Insoluble State Research Articles

Characterizing mineralogy and redox reactivity in potential host rocks for a UK geological disposal facility

AbstractGeological disposal facilities (GDF) are intended to isolate and contain radioactive waste within multiple protective barriers, deep underground, to ensure that no harmful quantities of radioactivity reach the surface environment. The last line of defense in a multi-barrier GDF is the geosphere, where iron is present in the host rock mineralogy as either Fe(II) or Fe(III), and in groundwater as Fe(II) under reducing conditions. The mobility of risk-driving radionuclides, including uranium and technetium, in the environment is affected significantly by their valence state. Due to its low redox potential, Fe(II) can mediate reduction of these radionuclides from their oxidized, highly mobile, soluble state to their reduced, insoluble state, preventing them from reaching the biosphere. Here a study of five types of potential host rocks, two granitoids, an andesite, a mudstone and a clay-rich carbonate, is reported. The bulk rocks and their minerals were analysed for iron content, Fe(II/III) ratio, and for the speciation and fine-grained nature of alteration product minerals that might have important controls on groundwater interaction. Total iron content varies between 0.9% in clays to 5.6% in the andesite. X-ray absorption spectroscopy reveals that Fe in the granitoids and andesite is predominantly Fe(II), and in mudstones, argillaceous limestone and terrestrial sandstone is predominantly Fe(III). The redox reactivity of the potential host rocks both in the presence and absence of Fe(II)-containing 'model' groundwater was investigated using an azo dye as a probe molecule. Reduction rates as determined by reactivity with the azo dye were correlated with the ability of the rocks to uptake Fe(II) from groundwater rather than with initial Fe(II) content. Potential GDF host rocks must be characterized in terms of mineralogy, texture, grain size and bulk geochemistry to assess how they might interact with groundwater. This study highlights the importance of redox reactivity, not just total iron and Fe(II)/(III) ratio, when considering the host rock performance as a barrier material to limit transport of radionuclides from the GDF.

Aggregation of MBP in chronic demyelination.

ObjectivesMisfolding of key disease proteins to an insoluble state is associated with most neurodegenerative conditions, such as prion, Parkinson, and Alzheimer’s diseases. In this work, and by studying animal models of multiple sclerosis, we asked whether this is also the case for myelin basic protein (MBP) in the late and neurodegenerative phases of demyelinating diseases.MethodsTo this effect, we tested whether MBP, an essential myelin component, present prion-like properties in animal models of MS, as is the case for Cuprizone-induced chronic demyelination or chronic phases of Experimental Autoimmune Encephalomyelitis (EAE).ResultsWe show here that while total levels of MBP were not reduced following extensive demyelination, part of these molecules accumulated thereafter as aggregates inside oligodendrocytes or around neuronal cells. In chronic EAE, MBP precipitated concomitantly with Tau, a marker of diverse neurodegenerative conditions, including MS. Most important, analysis of fractions from Triton X-100 floatation gradients suggest that the lipid composition of brain membranes in chronic EAE differs significantly from that of naïve mice, an effect which may relate to oxidative insults and subsequently prevent the appropriate insertion and compaction of new MBP in the myelin sheath, thereby causing its misfolding and aggregation.InterpretationPrion-like aggregation of MBP following chronic demyelination may result from an aberrant lipid composition accompanying this pathological status. Such aggregation of MBP may contribute to neuronal damage that occurs in the progressive phase of MS.

Trace elements pollution and toxicity of airborne PM10 in a coal industrial city

Trace elements in particulate matter associated with coal industries hold high risk to human health. Understanding the contents and occurrences of modes of these elements as well as their contribution to particulate toxicity is significant both environmentally and pathologically. A total of 24 PM10 samples were collected in Pingdingshan City, a coal industrial city in North China, in both winter and summer of 2008. An inductively coupled plasma mass spectrometry (ICP–MS) was used to determine the concentrations of 12 trace elements associated with coal industries (Cr, Ni, Cu, Zn, As, Mo, Cd, Sn, Sb, Tl, Pb, and Bi) in PM10 samples. The results indicated that the trace element concentrations were higher in winter than in summer; due mainly to more coal combustion during winter and to the different meteorological conditions of these two seasons. The soluble proportions of these trace elements compared with total values of intact whole samples were higher in winter than in summer, and this difference was attributed mainly to more SO2 reacting with pre–existing particles to form soluble particles in winter. Of all the analyzed elements, Ni, Tl, Sb, Mo, and Cd occurred mainly in the soluble state (>50% in the soluble fraction), Cr, Cu, Zn, and As occurred in both the soluble and insoluble state (20% to 50% in the soluble fraction), and Sn, Pb, and Bi occurred mainly in the insoluble state (<20% in the soluble fraction). A plasmid DNA assay indicated that winter samples had higher toxicity than summer samples. The correlation of PM10 toxicity (TD50 value) with the contribution of various trace elements to DNA damage (trace element concentration) was further analyzed, and the results indicated that PM10 toxicity was caused mainly by the soluble fractions of trace elements, including those of Ni, Pb, Cu, Cd, As, Zn, Cr, and Tl, which were the major toxic trace elements in Pingdingshan PM10.

Cation Effects on the Phase Transition of N-isopropylacrylamide Hydrogels.

Polymers formed from N-isopropylacrylamide (NIPAM) are highly water soluble and undergo a temperature-induced phase transition to an insoluble state. The phase behavior is determined by competing hydrophilic and hydrophobic forces. In this report, additional insight regarding the effect soluble metals have on the phase transition process is provided by showing that cation solvation aids with stabilization of hydrophobic forces. This reduces barriers to rehydration and decreases thermodynamic entropy and enthalpy, obtained with variable-temperature 1H nuclear magnetic resonance spectroscopy of NIPAM hydrogels in D2O, NaCl, MgCl2, and CaCl2. For the series of cations studied, it is observed that the order of increasing effect to facilitate the phase transition is Ca2+ < Mg2+ < Na+. NaCl and MgCl2 exhibited similar effects on the thermodynamics of the collapsing process. However, significant differences in the phase transition thermodynamics are observed between MgCl2 and CaCl2 salt solutions. The influence on Stage 1 enthalpy and entropy values for CaCl2 solutions is approximately half that of the MgCl2 solutions. This difference is likely related to their charge density of Ca2+, which is approximately half that of Mg2+.

Native and thermally modified protein-polyphenol coassemblies: lactoferrin-based nanoparticles and submicrometer particles as protective vehicles for (-)-epigallocatechin-3-gallate.

The interactions between native, thermally modified lactoferrin (LF) and (-)-epigallocatechin-3-gallate (EGCG) at pH 3.5, 5.0, and 6.5 were investigated. Turbidity, particle size, and charge of LF-EGCG complexes were mainly dominated by pH value and secondary structure of protein. At pH 3.5 and 5.0, LF-EGCG complexes were nanoparticles which had high ζ-potential, small size, and soluble state. At pH 6.5, they were submicrometer particles which exhibited low ζ-potential, large size, and insoluble state. The infrared spectra of freeze-dried LF-EGCG complexes showed that they were different from LF and EGCG alone. Far-UV CD results indicated that heat denaturation might irreversibly alter the secondary structure of LF and EGCG induced a progressive increase in the proportion of α-helix structure at the cost of β-sheet and unordered coil structure of LF at pH 3.5, 5.0, and 6.5. EGCG exhibited a strong affinity for native LF but a weak affinity for thermally modified LF at pH 5.0 and 6.5. An inverse result was observed at pH 3.5. These results could have potential for the development of food formulations based on LF as a carrier of bioactive compounds.

Can bioavailability of trace nutrients be measured in anaerobic digestion?

Trace nutrients significantly affect the microbial metabolic activity within anaerobic digestion processes but always imply the risk of overdosing of heavy metals. In this study the applicability of a sequential extraction scheme established for soil and sediment samples on biogas slurries with different compositions was tested and compared to an adapted version of this extraction method. The analytical results proved the successful applicability of the developed analytical technique for the speciation of trace nutrients in anaerobic digestion systems. The procedure fulfills the basic requirements of reproducible data, a time-saving analytical approach and economic feasibility. Recovery rates of 90–110% were obtained for the most important trace elements Fe, Co, Cu, Mo, Ni and Zn. However, it was demonstrated that the adapted method provides more reliable information about the bioavailable fractions and it is considered the more appropriate approach. Data on fractionation indicated that up to 76% of these essential trace nutrients might be present in an insoluble state. Depending on the specific trace element a significant fraction, from 30% to more than 70%, is not directly bioavailable. This important aspect should be considered to guarantee sufficient supply of the microbial consortium with trace elements and at the same time to avoid overdosage.

The fusions of elastin-like polypeptides and xylanase self-assembled into insoluble active xylanase particles

We fused the genes of elastin-like polypeptides (ELPs) and xylanase and then expressed them in Escherichia coli. Unexpectedly, the fusion proteins self-assembled into insoluble active particles as the ELPs underwent a hardly reversible phase transition. The specific activity of the particles was 92% of the native counterparts, which means it can act as a pull-down handler for converting soluble proteins into active aggregates. We evaluated the characterizations of the insoluble active xylanase particles in detail and the results were encouraging. The pH optimum (6.0) of the particles was the same as the free one, but the optimum pH range was 5–7, while the free xylanase was 6–7. The free xylanase had an optimum temperature of 50°C, whereas the insoluble active xylanase particles shifted to 70°C. The pH stability, thermostability and storage stability of the xylanase particles increased significantly when compared with the free xylanase. We also observed an increase of the Km values of the free xylanase from 0.374gL−1 to 0.980gL−1 at the insoluble state. The considerable higher activity and stability of the xylanase particles were much like immobilized xylanases and could be valuable for its industrial application.

Degradation mechanisms of the Tomato yellow leaf curl virus coat protein following inoculation of tomato plants by the whitefly Bemisia tabaci

Tomato yellow leaf curl virus (TYLCV) is a begomovirus infecting tomato cultures worldwide. TYLCV is transmitted to plants by the whitefly Bemisia tabaci. Once in the plant, the virus is subjected to attack by the host-plant defences, which may include sequestration in aggregates, proteolysis, ubiquitination, 26S proteasome degradation and autophagy. Elucidating how the virus avoids destruction will make it possible to understand infection and possibly devise countermeasures. The accumulation of viral coat protein (CP) and of viral DNA in plants is a marker of a successful virus transmission by B. tabaci. In response to infection, tomato tissues display multiple ways of degrading TYLCV proteins and DNA. In this study it is shown that CP (in soluble and insoluble states) is the target of protease digestion, 26S proteasome degradation and autophagy. The highest degradation capacity was detected among soluble proteins and proteins in large aggregates/inclusion bodies; cytoplasmic extracts displayed higher activity than nuclear fractions. The very same fractions possessed the highest capacity to degrade viral genomic DNA. Separately, 26S proteasome degradation was associated with large aggregates (more pronounced in the nuclear than in the cytoplasmic fractions), which are indicators of a successful abduction of plants by viruses. Autophagy/lysosome/vacuole degradation was a characteristic of intermediate aggregates, sequestering the CP in the cytoplasm and retarding the development of large aggregates. Chloroplast proteases were active in soluble as well as in insoluble protein extracts. To the best of the authors' knowledge, this study is the first attempt to identify elements of the virus-targeted degradation machinery, which is a part of the plant response to virus invasion.

γ-Secretase Modulators and Presenilin 1 Mutants Act Differently on Presenilin/γ-Secretase Function to Cleave Aβ42 and Aβ43

Deciphering the mechanism by which the relative Aβ42(43) to total Aβ ratio is regulated is central to understanding Alzheimer disease (AD) etiology; however, the mechanisms underlying changes in the Aβ42(43) ratio caused by familial mutations and γ-secretase modulators (GSMs) are unclear. Here, we show in vitro and in living cells that presenilin (PS)/γ-secretase cleaves Aβ42 into Aβ38, and Aβ43 into Aβ40 or Aβ38. Approximately 40% of Aβ38 is derived from Aβ43. Aβ42(43) cleavage is involved in the regulation of the Aβ42(43) ratio in living cells. GSMs increase the cleavage of PS/γ-secretase-bound Aβ42 (increase k(cat)) and slow its dissociation from the enzyme (decrease k(b)), whereas PS1 mutants and inverse GSMs show the opposite effects. Therefore, we suggest a concept to describe the Aβ42(43) production process and propose how GSMs act, and we suggest that a loss of PS/γ-secretase function to cleave Aβ42(43) may initiate AD and might represent a therapeutic target.

Selective Synthesis of Glycerol Monoester with Heteropoly Acid as a New Catalyst

In this work, we were study the selective synthesis of GME from oleic acid and glycerol using two types of solid heteropoly acid catalysts, namely silicotungstic acid bulk (STAB) and STA-silica sol gel (STA-SG). The performance and selectivity of STAB and STA-SG in the esterification reaction have been investigated and compared to the sulphuric acid (H2SO4) as conventional homogeneous catalyst. The catalysts were then characterized their physical and chemical properties using BET, XRD, TEM and XPS. XPS analyses were shown a significant formation of W-O-Si, W-O-W and Si-O-Si bonding in STA-SG compared to that in STAB. The main spectra of O1s (90.74 %, 531.5 eV) followed by other O1s peak (9.26 %, 532.8 eV) were due to the presence of W-O-W and W-O-Si bonds, respectively. The STA-SG catalyst was found to be the more environmentally benign solid acid catalyst for the esterification reaction between oleic acid and glycerol due to its lower toxicity in terms of the relatively lower pH value (pH 3.7) than the STAB (pH 2.8). In addition, the ease of separation for STA-SG catalyst was attributed to its insoluble state in the product phase. The esterification products were then analysed by FTIR and HPLC. Both the H2SO4 and the STAB gave high conversion of 100 % and 98 %, but at a lower selectivity of GME with 81.6% and 89.9%, respectively. On the contrary, the STA-SG enabled a conversion of 94 %, but with a significantly higher GME selectivity of 95 % rendering it the more efficient solid acid catalyst.

Biotechnological Mineral Composites via Vaterite Precursors

ABSTRACTVaterite is one of the thermodynamically less stable polymorphs of calcium carbonate. Under ambient conditions it transforms into calcite, the most stable form of calcium carbonate. Organisms are able to stabilize minerals such as vaterite by means of organic molecules. The exact mechanisms how biomineralization proteins interact with metastable mineral phases are, however, less well understood. Many in vitro studies were performed using calcite as a model system. A deeper understanding of the interaction of organic molecules with metastable mineral phases would make them useful as a tool to control mineralization processes in vitro. In this study, we report on the co-precipitation of a natively soluble histidine-tagged GFP (green fluorecent protein) with a metastable vaterite phase and the subsequent insolubility of the fluorescent organic matrix in a 30μl calcium carbonate precipitation assay. The intrinsic fluorescence of GFP is conserved during the interaction with the mineral phase, indicating proper folding even in the insoluble state. This experiment can be extended to obtain deeper insights into some mechanistic models of biomineralization proteins by tracking native and modified GFP proteins microscopically during various stages of mineral precipitation and dissolution.

Synthesis and Characterization of Silicotungstic Acid Nanoparticles via Sol Gel Technique as a Catalyst in Esterification Reaction

Glycerol monooleate (GMO) as an esterification product of oleic acid and glycerol is highly potential as an anti-friction substance in the engine lubricant. The purpose of this work is to study the synthesis, characterization and catalytic performance of solid heteropoly acid catalysts, namely silicotungstic acid bulk (STAB) and STA-silica sol gel (STA-SG). The activity and selectivity of STAB and STA-SG in the esterification reaction have been investigated and compared to the homogeneous catalyst i.e. sulphuric acid (H2SO4). The synthesized catalysts were characterized by BET, XRD, TEM, XPS and TPD-NH3. BET analyses shown that the STA-SG catalyst is very high in surface area compared to STAB of 460.11 m2/g and 0.98 m2/g, respectively. From the XPS analyses, there was a significant formation of W-O-Si, W-O-W and Si-O-Si bonding in STA-SG compared to that in STAB. The main species of O1s (90.74 %, 531.5 eV) followed by other O1s peak (9.26 %, 532.8 eV) were due to the presence of W-O-W and W-O-Si bonds, respectively. In addition, the ease of separation for STA-SG catalyst was attributed to its insoluble state in the product phase. The esterification products were then analysed by FTIR and HPLC. Both the H2SO4 and the STAB gave high conversion of 100% and 98%, while lower selectivity of GME with 81.6% and 89.9%, respectively. On the contrary, the STA-SG enabled a conversion of 94%, while significantly higher GME selectivity of 95% rendering it the more efficient solid acid catalyst.

Environmental insights from Langmuir adsorption site capacities

Adsorption by soil humic acids (HAs) regulates the bioavailability of nutrients and pollutants, and adsorption studies can lead to useful environmental models. Langmuir's model of reversible adsorption of a gas on a solid surface as it applies to adsorption of a dissolved substance B on insoluble adsorbent A to give insoluble state A· B is examined in terms of the measured site capacity v obsd (mmol solute/g adsorbent) at equilibrium concentration [ B], adsorbent site capacity v at infinite concentration [ B], and the maximum site capacity of the adsorbent, v max. The relationship v = S m f v max = S/〈 M w 〉, where S is the number of solute entities per adsorbent site, m f is the mass fraction of adsorption sites and 〈 M w 〉 is the adsorbent's mass-average molar mass, is discussed. This relationship is applied to analysis of (1) different adsorption sites on a solid, municipal compost-derived HA and on a proposed HA building block, (2) adsorption of herbicide acifluorfen on a solid HA and (3) adsorption of herbicides alachlor and metachlor on activated charcoals. Practical applications of the relationship are listed.

The Role of Mn(II) in Silk Fibroin Based on EPR and NMR Spectroscopy

ABSTRACT The formation of the natural silk fiber depends on the conformational transition of silk fibroin (SF) from a soluble random coil and/or helix state (Silk I) to an insoluble β-sheet state (Silk II). Previous investigations have shown that many factors including H+, K+, Na+, Ca2+, Cu2+, and Fe3+ have an effect on the transition. In the present investigation, we report that 13C nuclear magnetic resonance (NMR) spectroscopy indicates that Mn2+ ion does not promote the conformational transition of silk fibroin. Using electron paramagnetic resonance (EPR), we show that Mn(II) ions are likely to exist in the silk fibroin molecules in two states: a six-coordination Mn(II)/SF complex and a complex. The binding of Mn(II) to the amino acid residues located in the hydrophilic spacers of the silk fibroin heavy chain might favor the Silk II state, while the complex might counter this effect by stabilizing the Silk I state. pH change has no considerable influences on the conformation transition of the silk fibroin because of the complex existence.

Influence of chelating ligands on bioavailability and mobility of iron in plant growth media and their effect on radish growth

In this study, the effects of chelating ligands on iron movement in growth medium, iron bioavailability, and growth of radish sprouts ( Raphanus sativus) were investigated. Iron is an important nutrient for plant growth, yet the insoluble state of iron hydroxides in alkaline conditions decreases its bioavailability. Iron chelates increase iron uptake and have been used in agriculture to correct iron chlorosis. While previous studies have reported the effects of chelating ligands on iron solubility and bioavailability, the present study elucidates the pattern of iron movement by chelating ligands in plant growth medium. The apparent mobility of iron in growth medium was calculated using a ‘4-box’ model. Ethylenediaminedisuccinic acid (EDDS) and hydroxy-iminodisuccinic acid (HIDS) produced the highest apparent mobility of iron from the bottom layer of the medium (initially 10 −4 M Fe(III)) to the upper layer (no iron), followed by glutamatediacetic acid (GLDA), ethylenediaminetetraacetic acid (EDTA), methylglycinediacetic acid (MGDA), and iminodisuccinic acid (IDS). Iron movement in the growth medium was influenced by the chelating ligand species, pH, and ligand exposure time. The iron uptake and growth of radish sprouts were related to the iron mobility produced by the chelating ligands. These results suggest that, in alkaline media, chelating ligands dissolve the hardly soluble iron hydroxide species, thus increasing iron mobility, iron uptake, and plant growth. HIDS, which is biodegradable, was one of the most effective ligands studied; therefore, this compound would be a good alternative to other environmentally persistent chelating ligands.

Synthesis, Characterization and Catalytic Performance ofH3SiW12O40/SiO2Prepared by Sol-Gel Technique

The purpose of this work is to study the synthesis, characterization, and catalytic performance of two types of solid heteropoly acid catalysts, namely, silicotungstic acid bulk (STAB) and STA-silica sol-gel (STA-SG) compared with sulfuric acid. From the XPS analyses, there was a significant formation of W-O-Si, W-O-W, and Si-O-Si bonding in STA-SG compared to that in STAB. The main spectra of O1s (90.74%, 531.5 eV) followed by other O1s peak (9.26%, 532.8 eV) were due to the presence of W-O-W and W-O-Si bonds, respectively. The STA-SG catalyst was found to be the more environmentally benign solid acid catalyst for the esterification reaction between oleic acid and glycerol due to its lower toxicity supported by silica via sol-gel technique. In addition, the ease of separation for STA-SG catalyst was attributed to its insoluble state in the product phase. The esterification products were then analysed by FTIR and HPLC. Both the H2SO4and the STAB gave high conversion of 100% and 98% but at a lower selectivity of GME with 81.6% and 89.9%, respectively. On the contrary, the STA-SG enabled a conversion of 94% but with a significantly higher GME selectivity of 95%, rendering it the more efficient solid acid catalyst.

Phase behavior and self-assembly of PSn(P2VP-b-PAA)n multiarmed multisegmented star terpolymers with ampholytic arms

We report on the phase behavior and self-assembly phenomena of (polystyrene)n(poly(2-vinylpyridine)-b-poly(acrylic acid))n, PSn(P2VP-b-PAA)n, heteroarm star block terpolymers, in DMF/water solvent mixture. The stars were prepared using a one-pot, four-step anionic polymerization method following the “in–out” approach. These multiarmed, multisegmented star terpolymers self-assemble in selective media exhibiting pH responsiveness owing to the ampholytic nature of their diblock copolymer arms. Two soluble and two insoluble states were observed depending upon the pH of the medium. At low pH (pH 1.0), core–shell unimolecular micelles, with a hydrophobic PS core and P2VP, PAA segments in the shell, were formed. At pH 3.0, H-bonding and electrostatic interactions between the P2VP and PAA segments led to compact spheres. Finally, at pH 8.0 the stars were transformed from a bis-hydrophilic state to bis-hydrophobic state, leading to a network-like intermicellar assembly.

NMR assignments of the N-terminal domain of Nephila clavipes spidroin 1

The building blocks of spider dragline silk are two fibrous proteins secreted from the major ampullate gland named spidroins 1 and 2 (MaSp1, MaSp2). These proteins consist of a large central domain composed of approximately 100 tandem copies of a 35-40 amino acid repeat sequence. Non-repetitive N and C-terminal domains, of which the C-terminal domain has been implicated to transition from soluble and insoluble states during spinning, flank the repetitive core. The N-terminal domain until recently has been largely unknown due to difficulties in cloning and expression. Here, we report nearly complete assignment for all (1)H, (13)C, and (15)N resonances in the 14 kDa N-terminal domain of major ampullate spidroin 1 (MaSp1-N) of the golden orb-web spider Nephila clavipes.

Partially Folded Aggregation Intermediates of Human γD-, γC-, and γS-Crystallin Are Recognized and Bound by Human αB-Crystallin Chaperone

Human γ-crystallins are long-lived, unusually stable proteins of the eye lens exhibiting duplicated, double Greek key domains. The lens also contains high concentrations of the small heat shock chaperone α-crystallin, which suppresses aggregation of model substrates in vitro. Mature-onset cataract is believed to represent an aggregated state of partially unfolded and covalently damaged crystallins. Nonetheless, the lack of cell or tissue culture for anucleate lens fibers and the insoluble state of cataract proteins have made it difficult to identify the conformation of the human γ-crystallin substrate species recognized by human α-crystallin. The three major human lens monomeric γ-crystallins, γD, γC, and γS, all refold in vitro in the absence of chaperones, on dilution from denaturant into buffer. However, off-pathway aggregation of the partially folded intermediates competes with productive refolding. Incubation with human αB-crystallin chaperone during refolding suppressed the aggregation pathways of the three human γ-crystallin proteins. The chaperone did not dissociate or refold the aggregated chains under these conditions. The αB-crystallin oligomers formed long-lived stable complexes with their γD-crystallin substrates. Using α-crystallin chaperone variants lacking tryptophans, we obtained fluorescence spectra of the chaperone–substrate complex. Binding of substrate γ-crystallins with two or three of the four buried tryptophans replaced by phenylalanines showed that the bound substrate remained in a partially folded state with neither domain native-like. These in vitro results provide support for protein unfolding/protein aggregation models for cataract, with α-crystallin suppressing aggregation of damaged or unfolded proteins through early adulthood but becoming saturated with advancing age.

Proteome Changes in Bovine Longissimus Thoracis Muscle during the First 48 h Postmortem: Shifts in Energy Status and Myofibrillar Stability

Changes in the insoluble protein fraction of bovine longissimus thoracis muscle from eight Norwegian Red (NRF) dual-purpose young bulls during the first 48 h postmortem were investigated by two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF MS/MS). Significant changes were observed in a total of 35 proteins, and of those, 26 were identified and divided into three different groups: metabolic enzymes, cellular defense/stress proteins, and structural proteins, according to their predicted function. The majority of the metabolic enzymes identified are involved in the energy metabolism of the cell, while the cellular defense/stress proteins can be related to regulation and stabilization of the myofibrillar proteins. Both easily soluble proteins as well as structural proteins were identified in the insoluble protein fraction. We have studied the changes in solubility during postmortem storage by comparing the postmortem changes in protein composition between the soluble and insoluble protein fractions. We have identified two metabolic enzymes (2,3-bisphosphoglycerat mutase and NADH dehydrogenase) and one protein involved in the stress responses/apoptosis of the cell (Hsp70) that have not been identified previously in the insoluble protein fraction. The occurrence of these easily soluble proteins in the insoluble protein fraction could be due to precipitation or aggregation, thereby going from a soluble to an insoluble state.