Identification of species in cooked crabmeat by thin layer isoelectric focusing.

Thin layer polyacrylamide gel isoelectric focusing (TLIEF) is described for characterizing the species-specific, heat-denatured proteins of 8 species of crab: red (Geryon quinquedens), rock (Cancer irroratus), Jonah (Cancer borealis), blue (Callinectes sapidus), king (Paralithodes camtschatica), snow (Chionoectes spp.), European edible (Cancer pagurus), and dungeness (Cancer magister). Protein pattern differences are shown not only among species, but also between 2 modes of heat processing of the crabmeat. Individual variation within the species as to sex, size and maturity, length of frozen storage, and body parts chosen for sampling do not alter the species banding pattern. The reproducible species-specific fingerprint obviates the need to analyze authenticated samples simultaneously with the unknown crabmeat.     

Evaluation of Novel Precast SDS‐PAGE Gels for Separation of Sorghum Proteins

Precast polyacrylamide gels using novel buffer chemistry for enhanced resolution and shelf life stability of the gels were evaluated for separating sorghum proteins. Two gels with different acrylamide concentrations, 12 and 4–12%, were tested with two different buffer systems. Gels were evaluated for separation resolution, as well as protein solubility problems such as streaking. High‐resolution separations were obtained for all the major classes of kernel proteins using these gels. Run times were typically 45–60 min, producing relatively rapid separations. Resolution was significantly affected by the buffer system used. The use of precast gradient gels eliminates the need for casting gradient gels for routine analysis of sorghum proteins and avoids handling the toxic acrylamide monomer. This system will be useful for routine separations of sorghum proteins as well as for research programs using SDS‐PAGE to screen sorghum lines for digestibility or for other protein‐related quality factors.     

Preliminary study on puffer fish proteome-species identification of puffer fish by two-dimensional electrophoresis

The aims of this work were to determine the differential characterization of the urea soluble protein components of puffer fish species and to establish a preliminary proteomic database using an immobilized pH gradient two-dimensional electrophoresis (2DE) technique. The puffer fish muscle proteins resolved into 171-260 spots in the 2DE gels, with a pI range of 3-10 and molecular mass range of 7.4-205.0 kDa, following Comassie blue staining. Puffer fish muscle proteins fell in the region with pI values of 3.5-7.0, and molecular masses of 7.4-45.0 kDa were well-resolved and were good for species comparison. The more acidic proteins of lower molecular masses showed species specific characteristics. Therefore, the species of puffer fish can be differentiated from the comparison of the characteristic 2DE protein patterns.     

Characterization of storage proteins in wild (Glycine soja) and cultivated (Glycine max) soybean seeds using proteomic analysis

A combined proteomic approach was applied for the separation, identification, and comparison of two major storage proteins, beta-conglycinin and glycinin, in wild (Glycine soja) and cultivated (Glycine max) soybean seeds. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with three different immobilized pH gradient (IPG) strips was an effective method to separate a large number of abundant and less-abundant storage proteins. Most of the subunits of beta-conglycinin were well-separated in the pH range 3.0-10.0, while acidic and basic glycinin polypeptides were well-separated in pH ranges 4.0-7.0 and 6.0-11.0, respectively. Although the overall distribution pattern of the protein spots was similar in both genotypes using pH 3.0-10.0, variations in number and intensity of protein spots were better resolved using a combination of pH 4.0-7.0 and pH 6.0-11.0. The total number of storage protein spots detected in wild and cultivated genotypes was approximately 44 and 34, respectively. This is the first study reporting the comparison of protein profiles of wild and cultivated genotypes of soybean seeds using proteomic tools.     

Preparative fractionation of gliadins by electrophoresis at pH 3.1 (A-PAGE).

Purification of gliadin subclasses has been difficult since they share many biochemical and physicochemical properties. In this report, the optimization of a preparative electrophoretic method to fractionate gliadins is described. Separation was performed in preparative 7% polyacrylamide gels at pH 3.1. The separation performance was tested using analytical electrophoresis at pH 3.1 and capillary electrophoresis. Preparative gels of different lengths were employed. Using 5-cm preparative gels, several fractions of alpha-, beta-, and gamma-gliadins and fast-mobility and slow-mobility omega-gliadins were collected in 40 h of separation. Resolution was maintained at a protein load of up to 30 mg in each run. The highest efficiency of recovery was achieved using aluminum lactate as the collecting buffer. Fractionation with 10 cm in length gels improved resolution but increased operation times. Gels of 2 cm in length did not separate alpha/beta- and gamma-gliadins efficiently but were useful to separate the two main fractions of omega-gliadins in shorter times. In conclusion, preparative electrophoresis at low pH allowed the separation of alpha-, beta-, gamma-, and omega-gliadin fractions from crude material under nondenaturing conditions.     

Protein recovery from rainbow trout (Oncorhynchus mykiss) processing byproducts via isoelectric solubilization/precipitation and its gelation properties as affected by functional additives

Solubility of rainbow trout proteins was determined between pH 1.5 and 13.0 and various ionic strengths (IS). Minimum solubility occurred at pH 5.5; however, when IS = 0.2, the minimum solubility shifted toward more acidic pH. Isoelectric solubilization/precipitation was applied to trout processing byproducts (fish meat left over on bones, head, skin, etc.), resulting in protein recovery yields (Kjeldahl, dry basis) between 77.7% and 89.0%, depending of the pH used for solubilization and precipitation. The recovered protein contained 1.4-2.1% ash (dry basis), while the trout processing byproducts (i.e., starting material) 13.9%. Typical boneless and skinless trout fillets contain 5.5% ash, and therefore, the isoelectric solubilization/precipitation effectively removed impurities such as bones, scales, skin, etc., from the trout processing byproducts. The recovered proteins retained gel-forming ability as assessed with dynamic rheology, torsion test, and texture profile analysis (TPA). However, the recovered proteins failed to gel unless beef plasma protein (BPP) was added. Even with BPP, the recovered protein showed some proteolysis between 40 and 55 degrees C. Addition of potato starch, transglutaminase, and phosphate to the recovered proteins resulted in good texture of trout gels as confirmed by torsion test and TPA. Higher ( P < 0.05) shear stress and strain were measured for gels developed from basic pH treatments than the acidic counterparts. However, proteins recovered from acidic treatments had higher ( P < 0.05) lipid content than the basic treatments. This is probably why the gels from acidic treatments were whiter ( L* - 3 b*) ( P < 0.05) than those from the basic ones. Our study demonstrates that functional proteins can be efficiently recovered from low-value fish processing byproducts using isoelectric solubilization/precipitation and subsequently be used in value-added human foods.     

Identification of trout and salmon bloods by simple immunological technique and by electrofocusing patterns of red cell enzyme superoxide dismutase

Species identification of animal bloods is readily achieved by immunological tests. Differentiation among fish species on this basis is more difficult although considerable success has been achieved on the basis of both inter- and intra-specific differences in their serum proteins. This report describes a method for the identification of the different species of fish within the Salmonidae family and some coarse fish families on the basis of an immunological test and electrofocusing patterns of the enzyme superoxide dismutase from the red cell. The immunological technique relies on the development of a specific anti-trout (Salmonidae) serum which is used initially to differentiate the blood of a Salmonidae from other freshwater fish. Further discrimination, within the Salmonidae, is made on the basis of the different polymorphic forms of the enzyme superoxide dismutase separated in a pH 2.5 to 8 gradient. Using this technique, it is possible to differentiate among salmon, sea/brown trout, char, cheetah trout, and a number of varieties of rainbow trout.     

Identification of whale species by thin-layer isoelectric focusing of sarcoplasmic proteins.

Thin-layer isoelectric focusing was applied to the identification of whale (Cetacea) species by using water-soluble sarcoplasmic proteins of skeletal muscles. Twenty-eight samples consisting of 4 species (10 samples) of baleen whales (Mysticeti) and 8 species (18 samples) of toothed whales (Odontoceti) were analyzed. Each sample (approximately 1 g) was electrophoresed with Ampholine PAGplate, pH 3.5-9.5. The electrophoretic profiles were species-specific on the 4 toothed whale species that did not have a marked intra-species difference, and all 4 baleen whale species. However, the profiles were not specific on the 4 other dolphin species, even though they were discriminable from the other 4 toothed whale species. Numerical values of pIs and relative peak heights were obtained by densitometric analysis of the isoelectro-focused protein bands. The bands were also species-specific for the 8 toothed whale species mentioned. The values may be applicable to species identification without the need for a standard sample, which may not be readily obtainable. Experiments on test samples of minke and sel whales showed that bloodletting with ice water made the densities of isoelectro-focused bands thinner, although species identification was still possible by using the inside part of muscles. Heat treatment at below 60 degrees C for 10 min caused little denaturation; at higher temperatures the protein bands were diminished in a temperature-dependent fashion. Therefore, the present isoelectric focusing analysis should be applicable to small samples of whale meat, excluding several species of dolphins.     

Factors Affecting Herbaceous Richness and Biomass Accumulation Patterns of Reclaimed Coal Mines

Open‐cast mining reclamation strategies are focused on the identification of the environmental factors at different scales that facilitate the vegetation establishment and development. Here, we characterised the environmental factors at macro‐scale and micro‐scale that influenced the herbaceous richness and biomass accumulation patterns trough a 32‐year chronosequence. Herbaceous richness and biomass were influenced at macro‐scale by successional and soil development gradients whereas at micro‐scale by shrub cover and coarseness gradients. Indeed, certain environmental factors at macro‐scale and micro‐scale contributed simultaneously to determine these gradients. Explicitly, the successional gradient was related to carbon and nitrogen ratio, grazing intensity and Shannon diversity. Across this successional gradient, total herb biomass and Fabaceae biomass were reduced as well as main taxonomical groups richness. Soil development gradient was related to total nitrogen, pH and erosion severity. This gradient only influenced species richness and produced a richness reduction when pH and erosion severity increased. At micro‐scale, the shrub cover gradient was related to organic matter thickness, producing a Poaceae biomass and bryophytes cover increase when shrub cover and organic matter increased. The coarseness gradient was related to the cover of rocks and bare soil, producing a reduction of herb biomass and richness when rocks and bare soil increased. These results emphasise the need to incorporate in the management plans the influence of soil development, successional, shrub cover and coarseness gradients over herbaceous richness and biomass to improve mine reclamation strategies. Copyright © 2013 John Wiley & Sons, Ltd.     

Isoelectric focusing of β-glucosidase humic-bound activity in semi-arid Mediterranean soils under management practices

Changes in β-glucosidase enzyme–humic complexes and conventional parameters (pH, total organic C, total N, water-soluble C, and bulk density) were studied in an almond-cropped soil prone to erosion under a rehabilitation practice. The experimental plan included three soil slopes (0%, 2%, and 6%) and two type of fertilization (organic and mineral), with sampling of rhizosphere and inter-row soils. The enzyme humic complexes were extracted by pyrophosphate, purified by ultrafiltration of the organic extracts on molecular mass exclusion membranes (mol wt > 10⁴) and fractionated by isoelectric focusing technique (IEF). The IEF on polyacrylamide rod gels with a restricted pH gradient ranging between 6.0 and 4.0 gave five humic bands on the basis of the little differences of their electric charges (pI). Under both organic and mineral fertilization, β-glucosidase activity bound to the fractionated humic substances, especially in the pH range 4.5–4.2 of the rhizosphere soil, was higher than that of the inter-row soil. This also occurred in 6% slope where the enzyme activity was lower than in soil with lower slopes. The higher number of the β-glucosidase active humic bands in rhizosphere than inter-row soil, particularly for the 0% slope, may be due to the presence of humic molecules capable of preserving the enzyme molecules in the active form, other than to the higher microbial activity synthesizing and releasing the tested enzymes.     

Comparison of Endoproteinases of Various Grains

Two‐dimensional isoelectric focusing (IEF) × PAGE gels were used to compare the endoproteolytic (gelatinase) activities of germinated barley with those of bread and durum wheat, rye, triticale, oat, rice, buckwheat, and sorghum. Barley was used as the standard of comparison because its endoproteinase complement has been studied previously in the greatest detail. The characteristics of the grain proteases were appraised from their migration patterns and by how they were affected by pH levels. All of the germinated grains contained multiple enzyme activities and their separation patterns and pH levels were at least similar to those of barley. The proteinases of the bread and durum wheats, rye, oat, and sorghum were most similar to those of barley, whereas the other grains provided more varied patterns. The rice and buckwheat proteinases developed much more slowly than those of the other grains. The activity patterns of the triticale resembled those of the parents, wheat and rye, but the triticale contained many more activities and higher overall proteolytic activities than any of the other species. These results should be applied to scientific or commercial procedures with caution because grains contain potent endogenous proteinase inhibitors that could inactivate some of these enzymes in various tissues or germination stages.     

Efficient analysis of egg yolk proteins and their thermal sensitivity using sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing and nonreducing conditions

The multiple functional properties of egg yolk are mostly influenced by its complex protein composition. The high lipid content of egg yolk as well as the low solubility of delipidated egg yolk lipoproteins make analysis by conventional chromatographic or electrophoretic techniques a difficult task. This work describes a method to profile egg yolk proteins after delipidation with acetone using sodium dodecyl sulfate polyacrylamide gel electrophoresis on precast 8-18% T polyacrylamide gradient gels. Twenty bands were obtained for the whole egg yolk profile with molecular weights ranging between 5 and 221 kDa. The bands were identified based on their molecular weight and by comparison with isolated egg yolk subfractions. The dissociation behavior under reducing and nonreducing conditions provided additionally helpful information for identification and characterization of the yolk proteins. The method presented is very well suited for assaying the thermal sensitivity of whole yolk and its components and thus for the characterization of heat treatment processes.     

Study of some physicochemical and functional properties of quinoa (chenopodium quinoa willd) protein isolates

The amino acid composition and the physicochemical and functional properties of quinoa protein isolates were evaluated. Protein isolates were prepared from quinoa seed by alkaline solubilization (at pH 9, called Q9, and at pH 11, called Q11) followed by isoelectric precipitation and spray drying. Q9 and Q11 had high levels of essential amino acids, with high levels of lysine. Both isolates showed similar patterns in native/SDS-PAGE and SEM. The pH effect on fluorescence measurements showed decreasing fluorescence intensity and a shift in the maximum of emission of both isolates. Q9 showed an endotherm with a denaturation temperature of 98.1 degrees C and a denaturation enthalpy of 12.7 J/g, while Q11 showed no endotherm. The protein solubility of Q11 was lower than that of Q9 at pH above 5.0 but similar at the pH range 3.0-4.0. The water holding capacity (WHC) was similar in both isolates and was not affected by pH. The water imbibing capacity (WIC) was double for Q11 (3.5 mL of water/g isolate). Analysis of DSC, fluorescence, and solubility data suggests that there is apparently denaturation due to pH. Some differences were found that could be attributed to the extreme pH treatments in protein isolates and the nature of quinoa proteins. Q9 and Q11 can be used as a valuable source of nutrition for infants and children. Q9 may be used as an ingredient in nutritive beverages, and Q11 may be used as an ingredient in sauces, sausages, and soups.     

猪肌肉组织双向电泳分离条件的建立及常见问题分析

为建立和优化猪肌肉组织蛋白质双向电泳技术,从提取方法、加样量、IPG胶条的选择、SDS-PAGE胶浓度、染色方法等多个方面对双向电泳分离效果进行比较研究.结果显示,液氮研磨+超声破碎法提取猪肌肉组织蛋白效果优于液氮研磨法,前者细胞破碎彻底蛋白质溶解性好且核酸污染少,图谱质量较好;18cm pH 4～7的IPG胶条分离效果比pH 3～10非线性IPG胶条好;对于银染,18 cm pH 4～7的IPG胶条150μg上样量比较适宜;同一肌肉样品的三块胶的重复性可达70％以上;同时,对双向电泳过程中的典型问题作了详尽分析.研究结果表明,通过优化的双向电泳条件获得了较高分辨率和较好重复性的猪肌肉组织双向电泳图谱,可用于后续蛋白质组学分析.     

Detection of giant myofibrillar proteins connectin and nebulin in fish meat by electrophoresis in 3-5 gradient sodium dodecyl sulfate polyacrylamide slab gels

An improved method was investigated for sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (SDS-PAGE) to facilitate the analysis of the giant myofibrillar proteins, connectin and nebulin, in fish meat by using jack mackerel (Trachurus japonicus) as the sample fish. It was established that separation of the alpha-connectin band from the beta-connectin band by SDS-PAGE could be achieved by using 3-5% gradient gels with glycerol to facilitate the formation of a gradient with polymerization at 35 degrees C. SDS-PAGE samples of white dorsal muscle from the jack mackerel were homogenized with a 2% SDS solution containing an inhibitor mixture (1 microg/mL of phenylmethanesulfonyl fluoride, 1 microg/mL of leupeptin, and 1 microg/mL of E-64) and heated at 50 degrees C for 20 min. Heating these samples at 100 degrees C for 2 min resulted in the disintegration of connectin but did not affect nebulin. A purified myofibril sample and a whole muscle sample showed similar changes in the overall rate of degradation of whole connectin and nebulin during the postmortem storage period, but it was clear that beta-connectin was cleaved from alpha-connectin during the preparation of myofibrils at the early stage postmortem. Storage of the SDS-PAGE samples at -85 degrees C was preferable to storage at -18 degrees C for a long period.     

Cathepsin L: a predominant heat-activated proteinase in arrowtooth flounder muscle

Characterization of the autolytic profile of arrowtooth flounder (ATF) muscle indicated the involvement of heat-activated proteinases active at both acidic and alkaline pH values. Further assay of fish extract exhibited the maximum activity at 60 degrees C against casein used as a substrate at both pH 5.5 and 8.0. The maximum activity shifted to lower temperatures by the addition of urea with two distinctive patterns: activity reduction at pH 5.5 and activity enhancement at pH 8.0. The highest inhibition by E-64 indicated the proteinase belongs to the cysteine proteinase class. At pH 5.5, the proteinase hydrolyzed Z-Phe-Arg-NMec and all types of protein substrates tested at higher rate than that at pH 8.0. Activity bands, observed on the activity-stained substrate gels, indicated similar proteinases are responsible for the proteolytic activity observed at both pH values. When proteins of fish extract were separated by HPLC-SEC, only one proteolytic peak was observed at the retention time of 26 min with an estimated molecular weight of 39800 Da. The results implied cathepsin L is a predominant proteinase responsible for autolysis of ATF muscle at elevated temperatures.     

Heat-induced gels of egg white/ovalbumins from five avian species: thermal aggregation, molecular forces involved, and rheological properties

Product processing (heating, pH change, etc.) usually alters protein structure, improves rheological properties, and gives a unique texture to foods. The thermal aggregation and structural properties of ovalbumins from five avian species were studied at different pH values by polyacrylamide gel electrophoresis (PAGE) and determinations of sulfhydryl group content and surface hydrophobicity. The results showed that sulfhydryl group content changed insignificantly in heat-denatured ovalbumins other than hen ovalbumin (pH-independent), and surface hydrophobicity markedly increased (pH-dependent) after heating, with a significant difference among species. Furthermore, it was demonstrated that the hydrophobic interaction and sulfhydryl-disulfide interchange reaction were necessary in the aggregation and cross-linking of gel networks. Creep tests were also used to characterize the gel network structures of various egg white/ovalbumins upon heating. The viscoelastic behavior of the ovalbumins of all species was dependent on pH values, and changed significantly with the phylogeny of these species. With increases in pH value (7.0-8.5), the heat-induced gels of ovalbumins gradually changed from turbid to translucent, the instantaneous modulus (E(0)) increased slightly and reached a nearly constant value, and the Newtonian modulus (etaN) increased significantly in each sample. The heated egg white from these five avian species also formed highly viscoelastic gels, with a good correlation of viscoelastic behavior between ovalbumin and egg white in corresponding species.     

Ultrafast capillary electrophoretic analysis of cereal storage proteins and its applications to protein characterization and cultivar differentiation

Free zone capillary electrophoresis conditions have been improved to allow rapid (2-8 min) separations of grain proteins from several cereals (wheat, oats, rice, barley, and rye) with high resolution and reproducibility. This new method utilized the isoelectric compound iminodiacetic acid (IDA) in conjunction with 20% acetonitrile and 0.05% hydroxypropylmethylcellulose. Cultivars of all cereals tested could be differentiated in 3 min, including wheat, using either prolamin or glutelin protein patterns. Resolution was similar to or higher than that of separations in other acidic buffers. Migration time repeatability was excellent with run-to-run variability <1% RSD, day-to-day <1.4% RSD, and capillary-to-capillary <3.3% RSD. Because larger inner diameter capillaries (50 microm) could be used with this buffer, sensitivity was improved and capillary rinse times could be reduced when compared to smaller capillaries (25 microm i.d.). This also served to reduce total separation time so that the majority of cereal storage protein from several types of cereals could be analyzed with total analysis times of 2-8 min with extremely high resolution and repeatability. This method would allow unattended, high-throughput ( approximately 180-400 samples/24 h) analysis of cereal proteins without the generation of much organic solvent waste as well as automated data analysis and storage.     

小麦幼穗蛋白质双向电泳条件的优化

本研究以温光敏小麦为试材,用TCA/丙酮和酚提取法提取小麦幼穗蛋白样品,进行了双向电泳优化分析,并对双向电泳过程中出现的问题进行了讨论。结果表明,用TCA/丙酮法提取小麦幼穗蛋白质其产率（浓度）高于酚提取法。SDS-PAGE电泳显示,用TCA/丙酮提取法提取的蛋白质能获得较清晰条带,分辨率较高,而酚提取法提取的蛋白质其条带模糊,分辨率低。对蛋白质纯化除盐可以提高分辨率,减少横竖纹,获得背景清晰的圆形蛋白点。通过ImageMasterTM 2D Platinum5.0软件分析凝胶图谱,结果显示纯化后可降低噪点,纯化后蛋白点数可从未纯化蛋白点数的216增加到583。显然,采用TCA/丙酮法可获得高浓度高质量的蛋白质,而进一步纯化、除盐离子可进一步获得背景清晰可高重复性的电泳图谱。在双向电泳实验过程中,观察到一些异常缺陷胶的出现,如双向电泳图谱中蛋白点扩散,蛋白聚集形成斑点串,没有点或点很少,出现纵纹横纹及图谱扭曲等影响图谱质量的严重问题,本研究对这些问题做了分析并提出了解决方案。     

Elektrochemisches Verhalten harnstofflöslicher Komponenten von Weizenkleberproteinen

Electrochemical Behavior of Urea Soluble Components of Gluten Proteins of Wheat Protein zones of urea soluble gluten proteins of wheat samples from different cultivars and locations were analyzed after 2 dimensional separation by sucrose-density-gradient isoelectric focusing and disk polyacrylamide-gel electrophoresis. More than 50% of the urea soluble proteins was focused in a range from pH 6,5–7,6. The major concentration in all samples was observed close to pH 7,0 as indicated by the main peak in all absorption diagrams. Electrophoretic separation of proteins of equal isoelectric characteristics yielded fractions differing mainly in charges. Two dimensional separation yielded more than 40 protein fractions. The pattern of these fractions was more influenced by environmental effects than by cultivars. No relation between protein pattern and baking quality was observed.