SDS-polyacrylamide Gel Electrophoresis Analysis Research Articles

Megalin contributes to the early injury of proximal tubule cells during nonselective proteinuria

Megalin, a member of the LDL receptor family, is expressed on the apical membrane of proximal tubules and serves as an endocytic scavenger of filtered proteins and hence might contribute to the tubule injury as a consequence of glomerular disease. To study its role, we crossed megalin knockout mosaic mice (lacking megalin expression in 60% of proximal tubule cells) with NEP25 mice (a transgenic line expressing human CD25 in the podocyte). Treatment of this transgenic mouse with the immunotoxin causes nephrotic syndrome, focal segmental glomerulosclerosis and tubule-interstitial injury. Following this treatment, the double transgenic mice had massive non-selective proteinuria and mild glomerular and tubular injury. Comparison of megalin-containing to megalin-deficient proximal tubule cells within each kidney showed that albumin, immunoglobulin light chain, IgA and IgG were preferentially accumulated in proximal tubule cells expressing megalin. Tubule injury markers such as heme-oxygenase-1, monocyte chemoattractant protein-1 and cellular apoptosis were also preferentially found in these megalin-expressing cells. These results show that megalin plays a pivotal role in the reabsorption of small to large molecular size proteins and provides direct in vivo evidence that reabsorption of filtered proteins triggers events leading to tubule injury.

Quantitative assessment of the kinetics of growth factors release from platelet gel

The time course of the release of growth factors from platelet (PLT) gels has not been thoroughly studied and should be elucidated for a better standardization of the clinical use of these products. Release of PLT-derived growth factor-AB (PDGF-AB), transforming growth factor-beta1 (TGF-beta1), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and insulin-like growth factor-1 (IGF-1) was determined 5, 60, 120, and 300 minutes after PLT gel formation. Control experiments where PLT gel was removed and, afterward, exogenous thrombin was added, were also performed. Protein profiles of the PLT concentrates and of the releasates were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Mean PDGF-AB, TGF-beta1, EGF, and VEGF concentration increased to 76, 114, 3.7, and 0.8 ng per mL, respectively, in the presence of the PLT gel, but remained at approximately 28, 30, 0.28, and 0.34 ng per mL, respectively, when the PLT gel was removed after formation. IGF-1 content remained unchanged (approx. 80 ng/mL). SDS-PAGE analysis showed that several PLT proteins disappear during PLT gel formation and that the protein patterns of the releasates were undistinguishable at the different time points. There is a gradual and fast release of PDGF-AB, TGF-beta1, EGF, and VEGF from PLT gel for at least 60 to 300 minutes after gel formation, whereas the IGF releasate concentration remains unchanged. This study may provide useful information to improve clinical applications of PLT gels and to design improved blood-derived biomaterials with controlled release of growth factors.

Functional analysis of protein Z (Arg255His) and protein Z‐dependent protease inhibitor (Lys25Arg and Ser40Gly) polymorphisms

Functional analysis of protein Z (Arg255His) and protein Z‐dependent protease inhibitor (Lys25Arg and Ser40Gly) polymorphisms

Ceramide Generated by Sphingomyelin Hydrolysis and the Salvage Pathway Is Involved in Hypoxia/Reoxygenation-induced Bax Redistribution to Mitochondria in NT-2 Cells

Ceramide functions as an important second messenger in apoptosis signaling pathways. In this report, we show that treatment of NT-2 neuronal precursor cells with hypoxia/reoxygenation (H/R) resulted in ceramide up-regulation. This elevation in ceramide was primarily due to the actions of acid sphingomyelinase and ceramide synthase LASS 5, demonstrating the action of the salvage pathway. Hypoxia/reoxygenation treatment led to Bax translocation from the cytoplasm to mitochondria and cytochrome c release from mitochondria. Down-regulation of either acid sphingomyelinase or LASS 5-attenuated ceramide accumulation and H/R-induced Bax translocation to mitochondria. Overall, we have demonstrated that ceramide up-regulation following H/R is pertinent to Bax activation to promote cell death.

Molecular cloning and characterization of nitrate reductase gene from non-heading Chinese cabbage

Nitrate reductase (NR) is a key enzyme that catalyzes the first step in plants nitrogen metabolization. A cDNA of a NR gene from non-heading Chinese cabbage ( Brassica campestris ssp. chinensis Makino ) cultivar ‘Suzhouqing’ was isolated by RT-PCR and (5′/3′)-RACE techniques. The full-length cDNA sequence of 3049 bp contained an open reading frame of 2733 bp encoding 910 amino acids, a 5′-untranslated region of 113 bp and a 3′-untranslated sequence of 203 bp with a poly (A) tail. This protein shares common structural features with NRs from other higher plants and eukaryotes. It was classified as NR by sequence alignment, motif search and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and consequently nominated Brassica campestris ssp. chinensis Makino NR ( BcNR). Southern blot analysis indicated that BcNR gene represented as 3 copies in the non-heading Chinese cabbage genome. Accumulation of BcNR mRNA was transiently induced by nitrate supply and correlated with nitrate concentration, the highest mRNA levels of BcNR was induced by 30 mM NO 3 − and reached a maximum at 4 h at this optimal concentration treatment, in contrast, NH 4 + cannot induce the accumulation of BcNR mRNA. The BcNR was heterologously expressed in Escherichia coli BL21 (DE3) as a fusion protein. There was a specific band at about 110 kDa in size by SDS-PAGE analysis which was according with the expected molecular weight of the recombinant protein. To further explore its biological function, BcNR under regulation of the cauliflower mosaic virus (CaMV) 35S promoter was transferred into Arabidopsis thaliana. The transgenic plants exhibited an enhanced level of NR and nitrate reductase activity (NRA) in leaves under NO 3 − inducement.

Effects of surface microstructure of hydroxyapatite on protein adsorption and biological performance of osteoblasts

The aim of this study was to investigate the effect of surface microstructure on the serum protein adsorption and the biological performance of osteoblasts cultured in vitro, when seeded onto the surface of ceramics with different grain size: conventional HA, micron-sized HA and nano-sized HA. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to comparatively analyze the protein adsorption solution. The content of alkaline phosphatase (ALP) was determined, and then by using wash way method, the adhesion ability was tested. XPS tests indicated that the content of N on the surface was significant different between the three groups ( P < 0.05). SDS-PAGE analysis indicated that all the materials in these three groups could adsorb a large amount albumin, while the material in the nHA group adsorbed more albumin than the other groups. There were significant differences among them on the levels of osteoblast proliferation and adhesion in vitro. The biocompatibility of nHA is the best and of conventional HA is the worst.

Changes in Amounts of Total Salivary Gland Proteins of &lt;I&gt;Lutzomyia longipalpis&lt;/I&gt; (Diptera: Psychodidae) According to Age and Diet

Saliva plays important roles in facilitation of a bloodmeal, lubrication of mouthparts, and parasite transmission for some vector insects. Salivary composition changes during the lifetime of an insect, and differences in the salivary profile may influence its functions. In this report, the amount and profile of salivary gland protein of the American visceral leishmaniasis vector Lutzomyia longipalpis (Lutz & Neiva, 1912) were analyzed at different times of insect development and diet. Protein content from unfed female sand flies increased significantly with age, and a significant difference was observed in sugar-fed females during the first 10 d of adult life. Salivary protein content sharply decreased 1 d after blood feeding, with gradual increase in concentration the following days. SDS-polyacrylamide gel electrophoresis analysis revealed that most polypeptides present in the saliva of sugar-fed also were present in the saliva of blood-fed females. Understanding changes in sand fly's saliva contents at distinct days after emergence and the influence of a bloodmeal in this aspect may reveal the role played by saliva during leishmaniasis transmission.

Highly-Purified Natural Rubber by Saponification of Latex: Analysis of Residual Proteins in Saponified Natural Rubber

Abstract Highly purified natural rubber (NR) was prepared by saponification of fresh latex (FL-latex) and preserved high-ammonia latex (HA-latex) in the presence of surfactant to reduce the residual proteins in resulting solid NR. Saponification of latex diluted to 30% DRC was carried out with 1–7% (w/v) sodium hydroxide at room temperature for 1–7 hr at 70 °C and coagulated with formic acid. The nitrogen content of NR obtained by coagulation of the saponified latex markedly decreased to less than 0.014% by centrifugation of the saponified latex or soaking the coagulum in aqueous sodium hydroxide solution. The nitrogenous compounds from saponified NR (SAP-NR) were extracted with sodium dodecyl sulphate (SDS) aqueous solution and subjected to SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis to check the molecular weight of extracts. The extract from SAP-NR and deproteinized NR by protease (DPNR) for comparison was subjected to the analysis of allergic protein by FIT Kit method, based on Enzyme-Linked Immunosorbent Assay (ELISA) method. No extractable protein was observed in SAP-NR, whereas DPNR contained 1.5 μg/ml proteins. The results from SDS-PAGE analysis and FIT Kit test demonstrated that NR free from allergic proteins is obtainable by saponification of FL-latex with 1.5% NaOH at 70 °C for 1 hr or at room temperature for 24 hr.

Gene Targeting of Mutant COL1A2 Alleles in Mesenchymal Stem Cells From Individuals With Osteogenesis Imperfecta

Mesenchymal stem cells (MSCs) are adult cells with the capacity to differentiate into multiple cell types, including bone, fat, cartilage, and muscle cells. In order to effectively utilize autologous MSCs in cell-based therapies, precise genetic manipulations are required to eliminate the effects of disease-causing mutations. We previously used adeno-associated virus (AAV) vectors to target and inactivate mutant COL1A1 genes in MSCs from individuals with the brittle bone disorder, osteogenesis imperfecta (OI). Here we have used AAV vectors to inactivate mutant COL1A2 genes in OI MSCs, thereby demonstrating that both type I collagen genes responsible for OI can be successfully targeted. We incorporated improved vector designs so as to minimize the consequences of random integration, facilitate the removal of potential antigens, and avoid unwanted exon skipping. MSCs targeted at mutant COL1A2 alleles produced normal type I procollagen and formed bone, thereby demonstrating their therapeutic potential.

Effects of sugar additives on protein stability of recombinant human serum albumin during lyophilization and storage

Few researches on the protein stabilization of recombinant human serum albumin (rHSA) have been done. In the present study, we assessed the impact of sugar lyoprotectants on the protein stability of lyophilized rHSA (65 KDa) in the solid state. For the assessment, rHSA was formulated with sucrose and trehalose, respectively, alone or in combination with mannitol, which were lyophilized and stored at 35 degrees C. Degradation and aggregation of the resulting lyophilized formulations was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Induction of amorphous state by the lyophilactants with rHSA was determined by differential scanning calorimetry (DSC). The protein secondary structure of the rHSA in the formulations was analyzed by Fourier transform infrared spectroscopy (FT-IR). Results from SDS-PAGE analysis displayed that mannitol formulation caused aggregation resulting in a few bands that were greater than 65 KDa, whereas sucrose and trehalose formulations revealed no such aggregation. However, the aggregation of the protein decreased when mannitol was combined with sucrose or trehalose. DSC measurement supported the electrophoresis data showing that sucrose and trehalose formed complete amorphous state, but mannitol induced a partial amorphous state. These data indicate during lyophilization the most effective protein protection against aggregation was provided by sucrose and trehalose. The protection lasted during 4 months storage at 35 degrees C. FT-IR analysis displayed that the sucrose formulation inhibited deamidation. In conclusion, our data suggest that sucrose and trehalose as additives seems to be sufficient to protect from lyophilization of rHSA protein and also maintain its stability in the solid state during storage.

Characterization, amino acid composition and in vitro digestibility of hemp ( Cannabis sativa L.) proteins

The protein constituents and thermal properties of hemp ( Cannabis sativa L.) protein isolate (HPI) as well as 11S- and 7S-rich HPIs (HPI-11S and HPI-7S) were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and different scanning calorimetry (DSC), and their amino acid composition and in vitro digestibility were also evaluated, as compared to soy protein isolate (SPI). SDS-PAGE analysis showed that the edestin (consisting of acidic and basic subunits, AS and BS) was the main protein component for HPI and HPI-11S, while HPI-7S was composed of the BS of edestin and a subunit of about 4.8 kDa. DSC analysis characterized thermal transition of the edestin component and the possible present form of different subunits. Except lysine and sulfur-containing amino acids, the essential amino acids of various HPIs met the suggested requirements of FAO/WHO for 2–5 year old infants. The proportion of essential amino acids to the total amino acids ( E/ T) for HPI (as well as HPI-11S) was significantly higher than that of SPI. In an in vitro digestion model, various protein constituents of various HPIs were much easily digested by pepsin plus trypsin, to release oligo-peptides with molecular weight less than 10.0 kDa (under reduced condition). Only after pepsin digestion, in vitro digestibility of HPIs was comparable to that of SPI, however after pepsin plus trypsin digestion, the digestibility (88–91%) was significantly higher than that (71%) of SPI ( P < 0.05). These results suggest that the protein isolates from hempseed are much more nutritional in amino acid nutrition and easily digestible than SPI, and can be utilized as a good source of protein nutrition for human consumption.

Overexpression and characterization of thermostable serine protease in Escherichia coli encoded by the ORF TTE0824 from Thermoanaerobacter tengcongensis

A novel extracellular serine protease derived from Thermoanaerobacter tengcongensis, designated tengconlysin, was successfully overexpressed in Escherichia coli as a soluble protein by recombination of an N-terminal Pel B leader sequence instead of the original presequence and C-terminal 6x histidine tags. The purified protein was activated by 0.1% sodium dodecyl sulfate (SDS) treatment but not by thermal treatment. The molecular weight of tengconlysin estimated by SDS-polyacrylamide gel electrophoresis analysis and gel filtration chromatography was 37.9 and 36.2 kDa, respectively, suggesting that the enzyme is monomeric. The N-terminal sequence of mature tengconlysin was LDTAT, suggesting that it is a preproprotein containing a 29 amino acid presequence (predicted from the SigP program) and a 117 amino acid prosequence in the N-terminus. The C-terminal putative propeptide (position 469-540 in the preproprotein) did not inhibit the protease activity. The optimum temperature for tengconlysin activity was 90 degrees C in the presence of 1 mM calcium ions and the optimum pH ranged from 6.5 to 7.0. Activity inhibition studies suggest that the protease is a serine protease. The protease was stable in 0.1% SDS and 1-4 M urea at 70 degrees C in the presence of calcium ions and was activated by the denaturing agents.

Purification and Characterization of Catechol 2,3-Dioxygenase from the Aniline Degradation Pathway ofAcinetobactersp. YAA and Its Mutant Enzyme, Which Resists Substrate Inhibition

Catechol 2,3-dioxygenase (C23O), a key enzyme in the meta-cleavage pathway of catechol metabolism, was purified from cell extract of recombinant Escherichia coli JM109 harboring the C23O gene (atdB) cloned from an aniline-degrading bacterium Acinetobacter sp. YAA. SDS-polyacrylamide gel electrophoresis and gel filtration chromatography analysis suggested that the enzyme (AtdB) has a molecular mass of 35 kDa as a monomer and forms a tetrameric structure. It showed relative meta-cleavage activities for the following catechols tested: catechol (100%), 3-methylcatechol (19%), 4-methylcatechol (57%), 4-chlorocatechol (46%), and 2,3-dihydroxybiphenyl (5%). To elevate the activity, a DNA self-shuffling experiment was carried out using the atdB gene. One mutant enzyme, named AtdBE286K, was obtained. It had one amino acid substitution, E286K, and showed 2.4-fold higher C23O activity than the wild-type enzyme at 100 microM. Kinetic analysis of these enzymes revealed that the wild-type enzyme suffered from substrate inhibition at >2 microM, while the mutant enzyme loosened substrate inhibition.

Diversity of Shewanella population in fish Sparus aurata harvested in the Aegean Sea

To study the diversity of Shewanella population in Sparus aurata fish harvested in the Aegean Sea, as well as to elucidate the influence of fish storage conditions on the selection in Shewanella strains. A total of 108 strains of Shewanella spp. were isolated from Sparus aurata during storage under various conditions. Conventional phenotypic analysis along with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell proteins and 16S rRNA sequence analysis were used for the characterization of the strains. Numerical analysis of whole cell protein profiles showed that the isolates were separated into two distinct clusters A and B with 47% similarity. Cluster B was further subdivided into two subclusters B1 and B2 with 70% similarity. One strain could not be assigned to any of these groups. The different ability of isolates to utilize deoxycholate, D-saccharate, D-glucuronate, N-acetyl-glycosamine, D-maltose, gluconate and citrate, as well as the different type of metabolism on the Hugh and Leifson medium distinguished the different Shewanella biogroups, as these were defined by the SDS-PAGE analysis. Representative strains from the three biogroups were further investigated by 16S rRNA sequence analysis and showed more than 99.4% similarity. Significant similarities between the isolates and the type strains of S. baltica, S. putrefaciens and S. oneidensis at both phenotypic and molecular level signalize that the new isolates are closely related with the above Shewanella species, but do not provide a clear evidence to which of these species they belong. The lack of information about the diversity of Shewanella population in Sparus aurata fish originated from Mediterranean Sea could be confronted using conventional phenotypic techniques, SDS-PAGE analysis of whole cell proteins and 16S rRNA sequencing.

Electron Paramagnetic Resonance Spectroscopy of Site-directed Spin Labels Reveals the Structural Heterogeneity in the N-terminal Domain of ApoA-I in Solution

Apolipoprotein A-I (apoA-I) is the major protein constituent of high density lipoprotein (HDL) and plays a central role in phospholipid and cholesterol metabolism. This 243-residue long protein is remarkably flexible and assumes numerous lipid-dependent conformations. Consequently, definitive structural determination of lipid-free apoA-I in solution has been difficult. Using electron paramagnetic spectroscopy of site-directed spin labels in the N-terminal domain of apoA-I (residues 1-98) we have mapped a mixture of secondary structural elements, the composition of which is consistent with findings from other in-solution methods. Based on side chain mobility and their accessibility to polar and non-polar spin relaxers, the precise location of secondary elements for amino acids 14-98 was determined for both lipid-free and lipid-bound apoA-I. Based on intermolecular dipolar coupling at positions 26, 44, and 64, these secondary structural elements were arranged into a tertiary fold to generate a structural model for lipid-free apoA-I in solution.

Biochemical and immunohistochemical characterisation of mucins in 8 cases of colonic disease--a pilot study.

To characterise mucins in cancer of the colon and compare these with controls using stringent biochemical measures to avoid endogenous proteolysis. Crude mucus scrapings were collected from 12 specimens obtained by colectomy. Specimens from 3 traumatic colectomies and 1 sigmoid volvulus were used as controls, and compared with 6 specimens from colons resected for adenocarcinoma and 2 irradiated colons. The median age of the 4 female patients was 76 years (range 49 - 82 years), and of the 8 male patients 46.5 years (range 16 - 74 years). The crude mucus scrapings in the 9 specimens ranged in weight from 353 mg to 7 697 mg (median 4 928 mg). The median of purified mucin in the 9 specimens was 0.72 microg/mg wet weight of scraped material. Eight samples gave non-extractable pellet material, and were treated with DTT to reduce disulphide bonds for further analysis. One of these 8 pellets was resistant to reduction and had to be digested with papain before analysis. Only 5 of these pellets had mucin. Gel filtration and SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) analysis revealed different populations of mucin based on size and extent of degradation. Western blotting and immunohistochemical analysis confirmed the presence of MUC2 in all samples, MUC5AC in 2 and MUC5B in 5 diseased specimens. Immunohistochemical analysis showed that there was no MUC1 in the normal specimens, MUC1 apoprotein (MUC1 core) in 2 cancer specimens and MUC1 in 1 cancer specimen. Histochemical analysis showed that normal tissue expressed neutral and acidic mucins and diseased specimens predominantly expressed acidic mucins. The electrophoretic behaviour of MUC2 in sigmoid volvulus was different from that in cancer of the colon.

The Inactivation of Cytochrome P450 3A5 by 17α-Ethynylestradiol Is Cytochromeb5-Dependent: Metabolic Activation of the Ethynyl Moiety Leads to the Formation of Glutathione Conjugates, a Heme Adduct, and Covalent Binding to the Apoprotein

17Alpha-ethynylestradiol (EE) inactivates cytochrome P450 3A5 (3A5) in the reconstituted system in a mechanism-based manner. The inactivation is dependent on NADPH, and it is irreversible. The inactivation of 3A5 by EE is also dependent on cytochrome b5 (b5). The values for the K(I) and k(inact) of the 7-benzyloxy-4-(trifluoromethyl)coumarin O-debenzylation activity of 3A5 are 26 microM and 0.06 min(-1), respectively. Incubation of 3A5 with EE resulted in a 62% loss of catalytic activity, 60% loss in the reduced CO difference spectrum, and 40% decrease in native heme with the formation of a heme adduct. The partition ratio was approximately 25, and the stoichiometry of binding was approximately 0.3 mol of EE metabolite bound/mol of P450 inactivated. Four major metabolites were formed during the metabolism of EE by 3A5. SDS-polyacrylamide gel electrophoresis analysis demonstrated that [3H]EE was irreversibly bound to 3A5 apoprotein. Liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) revealed that two glutathione (GSH) conjugates with m/z values of 620 were formed only in the presence of b5. These two conjugates are formed from the reaction of GSH with the ethynyl group with the oxygen being inserted into either the internal or terminal carbon. A heme adduct with the ion at m/z 927 and two dipyrrole adducts with ions at m/z 579 were detected by LC-MS/MS analysis. In conclusion, 3A5 can activate EE to a 17alpha-oxirene-related reactive species that can then partition the oxygen between the internal and terminal carbons of the ethynyl group to form heme and apoprotein adducts, resulting in the inactivation of P450 3A5.

INFLUENCE OF TRANSGLUTAMINASE-INDUCED CROSS-LINKING ON IN VITRO DIGESTIBILITY OF SOY PROTEIN ISOLATE

The influence of covalent cross-linking by microbial transglutaminase (MTGase) on the sequential in vitro pepsin and trypsin digestion process and the digestibility of soy protein isolate (SPI), was investigated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nitrogen release analyses. Various subunits of β-conglycinin and acidic subunits of glycinin were cross-linked by MTGase to form high molecular weight (MW) biopolymers, while basic subunits of glycinin were unaffected. SDS-PAGE analysis indicated that the cross-linking mainly affected in vitro pepsin digestion pattern of various subunits of β-conglycinin, while the trypsin digestion pattern of native SPI was nearly unaffected. Nitrogen release analysis showed that the in vitro pepsin or/and trypsin digestibility of native SPI (at the end of pepsin or trypsin ingestion) was significantly decreased (P ≤ 0.01) by the MTGase treatment (for more than 2 h). The cross-linking by MTGase also significantly decreased the in vitro digestibility of preheated SPI. These results suggest that the cross-linking by means of transglutaminase may negatively affect the nutritional properties of food proteins.

Rana catesbeiana virus Z (RCV-Z): a novel pathogenic ranavirus

A virus, designated Rana catesbeiana virus Z (RCV-Z), was isolated from the visceral tissue of moribund tadpoles of the North American bullfrog Rana catesbeiana. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis of viral proteins and sequence analysis of the amino terminal end of the major capsid protein showed that RCV-Z was similar to frog virus 3 (FV3) and other ranaviruses isolated from anurans and fish. However, analysis of restriction fragment profiles following digestion of viral genomic DNA with XbaI and BamHI indicated that RCV-Z was markedly different from FV3. Moreover, in contrast to FV3, RCV-Z contained a full-length copy of the viral homolog of eukaryotic initiation factor 2 alpha (eIF-2alpha). Experimental infection of bullfrog tadpoles with FV3 and RCV-Z demonstrated that RCV-Z was much more pathogenic than FV3, and that prior infection with FV3 protected them from subsequent RCV-Z induced mortality. Collectively, these results suggest that RCV-Z may represent a novel species of ranavirus capable of infecting frogs and that possession of a viral eIF-2alpha homolog (vIF-2alpha) correlates with enhanced virulence.

Effects of gamma radiation on β‐lactoglobulin: Oligomerization and aggregation

The conformational changes and aggregation process of beta-lactoglobulin (beta-LG) subjected to gamma irradiation are presented. Beta-LG in solutions of different protein concentrations (3 and 10 mg/ml) and in solid state with different water activities (a(w)) (0.22; 0.53; 0.74) was irradiated using a Cobalt-60 radiation source at dose level of 1-50 kGy. Small-angle X-ray scattering (SAXS) was used to study the conformational changes of beta-LG due to the irradiation treatment. The irradiated protein was also examined by high performance size exclusion chromatography (HPSEC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing and reducing conditions and fluorescence. SAXS analysis showed that the structural conformation of irradiated beta-LG in solid state at different a(w) and dose level was essentially the same as the nonirradiated beta-LG. The scattering data also showed that the irradiation of beta-LG in solution promoted the formation of oligomers. Interestingly, from the data analysis and model building, it could be shown that the formed oligomers are linear molecules, built by linear combinations of beta-LG dimers (tetramers, hexamers, etc). The formation of oligomers was also evidenced by SDS-PAGE analysis and HPSEC chromatograms, in which products with higher molecular mass than that of the dimeric beta-LG were detected. Formation of intermolecular cross-linking between tyrosyl radicals are proposed to be at least partially responsible for this occurrence. From the results it could be shown that the samples irradiated in solution presented some conformational changes under gamma irradiation, resulting in well ordered oligomers and aggregates formed by cross-linking of beta-LG dimers subunits, while the samples irradiated in the solid state were not modified.