Analysis of free amino acids in mammalian brain extracts

An optimized method for analysis of free amino acids using a modified lithium-citrate buffer system with a Hitachi L-8800 amino acid analyzer is described. It demonstrates clear advantages over the sodium-citrate buffer system commonly used for the analysis of protein hydrolysates. A sample pretreatment technique for amino acid analysis of brain extracts is also discussed. The focus has been placed on the possibility of quantitative determination of the reduced form of glutathione (GSH) with simultaneous analysis of all other amino acids in brain extracts. The method was validated and calibration coefficient (K _GSH) was determined. Examples of chromatographic separation of free amino acids in extracts derived from different parts of the brain are presented.     

Relationships between the osmoadaptation strategy, amino acid composition of bulk protein, and properties of certain enzymes of haloalkaliphilic bacteria

Haloalkaliphilic microorganisms isolated from soda lakes were compared in terms of the amino acid composition of bulk protein and the reaction of a number of key enzymes to salts and pH of the medium. In the extremely haloalkaliphilic bacterium Natroniella acetigena (selt-in osmoadaptation strategy), acidic amino acids (glutamic and aspartic) made up 30.91 mol % of the total of bulk protein amino acids. In the moderate haloalkaliphiles Tindallia magadiensis, Halomonas campisalis, and Halomonas sp. AIR-2 (compatible-solutes osmoadaptation strategy), the proportion of acidic amino acids (24.36, 23.15, and 23.58 mol %, respectively) was lower than in N. acetigena but higher than in the freshwater Acetobacterium paludosum (20.77 mol %). The excess of acidic amino acids over basic amino acids (lysine and arginine) increased with the degree of halophily. The enzymes of haloalkaliphiles proved to be tolerant to salts and high pH values, although the degree of tolerance varied. The activity of N. acetigena CO dehydrogenase was maximum in the presence of 0.7 M NaCl, but it was virtually independent of the NaHCO₃ concentration. The hydrogenase and CO dehydrogenase of T. magadiensis exhibited maximum activity in the absence of NaCl; the Co dehydrogenase was most active at 0.25 M NaHCO₃, and hydrogenase activity was only weakly dependent on NaHCO₃ in the concentration range of 0–1.2 M. The nitrate reductases of H. campisalis and Halomonas sp. AIR-2 were active in broad ranges of NaCl and KCl concentrations; the activity maxima were recorded at moderate concentrations of these salts. The pH optima of most of the studied enzymes of haloalkaliphiles were in the alkaline zone. Thus, it was shown that the amino acid composition of bulk protein is determined by the osmoadaptation strategy employed by the bacterium. A correlation was found between the salt tolerance of enzymes and the proportion of acidic amino acids in the bulk protein. The ability of enzymes to function at high pH values is one of the mechanisms of adaptation of microorganisms to high pH values.     

Isolation and quantitation of isotopically labeled amino acids from biological samples

To face the problem of simultaneous isolation and quantitation of isotopically labeled amino acids in biological samples, two semi-preparative chromatographic methods were developed. One method was especially designed to isolate radioactively labeled amino acids for which we used derivatization with the fluorophore o-phtaaldialdehyde (OPA), which is known to be easy and reliable. Isolation of amino acids labeled with stable isotopes required another approach as we wanted to use isotope ratio mass spectroscopy (IRMS), which can only be performed on pure, non-derivatized amino acids. Becuase the OPA probe cannot be removed after isolation of the derivative, we used 9-fluorenylmethylchloroformate (FMOC) instead. This probe is linked to an amino acid via a peptide bond which can easily be broken byb gas-phase acid hydrolysis (103% recovery after 5 h at 150°C: S.D = 3.5%, n = 14). Run time (injection to injection) was 60 min for the OPA method and 75 min for the FMOC method. Both fluorescence and UV absorbance detection can be employed. The coefficient of variation (C.V.) for peak area measurement was below 2% for most OPA amino acids and below 3% for most FMOC amino acids. At maximum, a total of 1000 μl could be injcted, representing approximately 200 μl of deproteinized plasma. The methods were linear up to injection of 0.5 μmol of all amino acids (OPA: r²=0.995−0.999; FMOC: r²=0.992−0.999). The C.V. of the IRMS measurement within the range which can be isolated maximally in one chromatographic run (50–500 nmol), was less than 3% above 100 mmol, indicating that chromatographic isolation fulfils the needs of the IRMS determination. The resulting methods are suitable for the isolation and quantitation of micromolar amounts of labeled amino acids from biological samples.     

Sequence determination of eglin c using combined microtechniques of amino acid analysis,peptide isolation,and automatic Edman degradation

The complete amino acid sequence of a proteinase inhibitor, eglin c (M_r 8100), has been determined with less than 150 μg of the protein using the following microtechniques: (a) amino acid analysis with a low-nanogram amount of protein hydrolysate using dimethylaminoazobenzene sulfonyl chloride, (b) peptide isolation at the picomole level using the dimethylaminoazobenzene isothyiocyanate (DABITC) precolumn derivatization method, and (c) automatic Edman degradation. One amino acid residue has been corrected for the previously reported sequence. The Contribution of each technique to the microsequencing is discussed. In addition, a new high-performance liquid chromatography system that gives a complete baseline separation of all phenylthiohydantoin-amino acids is described.     

Purification and characterization of high antioxidant peptides from duck egg white protein hydrolysates

The hydrolysate from duck egg white protein (DEWP) prepared by “SEEP–Alcalase” at degree of hydrolysis (DH) value of 21% (namely HSA₂₁) exhibited high antioxidant capacity in different oxidation systems. A consecutive chromatographic method was then developed for separation and purification of HSA₂₁, including ion-exchange chromatography, macroporous adsorption resin (MAR) and gel filter chromatography. The final peptides “P_21-3–75-B” were obtained with significantly enhanced antioxidant activity (p < 0.05). It was further confirmed that the product mainly consisted of five oligopeptides (Mr: 202.1, 294.1, 382.1, 426.3, and 514.4 Da). Furthermore, the antioxidant activity of P_21-3–75-B kept stable after in vitro digestive simulation. Antioxidant capacity of the purified peptides was closely related to the molecular mass, hydrophobic amino acid residues, acidic amino acid and some antioxidant amino acids. This research provided a valuable route for producing new natural-source peptides with strong antioxidant capacity and high nutritious value for our daily intake.     

Isolation of all proteins from the 50S subparticles of ribosomes of Escherichia coli

The paper proposes a method of preparative isolation of all proteins from the 50S subparticle of E. coli ribosomes. The method is based on (1) preliminary fractionation into protein groups and ribonucleoprotein particles by a consecutive treatment of the 50S particles with increasing LiCl concentrations, and (2) chromatographic separation of protein groups on DE- and CM-cellulose and gel-filtration of separate fractions. The method allows to obtain any protein required for studies in preparative amounts avoiding many chromatographic stages. A detailed scheme of isolation of all proteins is given together with quantitative data of yields of individual proteins calculated per 6 g of the 50S subparticles.     

Reverse-phase liquid chromatographic analysis of amino acids after reaction with o-phthalaldehyde

Precolumn formation of o-phthalaldehyde (OPA) derivatives of amino acids, followed by reverse-phase separation and fluorescent detection, provides rapid, sensitive amino acid analysis. Eighteen OPA-amino acid derivatives are resolved on a Micropack MCH 5 column and can be measured at picomole levels. Ease of derivative preparation and separation makes liquid chromatographic analysis of OPA-amino acids a convenient and improved technique for measuring or confirming the presence of low levels of amino acids in aqueous solutions. Use of the method was demonstrated by measuring low concentrations of amino acids released from zooplankters stressed by contaminants.     

Quantitative gas chromatographic analysis of amino acids on a short glass capillary column

A study of the quantitative gas chromatographic analysis of protein amino acids as their N-heptafluorobutyric amino acid n-propyl esters on a glass capillary column has been made. The analysis is completed within 35 min with good separation of the common protein amino acids in a single-column run.Hydrolyzed peptides have been analyzed. The analyses were performed with a precision varying between 1 and 6% (mean relative standard deviation) depending on the number of amino acid residues in the peptide. The amount taken for analysis was 20–300 μg. The results agree with the known sequences of the peptides and with the analyses by ion-exchange chromatography except for cysteine. This amino acid can be analyzed after modification as S-methylated cysteine.     

Preparative liquid chromatography of 1:1 adducts derived from the reaction of malondialdehyde with amino acids

Purification of 1:1 adducts (enaminals) formed from the reaction of methyl esters of amino acids with either malondialdehyde or methylmalondialdehyde was accomplished by preparative liquid chromatography on the stationary phase Amberlite XAD-4. The enaminal was easily and rapidly separated from the by-products and starting materials by a EtOH-H₂O mobile phase and then easily and rapidly recovered from the EtOH-H₂O at good yield and high purity. Depending on column diameter, up to 50 mg of enaminal could be purified in one chromatographic step. Adducts derived from l-Arg, l-His, l-Tyr, and l-Cys were purified.     

Efficient method for preparation of highly purified lipopolysaccharides by hydrophobic interaction chromatography

A method for the efficient preparation of highly purified lipopolysaccharides (LPSs) by hydrophobic interaction chromatography (HIC) has been developed. The procedure can be used for the purification of cell wall bound LPSs after hot phenol–water extraction and for the isolation of extracellular LPSs from the supernatant, respectively. The method described has been tested with artificial mixtures containing LPSs, polysaccharide, protein and RNA and subsequently employed for the preparative purification of two LPSs of different origin, namely the extracellular LPS secreted by Escherichia coli E49 into the culture medium, and the cell wall bound LPS from Pseudomonas aeruginosa VA11465/1. Compared to currently used methods for LPS purification such as enzymatic digestion and ultracentrifugation, the chromatographic separation reported here combines superior purity with minimal loss of LPS, high reproducibility and simple handling. The removal of contaminants such as protein, RNA and polysaccharides and the recovery of LPSs were monitored by appropriate assays.     

Analysis of adipimidate-crosslinked lysine

The chromatographic conditions for separation of N,N′-bislysyl(?-N)adipamidine and N-lysyl(?-N)adipamidinic acid, which were the products of acid hydrolysis of proteins treated with adipimidate esters, from other amino acids on an amino acid analyzer were established including their ninhydrin color values. Kinetics of decomposition of these lysine derivatives under the conditions of total acid hydrolysis of protein are also reported.     

A simplified and efficient procedure for the purification of apolipoprotein AI from human serum high-density lipoprotein-3 by preparative isoelectric focussing on polyacrylamide gel beads

An improved method is described for the purification of milligram amounts of apolipoprotein AI from serum apo-HDL₃ by isoelectric focussing on polyacrylamide gel beads. The procedure involves a single focussing over a narrow (1.3 unit) pH gradient, and permits isolation of apo-AI of exceptional purity and in high yield (75% recovery of HDL₃ protein, ca. 50% corresponding to pure apo-AI). The electrophoretic mobility, pI values, molecular weight, antigenicity and amino acid composition of such apo-AI were indistinguishable from those reported in the literature. A rabbit antiserum to apo-AI isolated by focusing exhibited similar immunological reactivity to one prepared from an antigen isolated by gel filtration chromatography; moreover, apo-AI purified by the respective procedures reacted identically with both antisera. We conclude that isoelectric focussing on a support of polyacrylamide gel beads (as Bio-Gel P60) presents certain advantages for the isolation of highly purified apo-AI over both conventional chromatographic procedures and isoelectric focussing on a Sephadex support.     

Characterization of amino acid profiles of culture media via pre-column 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization and ultra performance liquid chromatography

The AccQ.Tag? method, as a well-established protocol for amino acid analysis combining derivatization procedure, dedicated HPLC separation and fluorescence detection, was properly transferred and accordingly optimized for the application on ultra performance liquid chromatography (UPLC?) and UV detection. Capitalizing on sub-2 μm particles, this newly established UV-UPLC? technique facilitated efficient chromatographic separation of 21 amino acid derivatives within 12 min. In addition, UPLC? demonstrated significant improvements due to superior performance and reduced run times compared with the former 35 min of the original HPLC protocol. Using UV instead of fluorescence detection, amino acid quantification after pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) yielded appropriate sensitivities within the low pmol range with corresponding detection limits varying from 0.11 to 0.57 pmol per injection. Moreover, the UPLC? method was applied to characterize changes in the free (FAA) as well as total amino acid (TAA) profiles specific to culture media at three distinctive stages of fermentation: fresh medium, fermentation broth after cell mass production prior to induction and after product expression at the end of fermentation. Amino acid profiles intrinsic to the fresh, sterilized medium indicated free, hence more bioavailable, amino acids at a FAA/TAA ratio of 40%, whereas ongoing fermentation implied a rather specific, successive decline in selective FAA concentrations. Thereby, the most distinctive variations in FAAs were highlighted by aspartic acid, serine and threonine, each exhibiting an almost complete uptake from the culture media (?96% to ?99%), with remaining FAA/TAA ratios of 1%, 8%, and 1%, respectively. This indeed may indicate limitations and shortages within the nutrient broth. Thus, amino acid monitoring utilizing high-throughput chromatography, such as UPLC?, can be considered as a valuable tool to facilitate rapid adjustments of fermentation broths and to optimize culture media to specific requirements.     

Subunit analysis of bovine heart complex I by reversed-phase high-performance liquid chromatography, electrospray ionization-tandem mass spectrometry, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry

An effective method was developed for isolation and analysis of bovine heart complex I subunits. The method uses C18 reversed-phase high-performance liquid chromatography (HPLC) and a water/acetonitrile gradient containing 0.1% trifluoroacetic acid. Employing this system, 36 of the 45 complex I subunits elute in 28 distinct chromatographic peaks. The 9 subunits that do not elute are B14.7, MLRQ, and the 7 mitochondrial-encoded subunits. The method, with ultraviolet (UV) detection, is suitable for either analytical (<50 μg protein) or preparative (>250 μg protein) applications. Subunits eluting in each chromatographic peak were initially determined by matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) with subsequent positive identification by reversed-phase HPLC-electrospray ionization (ESI)/tandem mass spectrometry (MS/MS) analysis of tryptic digests. In the latter case, subunits were identified with a 99% probability using Mascot for database searching and Scaffold for assessment of protein identification probabilities. The reversed-phase HPLC subunit analysis method represents a major improvement over previous separation methods with respect to resolution, simplicity, and ease of application.     

Purification and Amino Acid Sequence of MP-III 4R D49 Phospholipase A2 from Bothrops pirajai Snake Venom, a Toxin with Moderate PLA2 and Anticoagulant Activities and High Myotoxic Activity

MP-III 4R PLA₂ was purified from the venom of Bothrops pirajai venom (Bahia's jararacussu) after three chromatographic steps which started with RP-HPLC. The complete amino acid sequence of MP-III 4R PLA₂ from Bothrops pirajai was determined by amino acid sequencing of reduced and carboxymethylated MP-III 4R and the isolated peptides from clostripain and protease V8 digestion. MP-III 4R is a D49 PLA₂ with 121 amino acid residues and has a molecular weight estimated at 13,800 Da, with 14 half-cysteines. This protein showed moderate PLA₂ and anticoagulant activity. This PLA₂ does not have a high degree of homology with other bothropic PLA₂-like myotoxins (~75%) and nonbothropic myotoxins (~60%). MP-III 4R is a new PLA₂, which was isolated using exclusively analytical and preparative HPLC methods. Based on the N-terminal sequence and biological activities, MP-III 4R was identified as similar to piratoxin-III (PrTX-III), which was isolated by conventional chromatography based on molecular exclusion ion exchange chromatography. Clinical manifestations indicate that at the site of toxin injection, there may be pain of variable intensity, because animals continue to lick the limb. No clinical sign indicating general toxicity was noticed. Myotoxicity was observed in gastrocnemius muscle cells after exposure to MP-III 4R, with a high frequency (70%) of affected muscle fibers.     

Analysis of all protein amino acids as their tert.-butyldimethylsilyl derivatives by gas-liquid chromatography

A gas chromatographic method for the separation and quantitation of the 20 protein amino acids is described using N-methyl-N(tert.-butyldimethylsilyl)trifluoroacetamide, with 1% tert.-butyldimethylchlorosilane as catalyst, to prepare the tert.-butyldimethylsilyl amino acid derivatives. Alkylsilylation of amino acids proceeds at 140 degrees C in 20 min. The derivatives formed in the one-step reaction are used directly for gas-liquid chromatographic analysis, using a flame-ionization detector, without prior isolation or purification. Complete separation and quantitation of all protein amino acids are readily achieved using a 15-m DB-5 capillary column. Strict linearity extends from less than 15 to about 100 ng for all amino acids except Arg, which has a linear range from 50 to 300 ng. The limits of detection, however, range from one to several hundred nanograms. The method was used to analyze the free amino acid pool in carnation petals.     

High performance analysis of the cyanobacterial metabolism via liquid chromatography coupled to a LTQ-Orbitrap mass spectrometer: evidence that glucose reprograms the whole carbon metabolism and triggers oxidative stress

Cyanobacteria are environmentally important photosynthetic microorganisms attracting a growing attention in various areas of basic and applied researches. To better understand their metabolism, we presently report on the development of a robust and simple protocol for facile extraction and high throughput analysis of the metabolites of the widely-used strain Synechocystis PCC6803 through liquid chromatography coupled to high resolution mass spectrometry (LC/MS). Our analytical method was developed and tested with 102 reference compounds representative of the chemical diversity of polar cell metabolites, and Synechocystis cell extracts spiked with 37 reference compounds. These samples were analyzed with two chromatographic systems, each coupled to a LTQ-Orbitrap mass spectrometer: a liquid chromatographic system equipped with a pentafluorophenylpropyl column (the PFPP-LC/MS system), and an ultra-high performance liquid chromatographic system with a C₁₈-reversed phase column (the C₁₈-UHPLC/MS system). We showed that the PFPP-LC/MS method performs better than the C₁₈-UHPLC/MS method in terms of retention, separation and detection of metabolites. Consequently, we applied the PFPP-LC/MS method to analyze the metabolome of Synechocystis growing under various conditions of light and glucose, which strongly influence cell growth. We found that glucose increases glucose storage (synthesis of glycogen-like polysaccharide) and catabolism (oxidative pentose phosphate pathway and glycolysis), while it decreases the Calvin–Benson cycle that consumes photosynthetic electrons for CO₂ assimilation. Depending on light and glucose availabilities, this global metabolic reprogramming can generate an oxidative stress, likely through the recombination of the glucose-spared electrons with the photosynthetic oxygen thereby producing toxic reactive oxygen species.     

Detection of microgram quantities of carrier ampholytes in electrofocused proteins by thin-layer chromatography

A rapid and sensitive method for the detection of carrier ampholyte contamination in electrofocused proteins is described. Samples containing proteins and carrier ampholytes were applied to cellulose thin-layer chromatographic sheets and developed in 10% trichloroacetic acid. Proteins and large-molecular-weight carrier ampholytes were precipitated at the origin while 10% trichloroacetic acid-soluble carrier ampholytes migrated as a diffuse ninhydrin (nitrogen)-positive area at an R_f greater than 0.50. We found that 1.25 μg of carrier ampholytes contained enough 10% trichloroacetic acid-soluble components to be detected by thinlayer chromatography. Using this assay, we investigated techniques designed to remove carrier ampholytes from an electrofocused protein. Removal of large-molecular-weight components from carrier ampholytes by dialysis through a 3500 M_r cutoff membrane did not facilitate separation of carrier ampholytes from streptococcal pyrogenic exotoxin type C by dialysis or gel chromatography. Also, this protein binds irreversibly to mixed-bed ion-exchange resin. The best method for separating carrier ampholytes from streptococcal pyrogenic exotoxin type C was by electrodialysis at pH 4.0. Following electrodialysis, estimated carrier ampholyte contamination in this protein was less than 1 part in 500 parts (by weight).     

Rapid microdetermination of fatty acids in biological materials by gas-liquid chromatography

Total and free fatty acids in general ranging from lauric to nervonic acid were separated and quantitated based on an internal standard method as methyl esters by “on column” methylation with trimethyl-(α,α,α-trifluoro-m-tolyl) ammonium hydroxide (TMTFTH) in a gas chromatographic system. This study represents an application of a method published by MacGee and Allen and a change to an internal standard technique. For the determination of the total fatty acids the sampls were saponified with KOH-CH₃OH, acidified with H₂PO₄, and then the fatty acids were extracted into hexane. An aliquot of the hexane extract was then extracted with TMTFTH and chromatographed. For determination of free fatty acids the sample was acidified with H₃PO₄, immediately extracted with hexane and processed as described earlier. The relative standard deviation of 1.4 to 4.2% illustrates the precision of the method and the recovery of the fatty acids ranged from 88.5 to 100.5%. This method was applied to the determination of fecal fatty acids in conjunction with an interdepartmental study on “High protein diet in colon cancer” at the University of Missouri. In addition, the applicability of the analytical procedure (with small modifications) was shown for a wide variety of biological materials (serum, milk, skin tissue, fungal spores, food homogenates, beef tissues, and tumor cell cultures). The analyses were performed on different gas chromatographs by different analysts.     

Separation of lactobacilli bacteriocins from fermented broths using membranes

Current separation, isolation and purification techniques to obtain highly potent purified lactobacilli and lactococci bacteriocins include chemical precipitation, separation employing solvents and chromatographic techniques. These methods are arduous, costly, with limited scalability, offering low bacteriocin yields (<20%). To address these challenges, the alternatives of ultrafiltration and nanofiltration, as separation methods were tested. Three promising bacteriocin producing strains, Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB 8014 and Lactococcus lactis NCIMB 8586 were selected to investigate the applicability and feasibility of the method.To facilitate separation, the microorganisms were grown on specially developed low molecular weight medium (LMWM) mainly containing nutritive sources up to 4 kDa molecular weight. Bacterial cells were removed by centrifugation. The clarified broths were filtered using 4 and 1 kDa MWCO. Bacteriocin activity was determined by an antimicrobial activity test using nisin, which has an inhibitory effect on the growth of susceptible microorganisms. Recovery yields using filtration were found to range between 53 and 68%, a high recovery performance.The bacteriocin activity of crude extracts of all the three lactobacilli were between 95 and 105 IU ml^?1. When the substances were separated using ultrafiltration membrane (4 kDa MWCO) their activity was enhanced to 145–150 IU ml^?1, achieving a total potency yield of 44–53%. Further enhancement of yields up to 36% was attained employing nanofiltration (1 kDa MWCO) membranes with an activity increased up to 200 IU ml^?1.Bacteriocin isolation from crude extracts using filtration was found to be effective, offering high recovery yields, optimising their activity as well as presenting a realistic option towards the formulation of these as commercially available antibacterial agents.