Suppression of epimerization by cupric (II) salts in peptide synthesis using free amino acids as amino components.

An epimerization-free system for coupling N-protected peptides with free amino acids was developed. A number of inorganic substances were tested as epimerization suppressant additives during the coupling by various methods (carbodiimide plus additives, uronium salts, Woodward's reagent-K, isobutyl-chloroformate, etc.). Some of them (ZnCl2, RbClO4, LiCl, SnCl4, AlCl3, etc.) in combination with some coupling methods can guarantee coupling with minimal epimerization (D-epimer < 1%). But only a simultaneous use of 1-hydroxybenzotriazole and Cu2+ ions as additives in carbodiimide-mediated peptide couplings appeared to give a standard result (D-epimer < 0.1%). There was no epimerization even in the case when N-methyl amino acid (sarcosine) was used as an amino component, while in the absence of Cu2+ ions an unacceptable level of epimerization was observed (D-epimer, 22% for carbodiimide with the 1-hydroxybenzotriazole method). So far it has been considered that Cu2+ ions prevent obtaining peptides in high yields (< 90%) by various coupling methods. We have found that the use of 1-hydroxybenzotriazole, CuCl2 and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide instead of dicyclohexylcarbodiimide provides a possible method for obtaining the desired peptides in 90-99% yields without epimerization. All these results were shown by employing several model peptide couplings with free amino acids as amino components dissolved in an effective solvent system which readily dissolved them.     

Gas chromatographic determination of free amino acids and amino acids attached to transfer RNA in biological samples

A method was developed to analyze quantitatively free amino acids and amino acids attached to transfer RNA (tRNA) in tissue samples by gas chromatography. Free amino acids were purified by ion-exchange chromatography after deproteinization. Total cellular aminoacyl-tRNA was extracted from rabbit reticulocytes and liver by a modified phenol extraction method under conditions which were designed to prevent deacylation of the attached amino acids. After deacylation and separation from tRNA by pressure ultrafiltration, eighteen amino acids were determined by gas chromatography as their N-heptafluorobutyryl isobutyl derivatives.     

Automated GC-MS analysis of free amino acids in biological fluids

A gas chromatography-mass spectrometry (GC-MS) method was developed for the quantitative analysis of free amino acids as their propyl chloroformate derivatives in biological fluids. Derivatization with propyl chloroformate is carried out directly in the biological samples without prior protein precipitation or solid-phase extraction of the amino acids, thereby allowing automation of the entire procedure, including addition of reagents, extraction and injection into the GC-MS. The total analysis time was 30 min and 30 amino acids could be reliably quantified using 19 stable isotope-labeled amino acids as internal standards. Limits of detection (LOD) and lower limits of quantification (LLOQ) were in the range of 0.03-12 microM and 0.3-30 microM, respectively. The method was validated using a certified amino acid standard and reference plasma, and its applicability to different biological fluids was shown. Intra-day precision for the analysis of human urine, blood plasma, and cell culture medium was 2.0-8.8%, 0.9-8.3%, and 2.0-14.3%, respectively, while the inter-day precision for human urine was 1.5-14.1%.     

Taste attractiveness of free amino acids for juveniles of Persian sturgeon <Emphasis Type="Italic">Acipenser persicus</Emphasis>

Taste preferences towards 20 free amino acids (L-isomers, 0.1–0.0001 M) were determined in juveniles of Persian sturgeon Acipenser persicus. It was found that most amino acids (16) had a positive effect on extraoral gustatory reception (increased the frequency of catching artificial pellets by fishes). The most efficient amino acids were the following: threonine, histidine, arginine, asparagine, phenylalanine, cysteine, glutamine, and glycine. Fifteen amino acids were efficient for intraoral gustatory receptors: serine, arginine, cysteine, histidine, alanine, and some others. The presence of these amino acids in pellets increased the consumption. Amino acids decreasing the frequency of catching or consumption of pellets were not found. Highly significant positive correlation was found between the amino acid ranges mediated by the extraoral and intraoral gustatory reception. The fishes demonstrated maximum sensitivity to aspartic acid, 0.01 and 0.001 M, respectively, for intraoral and extraoral gustatory systems. The threshold concentrations of arginine and glycine were higher for the intraoral gustatory system (0.01 M) than for the extraoral one (0.1 M). The comparison of Persian sturgeon with sturgeon fishes (Russian sturgeon A. gueldenstaedtii, Siberian sturgeon A, baerii, and starred sturgeon A. stellatus) studied earlier confirmed high species specificity of intraoral taste preferences in representatives of Acipenser genus. Species specificity of extraoral taste ranges was less pronounced.     

WRF-TMH: predicting transmembrane helix by fusing composition index and physicochemical properties of amino acids

Membrane protein is the prime constituent of a cell, which performs a role of mediator between intra and extracellular processes. The prediction of transmembrane (TM) helix and its topology provides essential information regarding the function and structure of membrane proteins. However, prediction of TM helix and its topology is a challenging issue in bioinformatics and computational biology due to experimental complexities and lack of its established structures. Therefore, the location and orientation of TM helix segments are predicted from topogenic sequences. In this regard, we propose WRF-TMH model for effectively predicting TM helix segments. In this model, information is extracted from membrane protein sequences using compositional index and physicochemical properties. The redundant and irrelevant features are eliminated through singular value decomposition. The selected features provided by these feature extraction strategies are then fused to develop a hybrid model. Weighted random forest is adopted as a classification approach. We have used two benchmark datasets including low and high-resolution datasets. tenfold cross validation is employed to assess the performance of WRF-TMH model at different levels including per protein, per segment, and per residue. The success rates of WRF-TMH model are quite promising and are the best reported so far on the same datasets. It is observed that WRF-TMH model might play a substantial role, and will provide essential information for further structural and functional studies on membrane proteins. The accompanied web predictor is accessible at http://111.68.99.218/WRF-TMH/.     

Assemblies of free amino acids as possible prebiotic catalysts

Our understanding of how life emerged on Earth has much to do with speculations about the ways in which prebiotic catalysts could have been formed. Since enzymes, the contemporary biological catalysts, are polymers of amino acids, we looked at the possible activity of free amino acids as catalysts. In this study it is shown experimentally that mixtures of free amino acids exert catalytic activities of -galactosidase, carbonic anhydrase, and catalase. We also observed different levels of catalytic activty of individual amino acids: some were more efficient than others. Apparently, assemblies of amino acids which were formed around substrate molecules through weak interactions, could, in principle, catalyze many prebiotic reactions. This might have been one step in the emergence of biological enzymes.     

Engineering physicochemical and biological properties of proteins by chemical modification

Proteins can be modified by chemically binding synthetic or natural macromolecules to the surface of the molecules. This can counter some of the drawbacks of the native proteins and improves properties which could be important in their use as protein drugs and/or catalysts in bioreactors. The most difficult problem is endowing delicate proteins with suitable new properties by chemical modification without causing any loss of their functions.     

The free amino acids of muscle in the cephalochordate Branchiostoma,the cyclostome Myxine and some fishes

• 1.1. Branchiostoma and Myxine have the highest concentrations of amino acids (207 and 234 mM) of the five species investigated.
• 2.2. The predominant amino acids are glycine, proline, alanine, taurine, serine and valine, which form 83–98% of the total, except in Latimeria (60%).
• 3.3. Total amino acids are considered from the point of view of osmotic concentration in relation to other nitrogenous compounds of muscle.

     

Species-forming involution of pathogenic bacteria as a biological pattern (exemplified by bacteria of the genus Moraxella)

The author advances a hypothesis stating that a species disappearing as a result of new conditions evolving in a colonized animal or human body, unfavorable for the existence of this species, does not become extinct, but reverts into newly developing species due to the loss of its properties and acquiring new properties corresponding to new conditions. Thus the genus is preserved through the loss of species characteristics by individual organisms. The advanced hypothesis is substantiated by the analysis of the relevant processes observed in cases of infectious keratoconjunctivitis in humans and cattle, caused by bacteria of the genus Moraxella. The data indicating the possibility of such reversion in the genera Neisseria and Bordetella are presented.     

Osmolarity-sensitive release of free amino acids from cultured kidney cells (MDCK)

Summary The amino acid pool of MDCK cells was essentially constituted by alanine, glycine, glutamic acid, serine, taurine, lysine, -alanine and glutamine. Upon reductions in osmolarity, free amino acids were rapidly mobilized. In 50% hyposmotic solutions, the intracellular content of free amino acids decreased from 69 to 25mm. Glutamic acid, taurine and -alanine were the most sensitive to hyposmolarity, followed by glycine, alanine and serine, whereas isoleucine, phenylalanine and valine were only weakly reactive. The properties of this osmolarity-sensitive release of amino acids were examined using³H-taurine. Decreasing osmolarity to 85, 75 or 50% increased taurine efflux from 0.6% per min to 1.6, 3.5 and 5.06 per min, respectively. The time course of³H-taurine release closely follows that of the regulatory volume decrease in MDCK cells. Taurine release was unaffected by removal of Na⁺, Cl^– or Ca²⁺, or by treating cells with colchicine or cytochalasin. It was temperature dependent and decreased at low pH. Taurine release was unaffected by bumetanide (an inhibitor of the Na⁺/K⁺/2Cl^– carrier); it was inhibited 16 and 67 by TEA and quinidine (inhibitors of K⁺ conductances), unaffected by gadolinium or diphenylamine-2-carboxylate (inhibitors of Cl^– channels) and inhibited 50% by DIDS. The inhibitory effects of DIDS and quinidine were additive. Quinidine but not DIDS inhibited taurine uptake by MDCK cells.     

Capillary gas chromatographic determination of proteins and biological amino acids as N(O)-tert.-butyldimethylsilyl derivatives

Forty-seven biological amino acids containing all 22 protein amino acids were derivatized to N(O)-tert.-Butyldimethylsilyl (tBDMSi) derivatives by a single-step reaction with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide and successfully separated on an HP-1 capillary column. The relative standard deviations of the relative molar responses of most amino acids were <5%. Cystine seems to be partially converted into cysteine during derivatization. An increase in carrier gas flow-rate towards the end of the analysis by inlet pressure programming with electron pressure control avoided the peak broadening and adsorption of the derivatives with high boiling points on the column and especially increased sensitivity of cystine to 5 pmol. Glutamine was converted almost completely into pyroglutamic acid during prolonged storage of a standard solution prepared in 0.01 M HCl but not during derivatization. These results compared with those for the phenylthiocarbamyl derivatives analysed by HPLC and the analytical results reported in the literature on soybean hydrolysate showed good agreement except for cysteine. The results for the amino acid composition of bovine serum albumin also showed good agreement with results in the literature except for cysteine. In human urine, seventeen free amino acids were detected as tBDMSi derivatives.     

Thiophosphorylation of free amino acids and enzyme protein by thiophosphoramidate ions

In search of an activity-preserving protein thiophosphorylation method, with thymidylate synthase recombinant protein used as a substrate, potassium thiophosphoramidate and diammonium thiophosphoramidate salts in Tris- and ammonium carbonate based buffer solutions were employed, proving to serve as a non-destructive environment. Using potassium phosphoramidate or diammonium thiophosphoramidate, a series of phosphorylated and thiophosphorylated amino acid derivatives was prepared, helping, together with computational (using density functional theory, DFT) estimation of ³¹P NMR chemical shifts, to assign thiophosphorylated protein NMR resonances and prove the presence of thiophosphorylated lysine, serine and histidine moieties. Methods useful for prediction of ³¹P NMR chemical shifts of thiophosphorylated amino acid moieties, and thiophosphates in general, are also presented. The preliminary results obtained from trypsin digestion of enzyme shows peak at m/z 1825.805 which is in perfect agreement with the simulated isotopic pattern distributions for monothiophosphate of TVQQQVHLNQDEYK where thiophosphate moiety is attached to histidine (His²⁶) or lysine (Lys³³) side-chain.     

Chemical and catalytical properties of thermal polymers of amino acids (proteinoids)

The significance of thermal polyamino acids (proteinoids) as abiotic predecessors of proteins is reviewed on the basis of new experimental results. Most proteinoids yield only 50% to 80% amino acid upon acid hydrolysis. They contain 40% to 60% less peptide links than typical proteins, whereas their average nitrogen content is like that of proteins. The arrangement of amino acid residues is nonrandom. The degree of nonrandomness is difficult to determine because unusual crosslinks disturb most of the sequencing methods typically applied, in protein chemistry. The products obtained in a polymerization experiment are heterogeneous. They can be separated into a limited number of related fractions by chromatography or electrophoresis and other separation methods applied in protein chemistry. Their molecular weights are typically between 400 and 10 000. The number of free NH₂-groups, is usually smaller than in comparable proteins A significant fraction of NH₂-groups yields imidazole-type bases during the thermal polymerization. Optically active amino acids racemize during the same process. So far no helicity could be detected. Proteinoids are thus clearly distinct from proteins However, many of them exhibit weak catalytic activities and tend to undergo self-assembly into microstructures. Their properties of which only a few have been mentioned still support their role as possible candidates for ancestors of first proteins.     

Immobilization and mineralization of dissolved free amino acids by stream-bed biofilms

1. Radiolabelled (¹⁴C) amino acids were used to investigate the influence of sediment size as well as dissolved free amino acid (DFAA) concentration and composition on immobilization and mineralization of DFAAs by biofilms from a first-order stream. 2. Over time (240 min), biofilms on stony substrata immobilized a DFAA mixture more effectively than those on sandy substrata, however proportional mineralization of immobilized DFAAs was higher for sandy substrata (36 v 20%). 3. Using stony substrata, the DFAA mixture was immobilized more rapidly than glycine alone at ‘near-natural’ amino acid concentrations (c. 37 μgl^?1), as well as enriched concentrations (1 and 100 mg 1-^?1). Instantaneous rates of glycine immobilization and mineralization were not saturated at glycine enrichments of up to 980 mgl^?1. 4. With both the amino acid mixture and glycine alone, proportional mineralization of the immobilized amino acids increased on enrichment to Img 1-^?1 (DFAA mixture: from 25 to 37%; glycine alone: from 50 to 54%), but then fell on further enrichment to 100mgl^?1 (DFAA mixture: 11%; glycine alone: 7%). 5. Results are discussed in terms of the potential trophic utility of immobilized DFAAs as well as the apparent roles of biotic and abiotic immobilization mechanisms. Immobilization and mineralization responses to variables investigated in this study give an insight into potential variability of carbon immobilization and retention in stream-bed sediments. This is fundamental to an understanding of how DOC may become available to higher trophic levels.