Amino-Acid Composition and Biological Activity of Proteins from Psoralea drupacea Beans

Barley (Hordeum vulgare L.) is the fourth largest produced cereal worldwide. About two thirds of barley production is used to animal feed. When used to feed monogastric animals, the main shortcoming of barley grains is the deficiency of essential amino acids, especially lysine, threonine and methionine. The unbalanced amino acid composition is due to the main storage protein, the hordeins, which account for about 50% of total grain protein content. The nitrogen fertilization promotes C-hordein expression and accumulation, the hordein subgroup with the lowest content of essential amino acids, and the highest content of non-essential amino acids. Due to the importance of grain protein content and composition in the end use grain quality the key objective of the present study was to obtain a detailed insight into synthesis and accumulation of barley grain proteins and their relation to improved nutritional quality. An integrated proteomic and transcriptomic analysis have been undertaken in a set of transgenic antisense barley lines with the grain protein profile altered in comparison to the non-transgenic line cv. Golden Promise. The results were presented in three manuscripts in the thesis (chapters 2, 3 and 4). The first manuscript (chapter 2) reported a new grain protein extraction method combined with multi-method protein evaluation, including biochemical quantification, amino acid composition, sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) couple with mass spectrometry (MS) identification and a gel free shotgun MS identification and relative quantification. The results showed the changeability of proteins between protein groups and the importance of choosing an adequate proteomic-based method for protein identification according to the complexity of protein mixtures. In the second manuscript (chapter 3) a differential protein profile of non-transgenic barley cv. Golden Promise and the transgenic antisense C-hordein barley lines was achieved by two-dimensional gel electrophoresis (2-DE) for salt soluble proteins and the differentially expressed proteins were identified by MS. The key results showed that the suppression of Chordeins, the poor nutritional hordein subgroup, does not exclusively affects hordein synthesis and accumulation, and that the more balanced amino acid composition of these lines may be a consequence of distinct protein sources among different transgenic events, though a stable lysine-rich proteins upregulation occurs in all lines. In the third manuscript (chapter 4) the effects of nitrogen fertilization on hordein family at transcriptional and proteome level were assessed. The main results showed differential responses to N nutrition between nontransgenic and transgenic lines. In relation to C-hordein, specific C-hordein downregulation effect and in particular different responses to N were verified among subgroups of C-hordein multigene family in the transgenic line at transcriptional and proteomic level. In summary, the multi-method strategy used in the present work was successfully applied to obtain comprehensive information about barley grain proteins synthesis and accumulation and explain, at least in part, their relation to improved nutritional quality. These results can be useful in barley breeding programs aiming selective alterations of specific alleles/homologues to change amino acid composition by changing the relative proportions of the grain proteins in order to improve the barley grains nutritional quality.

The predictive limits of the amino acid composition for the secondary structural content (percentage of residues in the secondary structural states helix, sheet, and coil) in proteins are assessed quantitatively. For the first time, techniques for prediction of secondary structural content are presented which rely on the amino acid composition as the only information on the query protein. In our first method, the amino acid composition of an unknown protein is represented by the best (in a least square sense) linear combination of the characteristic amino acid compositions of the three secondary structural types computed from a learning set of tertiary structures. The second technique is a generalization of the first one and takes into account also possible compositional couplings between any two sorts of amino acids. Its mathematical formulation results in an eigenvalue/eigenvector problem of the second moment matrix describing the amino acid compositional fluctuations of secondary structural types in various proteins of a learning set. Possible correlations of the principal directions of the eigenspaces with physical properties of the amino acids were also checked. For example, the first two eigenvectors of the helical eigenspace correlate with the size and hydrophobicity of the residue types respectively. As learning and test sets of tertiary structures, we utilized representative, automatically generated subsets of Protein Data Bank (PDB) consisting of non-homologous protein structures at the resolution thresholds < or = 1.8A, < or = 2.0A, < or = 2.5A, and < or = 3.0 A. We show that the consideration of compositional couplings improves prediction accuracy, albeit not dramatically. Whereas in the self-consistency test (learning with the protein to be predicted), a clear decrease of prediction accuracy with worsening resolution is observed, the jackknife test (leave the predicted protein out) yielded best results for the largest dataset (< or = 3.0A, almost no difference to the self-consistency test!), i.e., only this set, with more than 400 proteins, is sufficient for stable computation of the parameters in the prediction function of the second method. The average absolute error in predicting the fraction of helix, sheet, and coil from amino acid composition of the query protein are 13.7, 12.6, and 11.4%, respectively with r.m.s. deviations in the range of 8.6 divided by 11.8% for the 3.0 A dataset in a jackknife test. The absolute precision of the average absolute errors is in the range of 1 divided by 3% as measured for other representative subsets of the PDB. Secondary structural content prediction methods found in the literature have been clustered in accordance with their prediction accuracies. To our surprise, much more complex secondary structure prediction methods utilized for the same purpose of secondary structural content prediction achieve prediction accuracies very similar to those of the present analytic techniques, implying that all the information beyond the amino acid composition is, in fact, mainly utilized for positioning the secondary structural state in the sequence but not for determination of the overall number of residues in a secondary structural type. This result implies that higher prediction accuracies cannot be achieved relying solely on the amino acid composition of an unknown query protein as prediction input. Our prediction program SSCP has been made available as a World Wide Web and E-mail service.

The similarity in collagen fiber diameter between the camel cornea (CC) and the human cornea, along with its relatively large size, positions the CC as a promising candidate for tissue engineering applications. This study aimed to evaluate the effectiveness of various decellularization methods on the CC, followed by assessing protein content similarities between the CC and the human cornea, highlighting its potential for human-related tissue engineering research. Camel corneas were extracted from freshly isolated camel eyeballs and subjected to multiple decellularization protocols, including sodium dodecyl sulfate (SDS)-based and formic acid (FA)-based methods. The decellularized camel corneas (dCCs) were then analyzed for histological properties, cell viability, collagen fiber integrity, ultrastructural characteristics, and susceptibility to bacterial contamination. Additionally, the amino acid (AA) composition of CC proteins was compared to that of the human cornea. Histological analyses confirmed successful decellularization of the CC using both SDS- and FA-based protocols. Cell viability significantly increased when cells were cultured on dCC scaffolds, with the highest improvement observed in the 0.5% SDS-Tris-treated group. OrgA and 0.5% SDS-Tris protocols preserved collagen fibers most effectively among the treatment groups. Ultrastructural assessments revealed consistent results across all groups. However, susceptibility to bacterial contamination was noted in the FA-treated group after one week of refrigerated storage. Lastly, the AA composition of CC proteins demonstrated a notable resemblance to the human cornea. This study demonstrates that the CC can be effectively decellularized using SDS- and FA-based methods while maintaining tissue morphology, ultrastructure, and collagen fiber integrity. Despite FA's efficacy in decellularization, its potential for causing bacterial contamination warrants caution in its application. The high degree of similarity in AA composition between CC and the human cornea further underscores the potential of CC as a scaffold for human-related tissue engineering studies.

Data on the amino acid composition of proteins having various functions from organisms representing different evolutionary levels (83 superfamilies) are used in order to elucidate the trends in protein molecular evolution. The interconnections evolutionary rate (rate of mutation acceptance) — amino acid composition, and evolutionary level of the organism — amino acid composition (in case of proteins of the same or very similar function) are studied. The amino acid compositions of proteins performing jointly an evolutionarily old functions are also juxtaposed. The mean contemporary protein composition is used as a basis for comparison. The obtained results are evidence in favour of the existence of a trend for an increase of the special amino acids (Met, Ile, Gln, His, Lys, Asn, Phe, Tyr, Trp, Cys) at the expense of the usual ones (Thr, Pro, Ala, Ser, Arg, Gly, Leu, Val, Glu, Asp). The tests of statistical significance of the obtained results (comparison of the mean compositions of proteins from low evolutionary level organisms with that of all sequenced proteins; comparison of the mean contemporary protein composition with that obtained after simulation of the evolutionary process) confirm and universalize the observed trend. The above results direct the attention to the concept of a smaller number of amino acids in the ancient proteins and respectively simpler genetic code. A fluctuation around the initial primitive level is suggested to explain the conservatism of proteins of the same function in evolutionarily low level organisms. The observed trend could be applied for designing new proteins.

The cuttlefish Sepia apama Gray (Mollusca: Cephalopoda) is a seasonally abundant food resource exploited annually by moulting albatrosses throughout winter and early spring in the coastal waters of New South Wales, Australia. To assess its nutritional value as albatross forage, we analysed S. apama for water, lipid protein, ash contents, energy density and amino acid composition. Because albatrosses consistently consume S. apama parts preferentially in the order of head, viscera and mantle, we analysed these sections separately, but did not identify any nutritional basis for this selective feeding behaviour. The gross energy value of S. apama bodies was 20.9 kJ/g dry mass, but their high water content (>83%; cf <70% for fish) results in a relatively low energy density of 3.53 kJ/g. This may contribute to a need to take large meals, which subsequently degrade flight performance. Protein content was typically >75% dry mass, whereas fat content was only about 1%. Albatrosses feed on many species of cephalopods and teleost fish, and we found the amino acid composition of S. apama to be comparable to a range of species within these taxa. We used S. apama exclusively in feeding trials to estimate the energy assimilation efficiency for Diomedea albatrosses. We estimated their nitrogen-corrected apparent energy assimilation efficiency for consuming this prey to be 81.82 ± 0.72% and nitrogen retention as 2.90 ± 0.11 g N kg(-1) d(-1). Although S. apama has a high water content and relatively low energy density, its protein composition is otherwise comparable to other albatross prey species. Consequently, the large size and seasonal abundance of this prey should ensure that albatrosses remain replete and adequately nourished on this forage while undergoing moult.

The amino acid and carbohydrate compositions of the larva of the raffia palm weevil were determined in this study. Nutritional components such as amino acid and carbohydrate analyses were carried out on the larva. In the larva, the amino acid profile of the protein and carbohydrate composition was determined using high performance liquid chromatography method. The amino acid composition reveals a total of 18 amino acids with glutamic acid (14.46g/100g) and aspartic acid (10.65g/100g) as the predominant amino acids. The amino acids have a total value of 96.06g/100g. The essential amino acids make up 53.08g/100g of the total amino acids. This value represents 55.26% of the total amino acid composition of the raffia palm weevil sample. The larva of the raffia palm weevil has high values of lysine (9.72g/100g), leucine (8.98g/100g) and arginine (6.55g/100g). The essential amino acids: lysine, leucine, isoleucine, threonine, valine, phenylalanine and tyrosine have higher values than the FAO/WHO/UNU reference values for the respective amino acids. The larva is rich in glucose (213.96mg/100g), fructose (42.47mg/100g), and sucrose (36.6mg/100g). The palm weevil larva represents a very good source of protein, a good complement of essential amino acids, and dietary energy.

Albumins, globulins, gliadins and glutenins presumably comprising 100 percent of the wheat seed proteins were sequentially extracted and electrophoresed on SDS-polyacrylamide gels. The SDS-electrophoretic patterns within each of the four fractions from T. boeotiaum, T. urartu, T. turgidum, T. timopheevii, T. aestivum, Ae. speltoides and Ae. squawosa were similar. They differed from one species to another only in a few minor components or density of certain components. Similarity in MW's of components, as indicated by the SDS-electrophoretic patterns, suggests that the wheats and Aegilops exhibit no variability for structural genes coding seed proteins. A minimum of 60 to 70 and a maximum of 360 to 420 structural genes with major or minor effects control the total seed protein in T. aestivum. Presumably, only one or the other homoeoallele was expressed in the polyploids. Different components of albumins and globulins presumably had distinct MW's and amino acid composition, while the components of gliadins and glutenins could be classified into a few groups each containing one or more components with the same MW and nearly identical amino acid composition. The genes for components with similar MW's and amino acid composition arose through multiplication of a single original gene and perhaps share the same regulatory mechanism. Seed protein content and quality in wheat might be improved through the incorporation of structural genes, coding for polypeptides with distinct MW's, from distantly related species, rather than by manipulation of the structural genes within the Triticum-Aegilops group. Regulatory mutants similar to opaque-2 of corn could be used to alter the proportion of gliadins in relation to albumins and globulins, to improve amino acid composition of wheat proteins.

A compact mitochondrial gene contains all essential information about the synthesis of mitochondrial proteins which play their roles in a small compartment of the mitochondrium. Almost no noncoding regions have been found through the gene, but a necessary set of tRNAs for the 20 amino acids is provided for biosynthesis, some of them coding different amino acids from those in a usual cell. Since the gene is so compact that the produced proteins would have some characteristic aspects for the mitochondrium, amino acid compositions of mitochondrial proteins (mt-proteins) were examined in the 20-dimensional composition space. The results show that compositions of proteins translated from the mitochondrial genes have a distinct character having more hydrophobic content than others, which is illustrated by a clustered distribution in the multidimensional composition space. The cluster is located at the tail edge of the global distribution pattern of a Gaussian shape for other various kinds of proteins in the space. The mt-proteins are rich in hydrophobic amino acids as is a membrane protein, but are different from other membrane proteins in a lesser content of Val. A good correlation found between the base and amino acid compositions for the mitochondria was examined in comparison to those of organisms such as thermophilic bacterium having an extreme G-C-rich base composition.

Analysis of amino acid composition in proteins of animal tissues and foods as pre-column o-phthaldialdehyde derivatives by HPLC with fluorescence detection.

Twelve pieces of Tilapia mossambicus fish samples of 28 cm average length and 52 g average weight, two from each of the six sampling locations were analyzed for Amino Acid composition using Applied Biosystems PTH Amino Acid Analyzer as described by standard methods. A proximate analysis of the fish samples was also carried out. Result showed that Glutamic acid and Aspartic acid are the most concentrated amino acid present in the fish which ranged between 11.96 to 15.14 g/100g protein and 7.69 to 9.42 g/100g protein respectively. The essential amino acid and protein composition of the fish were higher in the dams than in the rivers. The composition of the amino acid analyzed were within standard regulatory agency limits. For the proximate composition, moisture content varied between 71.05% - 77.71% while ash composition was 10.20%. The crude fat composition ranged between 8.59 – 11.67% during the dry season. The values obtained for the proximate composition where within the reported ranges with higher percentage in the case of ash. The concentration of the proximate components in the fish samples are within the reference value of FAO/WHO for Humans. Special attention is needed in monitoring gold mining activities which largely contribute to the reduction and pollution of the nutritional qualities of the fishes from the rivers.Keywords: amino acid, proximate composition, tilapia fish, water bodies

Ice recrystallization inhibition activity of pulse protein hydrolysates after immobilized metal affinity separation

BackgroundPlastids are an important component of plant cells, being the site of manufacture and storage of chemical compounds used by the cell, and contain pigments such as those used in photosynthesis, starch synthesis/storage, cell color etc. They are essential organelles of the plant cell, also present in algae. Recent advances in genomic technology and sequencing efforts is generating a huge amount of DNA sequence data every day. The predicted proteome of these genomes needs annotation at a faster pace. In view of this, one such annotation need is to develop an automated system that can distinguish between plastid and non-plastid proteins accurately, and further classify plastid-types based on their functionality. We compared the amino acid compositions of plastid proteins with those of non-plastid ones and found significant differences, which were used as a basis to develop various feature-based prediction models using similarity-search and machine learning.ResultsIn this study, we developed separate Support Vector Machine (SVM) trained classifiers for characterizing the plastids in two steps: first distinguishing the plastid vs. non-plastid proteins, and then classifying the identified plastids into their various types based on their function (chloroplast, chromoplast, etioplast, and amyloplast). Five diverse protein features: amino acid composition, dipeptide composition, the pseudo amino acid composition, Nterminal-Center-Cterminal composition and the protein physicochemical properties are used to develop SVM models. Overall, the dipeptide composition-based module shows the best performance with an accuracy of 86.80% and Matthews Correlation Coefficient (MCC) of 0.74 in phase-I and 78.60% with a MCC of 0.44 in phase-II. On independent test data, this model also performs better with an overall accuracy of 76.58% and 74.97% in phase-I and phase-II, respectively. The similarity-based PSI-BLAST module shows very low performance with about 50% prediction accuracy for distinguishing plastid vs. non-plastids and only 20% in classifying various plastid-types, indicating the need and importance of machine learning algorithms.ConclusionThe current work is a first attempt to develop a methodology for classifying various plastid-type proteins. The prediction modules have also been made available as a web tool, PLpred available at http://bioinfo.okstate.edu/PLpred/ for real time identification/characterization. We believe this tool will be very useful in the functional annotation of various genomes.

The analysis of the main methods of increasing the nutritional and biological value of wheat flour has been conducted. Considerable attention is paid to the issue of adjusting the protein content in flour due to additives. The relevance of improving the amino acid composition of flour protein is emphasized. The feasibility of comprehensive studies for the development of food system formulations with a balanced amino acid composition based on wheat flour and oilseed meal is substantiated. The amino acid content has been experimentally determined and the biological value of raw materials proteins for food systems which are sunflower and soybean meal has been calculated. The mixture composition of sunflower and soybean meal with an improved amino acid composition has been scientifically substantiated. It has been found that the protein composition of the meal mixture is maximally close to the reference in terms of the content of leucine, lysine and the amount of sulfur-containing amino acids (methionine, cystine). The score of isoleucine, tryptophan, phenylalanine and tyrosine in the protein composition of oilseed meal is 1.1–1.47 times higher than the reference. The amino acid composition has been calculated and the biological value of the protein of food systems containing 80–90 % wheat flour and 10–20 % composition of soybean and sunflower meal with improved amino acid composition has been determined. It has been found that the formulation of the food system containing 20 % of the composition of meal mixture and 80 % of wheat flour has the greatest biological value in comparison with wheat flour. In this food system, the limited amino acids – lysine and sulfur-containing (methionine and cystine) are the closest to the reference one and are 67.68 % and 70.12 %. The fatty acid composition has been experimentally determined. The biological effectiveness of the fats of the developed food systems has been calculated. The closest to the fatty acid ratio recommended by nutritionists is the food system with a ratio of meal mixture: wheat flour of 20:80. The resulting formulations of food systems will be useful in technologies of flour products with high biological value.

The amino acid composition and nutritional value of 5 clover varieties including 3 Polish ones ('Gloria', 'Hruszowska', 'Skrzeszowicka') and 2 of foreign origin ('Rotra' and 'Violetta') were investigated. No significant differences in the total protein content (19.2–20.0% of dry matter) as well as in qualitative amino acid composition were found among the clover varieties under examination. EAA index (Essential amino acid index) calculated according to Oser for 'Gloria' and 'Hruszowska' showed the highest nutritional value was – 40. The lowest value of EAA index was found for 'Violetta' cvar. – 32, intermediate values however for Rotra and Skrzeszowicka was 37 and 36.

We examined hair properties from determinations of diameter, amino acid composition, trace metal elements, and tensile strength of the hairs from protein-malnourished rats. The coarse or medium hair diameters of the experimental protein-malnourished rats had a tendency to decrease more than those of the control. Total contents of amino acids in the hairs had a tendency to decrease in the protein-deficient rats. Cystine content of rat hairs definitely decreased only in 5% wheat- and 5% rice-pattern amino acid mixture diet groups but not in 5% gluten diet and 5% casein diet groups. And many of the low-protein diet groups were significantly lower in methionine content than the control except for the 5% casein diet group. The Mg, Zn, and Fe contents in the hairs considerably increased in protein-deficient rats against the control. Tensile strength of coarse hairs in the experimental protein-malnourished rats was significantly lower than that of the control. The changes in amino acid composition of rat hair proteins are more likely to be influenced by various qualities of dietary protein or different compositions of amino acid mixtures in the diets at a similar dietary protein level. It should seriously be considered from our data whether the reduced cystine content in the hair can be regarded as an indicator of nutritional status.