Fluorometric determination of N-terminal prolyl dipeptides, proline and hydroxyproline in human serum by pre-column high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride

A highly sensitive HPLC method for the determination of prolyl dipeptides, Pro and Hyp in serum was developed. After deproteinization of serum and pretreatment with o-phthalaldehyde, the analytes were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70°C for 10 min. The fluorescent derivatives of prolyl dipeptides, Pro and Hyp, were separated on tandem reversed-phase columns by a gradient elution at 55°C and detected by fluorescence measured at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides were 2–5 fmol/injection (S/N=3). Pro–Hyp, Pro–Gly and Pro–Pro were identified as serum prolyl dipeptides. The within-day and between-day relative standard deviations were 1.5–7.9 and 2.4–10.8%, respectively. The recoveries were in the range of 90.8–97.3%. The concentrations of Pro–Hyp, Pro–Gly, Pro–Pro, Pro and Hyp in normal human serum (n=10) were 0.64±0.35, 0.078±0.047, 0.022±0.016, 177.0±43.0 and 11.1±3.5 μM, respectively. The concentrations of Pro–Hyp and Pro–Pro in serum of a patient with bone metastases of prostatic cancer were about three times and 50 times, respectively, higher than those in normal human serum.     

Highly sensitive determination of N-terminal prolyl dipeptides, proline and hydroxyproline in urine by high-performance liquid chromatography using a new fluorescent labelling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride

A highly sensitive pre-column HPLC method for simultaneous determination of prolyl dipeptides, Pro and Hyp in urine was developed. The analytes were labelled with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70°C for 20 min. The derivatives separated on tandem reversed-phase columns by a gradient elution and were monitored with fluorescence detection at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides, Pro and Hyp were 1–5 fmol/injection (S/N=3). Urine samples were treated with o-phthalaldehyde, followed by purification on a Bond Elut C₁₈ column before conducting the labelling reaction. Pro–Hyp, Pro–Gly and Pro–Pro were identified as prolyl dipeptides in urine. The within-day and between-day relative standard deviations were 1.5–4.8 and 1.7–5.8%, respectively. The concentrations of Pro–Hyp, Pro–Gly, Pro–Pro, Pro and Hyp in normal human urine were 97.6±28.2, 2.74±1.48, 2.08±1.13, 6.71±3.34 and 2.30±1.59 nmol/mg creatinine, respectively.     

Age determination based on amino acid racemization: A new possibility

Summary A method has been developed to determine the age of fossil bone samples based on amino acid racemization (AAR). Approximately one hundred fossil bone samples of known age from Hungary were collected and analysed for D- and L-amino acids. As the racemization of amino acids is affected by temperature, pH, metal content of the soil, and time passed since death, these factors were eliminated by comparing the estimated age to age determined by the radiocarbon method. Determining the D- and L-amino acid contents in samples of known age, determining the half life of racemization and plotting the D/L ratio as a function of time, calibration curves were obtained. These curves can be used for the age estimation of samples after determining their D- and L-amino acid content. The D/L ratio for 2 to 3 amino acids was determined for each sample and the mean value of estimated ages based on calibration curves was considered to estimate age of the fossil samples.     

The use of a new silica gel separation system in thin-layer chromatography

Summary A new method for the chromatography of amino acids is described in which D- or L-amino acids are separated on ICT-Empore thin-layers. The compounds are developed ascending by means of normally used solvent systems. An overloading of the plates is nearly impossible. On the other hand, hydrophilic amino acids are well separated. A second front, moving with these amino acids and emerging with ninhydrin stain, was not detectable.     

Chirally selective,intramolecular interaction observed in an aminoacyl adenylate anhydride

All earthly creatures use only L-amino acids in template directed protein synthesis. The reason for this exclusive use of the L-isomer is not yet apparent, although recent experiments by Usher and his colleagues have shown some stereoselctivity in the aminoacylation of di- and polynucleotides [1–3]. We have separately reported on intramolecular interactions between hydrophobic amino acid side chains and the adenine ring in aminoacyl adenylates [4]. There was a preferential association of Phe > Leu = Ile > Val with the adenine in these studies, but we made no attempts to address the question of D, L selectivity. Recently, in¹H NMR studies of N-acetylphenylalanyl adenylate anhydride, we noticed evidence that both D- and L-isomers of the amino acid were present and, furthermore, that one isomer seemed to be associating with the adenine ring more strongly than the other. Using HPLC, we have separated the two diastereoisomers and have enzymatically determined that the isomer which associates more strongly is the biologically important one, the L-isomer. We present those studies here and discuss the evolutionary significance of this finding.     

Total hydrolysis of proteins with strongly reduced racemization of amino acids

A new method has been devised for the complete hydrolysis of proteins with an extremely low level of racemization of amino acids. Proteins are incubated in 10 M HCl at a low temperature to obtain partial hydrolysis. They are then incubated with pronase and finally with leucine aminopeptidase and peptidyl-D-amino-acid hydrolase from Loligo vulgaris. The proposed method ensures the total hydrolysis of either purified proteins or proteins contained in a crude homogenate of animal or vegetable tissue. In both cases, the racemization of amino acids (expressed as rate of D form/D + L form X 100) was lower than 0.015% for aspartic acid and lower than 0.01% for other amino acids. D-Amino acids released from peptides or proteins were estimated with enzymatic methods based on the use of octopus D-aspartate oxidase or hog kidney D-amino acid oxidase; with these enzymes, 0.05 nmol of a D-amino acid was determined in the presence of up to 20 mumols of a mixture of L-amino acids (ratio %D/D + L = 0.00025). The method allows the determination of D-amino acids either in tissues in which they are present in high concentrations (as human cataract lenses, tooth enamel, etc.) or in those with low enantiomer content (as brain, erythrocytes, etc.). Using the method described, we hydrolyzed several synthetic peptides consisting of D- and L-amino acids and determined the amount of D-amino acids. In addition, we totally hydrolyzed all the nuclear proteins of human cataractous lenses. The amount of D-aspartic acid was 0.026 mumols/mg in lenses of women aged between 71 and 76 years and 0.0256 mumols/mg in lenses of men aged between 55 and 72 years. The D-aspartic acid measured corresponds to about 12% with respect to total aspartic acid.     

Determination of amino acids in human serum by capillary gas chromatography

A simple and rapid method for the determination of serum amino acids by gas chromatography (GC) has been developed. Following deproteinization of serum with perchloric acid, free amino acids in the supernatant were converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with flame ionization detection using a DB-17 capillary column. All the derivatives of the 22 protein amino acids were completely resolved as single peaks within 9 min by GC. The calibration curves were linear in the range 0.2–50 μg of each amino acid, and the correlation coefficients were above 0.998. By using this method, serum amino acids could be directly analysed without prior clean-up procedure such as ion-exchange column chromatography except for deproteinization of the samples, and without any interference from coexisting substances. Overall recoveries of amino acids added to serum samples were 88–108%. Analytical results for serum amino acids from normal subjects are presented.     

Determination of erythrocyte amino acids by gas chromatography

Erythrocyte amino acid levels were determined, by gas chromatography, in a group of 34 normal human adults. No significant sex or age correlations were noted.A method for the quantitative gas chromatographic analysis of free amino acids in erythrocytes is described. Following hemolysis and deproteinization the amino acids were isolated on a cation-exchange resin. Glutathione was removed from the amino acid mixture by adsorption on an anion-exchange resin. Following conversion to their N-acetyl-n-propyl esters, 19 amino acids were separated and quantitated by gas chromatography on a single column in 18 min. Typical reproducibility data indicate that a coefficient of variation of 2–5% is attainable.     

New enzymatic method of chiral amino acid synthesis by dynamic kinetic resolution of amino acid amides: use of stereoselective amino acid amidases in the presence of alpha-amino-epsilon-caprolactam racemase

D- and L-amino acids were produced from L- and D-amino acid amides by D-aminopeptidase from Ochrobactrum anthropi C1-38 and L-amino acid amidase from Pseudomonas azotoformans IAM 1603, respectively, in the presence of alpha-amino-epsilon-caprolactam racemase from Achromobacter obae as the catalyst by dynamic kinetic resolution of amino acid amides.     

Equilibrium of the Cis-Trans Isomerisation of the Peptide Bond with N-alkyl Amino Acids Measured by 2D NMR

The conformational cis-trans equilibrium around the peptide bond in model tripeptides has been determined by 2D NMR methods (HOHAHA, ROESY). The study was limited to three different N-substituted amino acids in position 2, namely Pro (proline), Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), and N-MePhe (N-methylphenylalanine). In all cases the amino acid in position 1 was tyrosine and in position 3, phenylalanine. The results of our studies show that the cis-trans ratio depends mostly on the configuration of the amino acids forming the peptide bond undergoing the cis-trans isomerisation. The amino acid following the sequence (in position 3) does not have much influence on the cis-trans isomerisation, indicating that there is no interaction of the side chains between these amino acids. The model peptides with the L-Tyr-L-AA-(L- or D-)Phe (where AA is N-substituted amino acid) chiralities give 80–100% more of the cis form in comparison to the corresponding peptides with the D-Tyr-L-AA-(L-or D-)Phe chiralities. These results indicate that the incorporation of N-substituted amino acids in small peptides with the same chirality as the precedent amino acid involved in the peptide bond undergoing the cis/trans isomerisation moves the equilibrium to a significant amount of the cis form.     

A new type of aminoacyltransferase from Saccharothrix sp. AS-2 favorable for the synthesis of D-amino acid-containing peptides

A unique enzyme with some properties favorable for the synthesis of D-amino acid-containing peptides has been purified from the culture broth of Saccharothrix sp. AS-2. The purification steps included ammonium sulfate fractionation, chromatographies on CM-Toyopearl 650M and ProtEx Butyl, and sucrose density-gradient isoelectric focusing. The enzyme, consisting of four subunits of 56 kDa, showed its maximum transfer activity at around pH 8.2 and 35 degrees C, and had an isoelectric point of 5.8. The enzyme yielded homooligomers from methyl esters of D-Asp(OMe), D-Met, D-Phe, D-Trp, D-Tyr, and L-Glu(OMe), but showed no hydrolytic activity toward any of the D- or L-amino acid methyl esters tested. The homooligomers were not formed from the corresponding free amino acids. The reaction of Ac-D-Phe-OMe with DL-Ala-NH(2), DL-Leu-NH(2), DL-Phe-NH(2), or DL-Trp-NH(2) was effectively catalyzed by the enzyme, both the DD- and DL-stereoisomers of the expected N-acetyldipeptide being yielded. The resulting dipeptides remained unhydrolyzed even after 48 h incubation. Also, it showed no detectable hydrolytic activity toward casein, diastereomers of diAla, diMet, and diPhe, D-/L-amino acid amides, or D-/L-amino acid p-nitroanilides, indicating that the enzyme had no peptidase activity leading to secondary hydrolysis of the growing peptide. The enzyme activity was strongly depressed by phenylmethanesulfonyl fluoride, but not by penicillin G or ampicillin, suggesting that the protein is a serine enzyme lacking penicillin-binding ability. These observations lead us to the conclusion that the enzyme from Saccharothrix sp. AS-2 characterized in this study is a new type of aminoacyltransferase with an amino acid ester as the acyl donor, and has potential utility as a catalyst for the synthesis of D-amino acid-containing peptides.     

Utilization of tyrosine- and histidine-containing dipeptides to enhance productivity and culture viability

Adequate supply of nutrients, especially providing a sufficient level of specific amino acids, is essential for cell survival and production. Complex raw materials such as soy hydrolysates or yeast extracts are the source for both free amino acids and peptides. However, typical chemically defined (CD) media provide amino acids only in free form. While most amino acids are highly soluble in media and can be provided at fairly high concentrations, certain amino acids such as tyrosine have poor solubility and thus, only a limited amount can be added as a media component. The limited solubility of amino acids in media can raise the risk of media precipitation and instability, and could contribute to suboptimal culture performance due to insufficient nutrient levels to meet cellular demands. In this study, we examine the use of chemically synthesized dipeptides as an alternative method for delivering amino acids to various monoclonal antibody producing cell lines. In particular, we focus on tyrosine-containing dipeptides. Due to their substantially higher solubility (up to 250-fold as compared with free tyrosine), tyrosine-containing dipeptides can efficiently provide large amounts of tyrosine to cultured cells. When tested in fed-batch processes, these supplemental dipeptides exerted positive effects, including enhanced culture viability and titer. Moreover, dipeptide-supplemented cultures displayed improved metabolic profiles including lower lactate and NH 4(+) production, and better pH maintenance. In bioreactor studies using two-sided pH control, a lactate spike occurring on Day 10 and the concomitant high levels of base addition could be prevented with dipeptide supplementation. These beneficial effects could be obtained by one-time addition of dipeptides during inoculation, and did not require further feeds during the entire 11-15-day process. Non-tyrosine-containing dipeptides, such as His-Gly, also showed improved productivity and viability over control cultures.     

Transport of the aromatic amino acids into isolated rat liver cells. Properties of uptake by two distinct systems.

The transport of the aromatic amino acids into isolated rat liver cells was studied. There was a rapid and substantial binding of the aromatic amino acids, L-alanine and L-leucine to the plasma membrane. This has important consequences for the determination of rates of transport and intracellular concentrations of the amino acids. Inhibition studies with a variety of substrates of various transport systems gave results consistent with aromatic amino acid transport being catalysed by two systems: a 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH)-insensitive aromatic D- and L-amino acid-specific system, and the L-type system (BCH-sensitive). The BCH-insensitive component of transport was Na+-independent and facilitated non-concentrative transport of the aromatic amino acids; it was unaffected by culture of liver cells for 24 h, by 48 h starvation, dexamethasone phosphate or glucagon. Kinetic properties of the BCH-inhibitable component were similar to those previously reported for the L2-system in liver cells. The BCH-insensitive component was a comparatively low-Km low-Vmax. transport system that we suggest is similar to the T-transport system previously seen only in human red blood cells. The results are discussed with reference to the importance of the T- and L-systems in the control of aromatic L-amino acid degradation in the liver.     

The complete amino acid sequences of four globins from the land leech Haemadipsa zeylanica var. japonica

The amino acid sequences of four globins from the land leech, Haemadipsa zeylanica var. japonica, were determined using nucleotide sequencing and protein sequencing. The mature globin-molecules were composed of 146 amino acid residues for M-1 globin, 156 for M-2 globin, 143 for D-1 globin, and 149 for D-2 globin. Alignment of the four kinds of globins by Clustal X revealed 22 invariant amino acids. The four globins were 26–33% identical. A striking feature of amino acid alteration was: the replacement of the E7 distal-His of D-1 globin by phenylalanine because histidine is conserved among the rest of the globins of H. zeylanica, those of other representative species (Lumbricus and Tylorrhynchus) of Annelida and most other hemoglobins. A phylogenetic tree constructed of 18 globin structures including two species of leeches, H. zeylanica (a land leech) and Macrobdella decora (a freshwater leech), T. heterochaetus (a representative species of polychaetes), L. terrestris (a representative species of oligochaetes), and human α and β globins strongly indicated that the leech globins first separated from globin lineage of annelids.     

Binding specificity of the periplasmic oligopeptide-binding protein from Escherichia coli.

The structural properties required for the binding of peptide substrates to the Escherichia coli periplasmic protein involved in oligopeptide transport were surveyed by measuring the ability of different peptides to compete for binding in an equilibrium dialysis assay with the tripeptide Ala-Phe-[3H]Gly. The protein specifically bound oligopeptides and failed to bind amino acids or dipeptides. Acetylation of the peptide amino terminus of (Ala)3 severely impaired binding, whereas esterification of the carboxyl terminus significantly reduced but did not completely eliminate binding. Peptides composed of L-amino acids competed more effectively than did peptides containing D-residues or glycine. Experiments with a series of alanyl peptide homologs demonstrated a decrease in competitive ability with increasing chain length beyond tripeptide. Competition studies with tripeptide homologs indicated that a wide variety of amino acyl side chains were tolerated by the periplasmic protein, but side-chain composition did affect binding. Fluorescence emission data suggested that this periplasmic protein possesses more than one substrate-binding site capable of distinguishing peptides on the basis of amino acyl side chains.     

Characterization of three aminopeptidases purified from maternal serum

The biochemical characteristics of aminopeptidase A (EC 3.4.11.7), oxytocinase (EC 3.4.11.3) and alanyl aminopeptidase (EC 3.4.11.2) purified from serum of pregnant women were compared. Aminopeptidase A hydrolysed only acidic amino acid derivatives, whereas oxytocinase and alanyl aminopeptidase had partially overlapping broad substrate specificities. Oxytocinase showed the highest Vmax value with LeuNA but the lowest Km value with ArgNA (Km 0.059 +/- 0.08 mmol/l). Alanyl aminopeptidase hydrolysed AlaNA most rapidly, but showed the highest affinity for LysNA (Km 0.054 +/- 0.006 mmol/l). The enzymes were sensitive to EDTA. Co2+, Ni2+ and Zn2+ were able to reactivate all suppressed enzymes, but Mn2+ reactivated only aminopeptidase A after EDTA inhibition. The alkaline earth metals were activators of aminopeptidase A, while Co2+ activated only alanyl aminopeptidase. This enzyme was the most sensitive to L-amino acids. Acidic amino acids inhibited aminopeptidase A but had no effect on the two other enzymes. Oxytocinase was most sensitive to thermal treatment. Amastatin did not inhibit oxytocinase, whereas aminopeptidase A was more resistant than alanyl aminopeptidase to this effector.     

An automatic analyzer for the specific determination of amino sugars

The techniques previously employed for the extraction and determination of amino acids from different matrices are not necessarily optimal for the determination of the amino sugars. An analytical system is described which is a hybrid between the conventional amino acid analyzer and the liquid chromatographic system for the detection of reducing sugars. The major, naturally occurring amino sugars are separated in about 40 min, with sensitivites lying under the nanomole range, without interference from other co-extracted compounds such as amino acids and sugars. The reagent employed is noncorrosive and stable over long periods of time. The amino sugar analyzer can be readily constructed by simple modification of a conventional phenylketonuria or amino acid analyzer.     

Chiral analysis of amino acids from conventional and transgenic yeasts

Autolysis of Saccharomyces cerevisiae yeast is the main source of molecules that contribute to the quality of sparkling wines made by the traditional method. In this work, a genetically modified yeast (LS11) is compared to its isogenic wild type strain (BY4741) after autolysis. Chiral micellar electrokinetic chromatography with laser-induced fluorescence detection (chiral-MEKC-LIF) is used to identify and quantify the main D- and L-amino acids from both strains after accelerated autolysis. The procedure includes amino acids extraction, derivatization with FITC and chiral-MEKC-LIF separation in a background electrolyte composed of 100 mM sodium tetraborate, 30 mM SDS, 20 mM beta-CD at pH 10.0. The D- and L-forms of Arg, Asn, Ala, Glu and Asp, corresponding to the major amino acids found in these samples, are separated in less than 30 min with efficiencies up to 800,000 plates/m and high sensitivity (i.e., LODs as low as 40 nM were obtained for D-Arg for a signal to noise ratio of three). From these results it is corroborated that the genetic modification brings a faster autolysis of the yeast, releasing a higher amount of L-amino acids to the medium in a short time. Interestingly, the pattern of release of D-amino acids was also different between the transgenic and the conventional yeast strains.     

Synthesis of optically active amino acids from alpha-keto acids with Escherichia coli cells expressing heterologous genes.

We describe a simple method for enzymatic synthesis of L and D amino acids from alpha-keto acids with Escherichia coli cells which express heterologous genes. L-amino acids were produced with thermostable L-amino acid dehydrogenase and formate dehydrogenase (FDH) from alpha-keto acids and ammonium formate with only an intracellular pool of NAD+ for the regeneration of NADH. We constructed plasmids containing, in addition to the FDH gene, the genes for amino acid dehydrogenases, including i.e., leucine dehydrogenase, alanine dehydrogenase, and phenylalanine dehydrogenase. L-Leucine, L-valine, L-norvaline, L-methionine, L-phenylalanine, and L-tyrosine were synthesized with the recombinant E. coli cells with high chemical yields (> 80%) and high optical yields (up to 100% enantiomeric excess). Stereospecific conversion of various alpha-keto acids to D amino acids was also examined with recombinant E. coli cells containing a plasmid coding for the four heterologous genes of the thermostable enzymes D-amino acid aminotransferase, alanine racemase, L-alanine dehydrogenase, and FDH. Optically pure D enantiomers of glutamate and leucine were obtained.     

Purification and characterization of a novel imidazole dipeptide synthase from the muscle of the Japanese eel Anguilla japonica

We have purified a novel enzyme from eel white muscle which catalyzes the syntheses of imidazole dipeptides, such as carnosine (β-alanyl-l-histidine), anserine (β-alanyl-π-methyl-l-histidine), and balenine (ophidine; β-alanyl-τ-methyl-l-histidine), directly from their precursors. The enzyme was purified 1130-fold from eel muscle by a series of column chromatographies. Although eel muscle contains a large amount of carnosine and only trace amounts of anserine and balenine, the anserine synthesizing activity was by far the highest. From gel permeation chromatography, the molecular mass of the enzyme was calculated to be 275 kDa. SDS-PAGE of the purified enzyme represented a band around 43 kDa, suggesting that the native enzyme is a hexamer or heptamer. The optimal pH and temperature were around 9.5 and 60 °C, respectively. K_m values for β-alanine and π-methyl-l-histidine were 44 and 89 mM, respectively. The enzyme was greatly activated by Zn²⁺ and inhibited by EDTA. The N-terminal amino acid sequence of 25 residues of the purified enzyme showed 52% amino acid identity to 38–62 residues of zebrafish haptoglobin precursor. The purified enzyme also exhibited hydrolytic activity against these imidazole dipeptides.