/MS analysis of the protein by MASCOT identified the protein as vimentin. Further, a three-fold decrease in mRNA expression of vimentin in periodontitis was obtained. A weak negative correlation was observed between Porphyromonas gingivalis load and mRNA levels of vimentin. ConclusionDifferentially expressed protein in 2D gel was identified as structural protein Vimentin and it was significantly downregulated at gene and protein level in periodontitis. This suggests the participation of vimentin in the pathogenesis of periodontitis.

A Re-Evaluation of Murine TFPI and Comparison to TFPI in Man: Murine Plasma TFPI Is Derived from TFPIγ

Tissue factor pathway inhibitor (TFPI) inhibits FXa and produces FXa-dependent feedback inhibition of FVIIa/TF activity. TFPI is expressed as isoforms α and β in man, and α, β and γ in the mouse. TFPIα consists of an acidic N-terminal peptide followed by three Kunitz-type protease inhibitory domains (K1K2K3) and a basic C-terminal peptide (CTP). TFPIβ lacks the K3 and CTP of TFPIα and instead contains a sequence that directs the attachment of a GPI membrane anchor to TFPIβ. Murine TFPIγ encodes a two-Kunitz domain TFPI with a short, unique C-terminal region.TFPI in murine plasma circulates at ~20 times the level in man and is not detectably affected by heparin treatment: ~31.5 + 3.7 nM before and 29.7 + 4.1 nM (n=10) 15 min after 2 units of heparin (IP). The major form of murine plasma TFPI migrates on SDS-PAGE as a ~41,000 MW protein but elutes from size-exclusion column chromatography with at an apparent MW of ~300,000. It binds to Cab-O-Sil and floats at 1.25 g/mL KBr consistent with an association with lipoproteins like the TFPI in human plasma. Mass spectroscopy (MS) analysis identifies this major form as a two-Kunitz domain form lacking detectible C-terminal isoform-specific sequences. In contrast, MS analysis of total TFPI isolated from murine plasma, detects both TFPIα-specific and TFPIγ-specific peptides, but no TFPIβ-specific peptides. This is the first identification of TFPI γ protein. MS analysis of human lipoprotein-associated TFPI was also found to be a two-Kunitz domain form lacking isoform-specific sequences. Analysis of total TFPI from human plasma detected TFPIα-specific, but no TFPIβ- and no TFPIγ-specific sequences.Sequences homologous with the mouse γ exon are present within the TFPI gene of many species. mRNA amplified by RT-PCR from a variety of human tissues contained the TFPIγ homologous sequence, but in each case it was part of TFPIβ 3‘-untranslated message. The 3‘ splice acceptor sequence for the TFPI γ exon in mouse, rat, Chinese hamster and squirrel is CAG, which is commonly used for splicing (71%), and rat ESTs encoding TFPIγ have been reported. The corresponding sequence in man and other primates, GAG, is rarely utilized (<1%) for splicing.TFPI α exon-deleted, β exon-deleted, β/γ exons-deleted and γ exon-disrupted mouse strains were generated using CRISPR/Cas9 technology. The KO mice, born at expected ratios, appear healthy. Using quantitative RT-PCR, validated with isoform-deleted mice, relative amounts of TFPI α, β, and γ mRNA in WT murine lung are 30%, 20%, and 50%, respectively. Plasma TFPI levels in TFPIα(-/-) and TFPIβ(-/-) mice are similar to WT mice (~100%). They are ~50% in TFPIγ(+/-) and TFPIβ/γ(+/-) mice and <2% in TFPIγ(-/-) and TFPIβ/γ(-/-) mice. Thus, the major form of murine plasma TFPI is derived form TFPI γ .Four samples of platelets isolated from 5-10 mice, revealed the blood level of TFPI in murine platelets (at 7.5 x 108/mL) was 74.6 + 16.0 pM. Present at WT levels in TFPIβ/γ KO mice, platelet TFPI is absent in TFPIα KO mice, confirming that in mice, like humans, platelet TFPI is TFPIα. Studies of mice with combined TFPIβ/γ(-/-) gene deletion (expressing α only) show a plasma level of TFPIα of 66.8 + 12.9 pM that increases 7.3 + 2.5-fold after heparin treatment. Our baseline TFPIα measurement represents a total value and the distribution of TFPIα derived “free” and lipoprotein-associated forms remains to be determined. The heparin-releasable TFPIα is likely endothelial-derived but this remains to be determined. Wild type mice presumably also release TFPIαwith heparin treatment but, in our experience, this response is masked due to the high circulating level of TFPIγ.Our results for TFPI in the m ouse differ from, and add to, previous reports in important respects: 1. The majority of TFPI circulating at high levels in mouse plasma is derived from TFPIγ, not α or β. 2. Sequences homologous to the mouse TFPIγ exon are present in many species, but the 3' acceptor splice site sequence in man and other primates is unfavorable for splicing. 3. Relative levels of TFPI α, β, and γ mRNA in mouse lung are 30%, 20%, and 50%, respectively. 4. Like in man, TFPIα circulates at a low level in mouse plasma, which increases several-fold following heparin treatment. 5. A two-Kunitz domain form of TFPI, lacking isoform-specific C-terminal sequences, is bound to plasma lipoproteins in both mouse and man. 6. Studies in TFPIγ deleted mice (expressing only α and β) should provide results most relevant to humans. DisclosuresNo relevant conflicts of interest to declare.

Reinjection flow field-flow fractionation method for nanoparticle quantitative analysis in unknown and complex samples

An analytical challenge that arises in environmental and food analysis is to quantify heterogeneous nanoparticles especially in polydisperse and complex samples. The method stated herein based on the reinjection asymmetrical flow field-flow fractionation (AF4 × AF4) coupled with inductively coupled plasma-mass spectrometer (ICP-MS) and statistical deconvolution allowed for identifying the molecular weight (Mw) and selenium abundance of the low Mw protein fractions (ca. < 132 kDa) in an unknown and complex sample (e.g., selenium-rich soybean protein isolates (Se-SPI)). A non-linear decay crossflow program was also developed to get better resolution and shorter elution time for both low and high Mw components. The concept of the reinjection method was based on the excellent ability for reducing sample complexity regarding the size fractionation, and peak reproducibility under the identical conditions of AF4 system. The standard protein mixture was used as a proof-of-principle sample. The results showed the underlying peaks predicted by the reinjection method were agreed with the separation result using the standard mixture (the relative standard deviation of peak locations < 1%), which indicated the reinjection method could provide an accurate assessment of the underlying peak number and location, and was promising to minimize the overfitting problem for statistic deconvolution. Interestingly, significant differences of Se abundance in protein fractions were observed in the low Mw range for Se-SPI, ranging from 0.28 to 1.66 cps/V with the Mw ranging from 13.75 kDa to 104.17 kDa, which indicated significant differences in the ability of binding Se for these selenium-rich proteins in Se-SPI.

Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates

Chinese hamster ovary (CHO) cells are the workhorse for obtaining recombinant proteins. Proteomic studies of these cells intend to understand cell biology and obtain more productive and robust cell lines for therapeutic protein production in the pharmaceutical industry. Because of the great importance of precipitation methods for the processing of samples in proteomics, the acetone, methanol-chloroform (M/C), and trichloroacetic acid (TCA)-acetone protocols were compared for CHO cells in terms of protein recovery, band pattern resolution, and presence on SDS-PAGE. Higher recovery and similar band profile with cellular homogenates were obtained using acetone precipitation with ultrasonic bath cycles (104.18 ± 2.67%) or NaOH addition (103.12 ± 5.74%), compared to the other two protocols tested. TCA-acetone precipitates were difficult to solubilize, which negatively influenced recovery percentage (77.91 ± 8.79%) and band presence. M/C with ultrasonic homogenization showed an intermediate recovery between the other two protocols (94.22 ± 4.86%) without affecting protein pattern on SDS-PAGE. These precipitation methods affected the recovery of low MW proteins (< 15 kDa). These results help in the processing of samples of CHO cells for their proteomic study by means of an easily accessible, fast protocol, with an almost complete recovery of cellular proteins and the capture of the original complexity of the cellular composition. Acetone protocol could be incorporated to sample-preparation workflows in a straightforward manner and can probably be applied to other mammalian cell lines as well. How to cite: Pérez-Rodriguez S, Ramírez OT, Trujillo-Roldán MA et al. Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates. Electron J Biotechnol 2020;48. https://doi.org/10.1016/j.ejbt.2020.09.006.

An evaluation of measurement techniques for algal-derived organic nitrogen

Algal-derived organic matter (AOM) from algal blooms in water supply systems contains dissolved organic nitrogen (DON) and dissolved organic carbon (DOC) among other constituents. The DON and DOC are disinfection by-product (DBP) precursor compounds, and must be well characterised to facilitate effective removal, thus minimising DBP formation during disinfection. While DOC character has been studied extensively, DON analysis suffers from inaccuracies due to sample pre-treatment and instrument sensitivities. A liquid chromatography method that combines size exclusion chromatography with highly sensitive organic carbon and nitrogen detectors (LC-OCND) has been widely adopted for DOC analysis; however, its potential for application for DON charactersation has been suggested as a viable alternative to existing DON characterisation techniquesnot been assessed despite its potential. Hence, the aim was to compare the effectiveness of conventional total dissolved N–dissolved inorganic N (TN–DIN), and LC-OCND methods for analysing DON in AOM. A suite of N-containing model compounds representative of DON and AOM extracted from Chlorella vulgaris CS-42/7 and Microcystis aeruginosa CS-555/1 were used to evaluate the techniques. The DON of both model compounds and AOM was first analysed using the conventional method and, then, via LC-OCND. It was observed that LC-OCND had a better precision for DON when TN contained more DIN. LC-OCND provided direct quantitative measurements for bulk and fractionated DON and DIN, with little interference caused by DIN. Additionally, LC-OCND provided information on MW distribution and protein content of the AOM. For example, LC-OCND results showed that M. aeruginosa AOM contained more HMW material than C. vulgaris AOM. However, as LC-OCND uses UV oxidation, it could not completely oxidise complex aromatic structures, and thus had a lower recovery for HMW model compounds and algal DON in comparison to the conventional method that used high temperature catalytic oxidation. Overall, it is advised that a combination of LC-OCND and TN analysis be used to provide a more detailed characterisation of N-containing AOM and other similar HMW aquatic NOM samples.

Interaction of Salmonella with Macrophages-Critical Roles of Salmonella SEp22, a Pathogenicity-related Protein, and Macrophage Reactive-Oxygen Intermediate Species (ROIs) on the Infection and Survival of Salmonella

Salmonella is a Gram-negative [Gram(-)] bacteria, distributed widely in such natural environments as soil, dust, or river water, causing food poisoning as well as oral infections such as Typhi or Paratyphi. Salmonella is highly tissue invasive, easily spreading throughout the whole body after initial growth in the phagocytic vesicles of macrophages as an intra-cellular parasite. Because there remain many unknown elements in the Salmonella-macrophage interaction, I started my study by focusing on the molecules and mechanisms underlying the interaction; for example, how Salmonella escapes natural biodefense systems armed by macrophages, and how macrophages surround and inactivate Salmonella. In addition, I developed insight into Salmonella survival in the face of both environmental stresses and immunological stresses, including attacks from macrophages, based on the idea that "pathogenicity" is not limited simply to an attack, but to both the attack and defense against hazards. In this study, I found a novel pathogenicity-related protein of Salmonella, SEp22, an iron-chelating protein of MW 18.7 kDa, to cope with reactive-oxygen intermediates (ROIs) generated by activated macrophages pre-treated with lipopolysaccharides (LPS), one of the major components of Salmonella outer membrane. We also showed that Salmonella attacks macrophages by a novel mechanism through the induction of apoptosis with large amounts of LPS and protein synthesis inhibition, in addition to the well-known mechanisms of type-three secretion system (TTSS)-induced cell damage, including InvA, an attacking, virulent factor of Salmonella. We showed that macrophages could escape from this type of cell death by LPS-induced macrophage activation and LPS-tolerance.

Biosynthesis of selenium nanoparticles mediated by fungus Mariannaea sp. HJ and their characterization

The biosynthesis of selenium nanoparticles (SeNPs) by Mariannaea sp. HJ was reported for the first time. Different cultural conditions, including SeO2 concentrations and pH were studied, 2 mM SeO2 and pH 10 were favorable for the removal of Se(IV), and extracellular SeNPs were observed at alkaline pH values. UV–vis spectroscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to characterize SeNPs. The results showed that removal of Se(IV) ions occurred during the late exponential phase of strain HJ. TEM and SEM images revealed that spherical nanoparticles were deposited on the cell wall and in the cytoplasmic region of strain HJ with a uniform size distribution. XRD pattern suggested the standard hexagonal phase of selenium crystals. FTIR, XPS and selective passivation experiments implicated that lipid and amide groups were involved in the formation process and Se(-I) or Se(-II) species were demonstrated to be functioned in this process. Based on the SDS-PAGE analysis, the up-regulated proteins of MW ∼91.6 and ∼37.4–57.2 kDa might promote SeNP formation, and a number of proteins adsorbed on the surface of nanoparticles, which may act as a natural stabilizing agent.

Reactions of Auranofin and Its Pseudohalide Derivatives with Serum Albumin Investigated through ESI-Q-TOF MS.

The reactions of auranofin and three pseudohalide derivatives with bovine serum albumin were explored by ESI-Q-TOF mass spectrometry; a detailed molecular description of the resulting adducts is achieved revealing even subtle differences in reactivity within this series of gold(I) complexes. Our study shows that this kind of investigative approach, formerly applied to the interactions of metal-based drugs with small model proteins of MW 10-15 kDa, e.g., cytochrome c and lysozyme, may now be extended with success to far larger proteins such as serum albumin (MW 66 kDa).

Toxicity of 32.2 kDa MW Escherichia coli Pili Adhesin Isolated from Infertile Male Semen in Reproductive System

Escherichia coli (E. coli) is the leading cause of male genital tract infection with no symptoms of infertility. Protein E. coli pili hemagglutinin isolated from infertile male sperm with 32.2 kDa MW acts as adhesion in spermatozoa. This study aimed to prove whether E. coli pili adhesin 32.2 kDa MW is toxic to male reproductive system. Samples consisted of spermatozoa of 30 guinea pigs divided into three groups: control, immunized with E. coli pili adhesin 32.2 kDa MW protein, and transurethral infected E. coli. Observations of sperm motility, vitality and morphology were performed under a microscope. MDA levels and sperm DNA damage were measured by a spectrophotometer and comet assay method and observed using a fluorescent microscope. There was no difference between control and immunization group of E. coli pili adhesin in motility (p=0.499), vitality (p=0.817) and morphology (p=0.176); between control and transuretral infection groups in motility (p=0.000), vitality (p=0.000) and morphology (p=0.000); and between control and both treatment groups in motility (p=0.001), vitality (p=0,000) and morphology (p=0.000). Histologic analysis showed E. coli pili adhesin of 32.2 kDa MW immunization group did not suffer from testicular tissue damage, while the positive group showed a deterioration of seminiferous tubular cells. MDA levels differed between immunization group E. coli pili, transurethral infection group, and control (p=0.024) and between transurethral and control (p=0.007) groups. However, between control and immunized group with E. coli pili protein showed no difference (p=0.251). DNA damage differed (p=0.000) between immunized group with E. coli pili, transurethral infection and control group; between control and transurethral infected group (p=0.000); and between transurethral infection group and E. coli pili protein immunization group (p=0.000). However, between control and E. coli pili immunization group showed no difference (p=0.600). In conclusion, E. coli pili adhesin 32.2 kDa MW protein is not toxic for sperm quality and the quality of sperm molecules.

Effect of milk proteins aggregation using Transglutaminase and Maillard reaction on Ca2+ milk gel

Effect of transglutaminase, Maillard reaction induced crosslinking and the combination of transglutaminase and Maillard reaction induced crosslinking between whey proteins and caseins in milk on calcium milk gel properties were investigated. Treatment of milk with transglutaminase, Maillard reaction, and transglutaminase + Maillard reaction cause to the appearance of new high MW protein bands. Water holding capacity, gel strength and sensory scores of gel samples increased and spontaneous whey separation decreased in calcium-induced milk gel made from transglutaminase and combination of transglutaminase and Maillard reaction treated milk compare with calcium-induced milk gel made from untreated milk alone.

Enrichment and Identification of the Most Abundant Zinc Binding Proteins in Developing Barley Grains by Zinc-IMAC Capture and Nano LC-MS/MS.

Background: Zinc accumulates in the embryo, aleurone, and subaleurone layers at different amounts in cereal grains. Our hypothesis is that zinc could be stored bound, not only to low MW metabolites/proteins, but also to high MW proteins as well. Methods: In order to identify the most abundant zinc binding proteins in different grain tissues, we microdissected barley grains into (1) seed coats; (2) aleurone/subaleurone; (3) embryo; and (4) endosperm. Initial screening for putative zinc binding proteins from the different tissue types was performed by fractionating proteins according to solubility (Osborne fractionation), and resolving those via Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) followed by polyvinylidene fluoride (PVDF) membrane blotting and dithizone staining. Selected protein fractions were subjected to Zn2+-immobilized metal ion affinity chromatography, and the captured proteins were identified using nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Results: In the endosperm, the most abundant zinc binding proteins were the storage protein B-hordeins, gamma-, and D-hordeins, while in the embryo, 7S globulins storage proteins exhibited zinc binding. In the aleurone/subaleurone, zinc affinity captured proteins were late abundant embryogenesis proteins, dehydrins, many isoforms of non-specific lipid transfer proteins, and alpha amylase trypsin inhibitor. Conclusions: We have shown evidence that abundant barley grain proteins have been captured by Zn-IMAC, and their zinc binding properties in relationship to the possibility of zinc storage is discussed.

Antioxidant profile, acetylcholinesterase inhibition, and platelet aggregation of polyphenols and proteins from germinating green gram (Vigna radiata)

ABSTRACTGerminating seeds tend to release a variety of bioactive compounds into the surrounding medium and their compositions vary depending on the type of seed and the temperature of the medium. During imbibition, green gram seeds exuded polyphenols and proteins, and their contents were higher at 45°C (warm seed) compared with that obtained from seeds soaked at room temperature. The total exudate from warm seeds also showed higher antioxidant activity compared with that of room-temperature exudate. Hence, the ethanol extract of the warm seed exudate was characterized for its health beneficial effects. Phenolic acids like gallic, ferulic, and sinapic acids, and flavonoids like vitexin and isovitexin were identified in the extract of the exudate. Among these, vitexin content was the highest compared to other bioactive compounds. DPPH radical scavenging activity and reducing power of the extract were comparable to those of butylated hydroxyanisole (BHA). Ethanol extract of warm seed exudate showed acetylcholinesterase (AChE) inhibition with an IC50 value of 9.6 µg GAE/ml and 72% inhibition of platelet aggregation at 0.11 mg GAE/ml concentration. High MW protein (>150kDa) of the exudate showed 83% inhibition of platelet aggregation at a concentration of 1 mg/ml. Low MW proteins of 29 kDa and 12 kDa showed 50% AChE inhibition at 8 μg/ml and 6.82 μg/ml concentrations, respectively.

Core-shell silica microsphere-based trypsin nanoreactor for low molecular-weight proteome analysis

Core-shell mesoporous silica (CSMS) microspheres with tunable mesopores in the shell are highly desired in various bioapplications. With novel CSMS microspheres that are synthesized using a convenient two-phase process, we report in this study the analysis of low molecular-weight (MW < 30 kDa) proteins by combining size-exclusion separation and enzyme immobilization. The obtained CSMS microspheres possess uniform diameter (1.3 μm with a shell thickness of 57 nm), large and tunable perpendicular mesopores (7.9 nm), high surface area (55.5 m2/g), large pore volume (0.12 cm3/g) and excellent water dispersibility. The CSMS microsphere-based enzyme nanoreactors have been fabricated by immobilizing trypsin on the pore channels of the CSMS microspheres using either physical absorption or covalent binding via thiol or aldehyde group with a high loading capacity of 11.8–6.1 mg/g. Due to the unique fibrous pore structure, low MW proteins can enter the channels in the shell to interact with immobilized trypsin, followed by analysis of the digestion products using MALDI-TOF MS or electrophoresis (CE) techniques. The properties and analytical performance of different trypsin-immobilized CSMS microspheres has been systematically evaluated. The results show that the peptide-sequence coverage of the smaller protein is enhanced by using trypsin-CSMS microspheres, indicating the size-dependent digestion which results from the size-exclusion interaction of the mesopores against the high-MW proteins. The present study would pave the way for further applications of mesoporous materials in proteome analysis.