Quantitative Amino Acid Analysis Research Articles

Structural Basis of Cellulosome Efficiency Explored by Small Angle X-ray Scattering

Cellulose, the main structural component of plant cell walls, is the most abundant carbohydrate polymer in nature. To break down plant cell walls, anaerobic microorganisms have evolved a large extracellular enzyme complex termed cellulosome. This megadalton catalytic machinery organizes an enzymatic assembly, tenaciously bound to a scaffolding protein via specialized intermodular "cohesin-dockerin" interactions that serve to enhance synergistic activity among the different catalytic subunits. Here, we report the solution structure properties of cellulosome-like assemblies analyzed by small angle x-ray scattering and molecular dynamics. The atomic models, generated by our strategy for the free chimeric scaffoldin and for binary and ternary complexes, reveal the existence of various conformations due to intrinsic structural flexibility with no, or only coincidental, inter-cohesin interactions. These results provide primary evidence concerning the mechanisms by which these protein assemblies attain their remarkable synergy. The data suggest that the motional freedom of the scaffoldin allows precise positioning of the complexed enzymes according to the topography of the substrate, whereas short-scale motions permitted by residual flexibility of the enzyme linkers allow "fine-tuning" of individual catalytic domains.

The Conformationally Dynamic C Helix of the RIα Subunit of Protein Kinase A Mediates Isoform-specific Domain Reorganization upon C Subunit Binding

Different isoforms of the full-length protein kinase A (PKA) regulatory subunit homodimer (R2) and the catalytic (C) subunit-bound holoenzyme (R2C2) have very different global structures despite similar molecular weights and domain organization within their primary sequences. To date, it has been the linker sequence between the R subunit dimerization/docking domain and cAMP-binding domain A that has been implicated in modulating domain interactions to give rise to these differences in global structure. The small angle solution scattering data presented here for three different isoforms of PKA heterodimer (deltaR-C) complexes reveal a role for another conformationally dynamic sequence in modulating inter-subunit and domain interactions, the C helix that connects the cAMP-binding domains A and B of the R subunit. The deltaR-C heterodimer complexes studied here were each formed with a monomeric N-terminal deletion mutant of the R subunit (deltaR) that contains the inhibitor sequence and both cAMP-binding domains. The scattering data show that type IIalpha and type IIbeta deltaR-C heterodimers are relatively compact and globular, with the C subunit contacting the inhibitor sequence and both cAMP-binding domains. In contrast, the type Ialpha heterodimer is significantly more extended, with the C subunit interacting with the inhibitor sequence and cAMP-binding domain A, whereas domain B extends out such that its surface is almost completely solvent exposed. These data implicate the C helix of RIalpha in modulating isoform-specific interdomain communication in the PKA holoenzyme, adding another layer of structural complexity to our understanding of signaling dynamics in this multisubunit, multidomain protein kinase.

Analysis of Acylcarnitine Ester for Neonatal Screening of Inborn Errors of Metabolism Using Tandem Mass-spectrometry

Tandem mass-spectrometry has been introduced worldwide into neonatal screening programs for the quantitative analysis of acylcarnitine species and amino acids for the diagnosis of organic acidurias, defects of fatty acid oxidation, and amino acidopathies, respectively. Since April 2002 more than 200000 newborn infants have been screened in Austria using tandem mass-spectrometry. In this cohort 37 infants with amino acidopathies and 38 infants with fatty acid oxidation defects or organic acidurias have been diagnosed. The overall incidence of these disorders is 0.036%. The analysis of acylcarnitine species using tandem mass-spectrometry has enabled the diagnosis of infants with inborn errors of metabolism prior to clinical presentation and to initiate therapy early enough to prevent long-term sequelae and to reduce mortality in these disorders.

Biosensors for Protein Detection: A Review

There is considerable demand for the rapid low‐cost determination of proteins, particularly in the food and beverage industry. The most widely used tests are based on colorimetric procedures in which proteins react to produce colored complexes. These methods (Lowry et al. 1951; Bradford 1976; Gornall et al. 1949; Smith et al. 1985) are dependent upon a number of factors other than absolute protein quantity, including amino acid composition, protein purity, and association state. The successful application of these methods relies on using representative calibration standards. Time‐consuming and complex methodologies such as the Kjeldahl technique and quantitative amino acid analysis procedures have been also reported. Apart from these methods, biosensors are interesting tools offering certain operational advantages over standard photometric methods, notably with respect to rapidity, ease‐of‐use, cost, simplicity, portability, and ease of mass manufacture. By appropriate recognition element selection, it is possible to detect either a particular target protein or a broad range of proteins. This review presents an overview of the different biological recognition elements and the various transduction systems that have been reported in the literature to design biosensors for protein detection. Examples are given to illustrate the different possibilities. It must be noticed that this review reports detection of proteins in solution and not proteins immobilized on membranes. Briefly, immobilized proteins can be detected by antibodies (Western blotting) or oligonucleotides such as aptamers (“Eastern blotting”) bearing reporter molecules, fluorescently labeled or radiolabeled.

Development of a new in vitro model of elastic fiber assembly in human pigmented epithelial cells

Objectives: We developed an in vitro model of elastic fiber assembly that provides a comparison of the efficiency of different tropoelastin molecules to organize into fibers. Design and methods: Recombinant tropoelastin was added to ARPE-19 cell culture medium. The elastic fiber assembly was evaluated by immunofluorescence staining, the quantitative analysis of cross-linking amino acids, and semi-quantitative analysis of matrix-associated tropoelastin. Results: We confirmed that ARPE-19 cells express fibrillin-containing microfibrils and lysyl oxidase, but they do not express tropoelastin. Immunofluorescence staining showed a dose- and time-dependent increase in the extracellular matrix. The quantity of cross-linking amino acids and matrix-associated tropoelastin also increased together with the matrix-associated elastin. Moreover, the analysis of a radioimmunoprecipitation assay (RIPA) buffer-soluble fraction indicated that tropoelastin interacted with microfibrils and cross-linked elastin was detected as a super molecular complex. Conclusion: These observations indicate that this in vitro model is especially useful for the analysis of mechanisms of elastic fiber formation.

Mutational Analysis of the Stator Subunit E of the Yeast V-ATPase

Subunit E is a component of the peripheral stalk(s) that couples membrane and peripheral subunits of the V-ATPase complex. In order to elucidate the function of subunit E, site-directed mutations were performed at the amino terminus and carboxyl terminus. Except for S78A and D233A/T202A, which exhibited V(1)V(o) assembly defects, the function of subunit E was resistant to mutations. Most mutations complemented the growth phenotype of vma4Delta mutants, including T6A and D233A, which only had 25% of the wild-type ATPase activity. Residues Ser-78 and Thr-202 were essential for V(1)V(o) assembly and function. The mutation S78A destabilized subunit E and prevented assembly of V(1) subunits at the membranes. Mutant T202A membranes exhibited 2-fold increased V(max) and about 2-fold less of V(1)V(o) assembly; the mutation increased the specific activity of V(1)V(o) by enhancing the k(cat) of the enzyme 4-fold. Reduced levels of V(1)V(o) and V(o) complexes at T202A membranes suggest that the balance between V(1)V(o) and V(o) was not perturbed; instead, cells adjusted the amount of assembled V-ATPase complexes in order to compensate for the enhanced activity. These results indicated communication between subunit E and the catalytic sites at the A(3)B(3) hexamer and suggest potential regulatory roles for the carboxyl end of subunit E. At the carboxyl end, alanine substitution of Asp-233 significantly reduced ATP hydrolysis, although the truncation 229-233Delta and the point mutation K230A did not affect assembly and activity. The implication of these results for the topology and functions of subunit E within the V-ATPase complex are discussed.

Comparison of assays for determination of peptide content for lyophilized thymalfasin*

Precise determination of the peptide content in drug substance samples depends highly upon the particular peptide compound and methodology used. Four independent methods were evaluated and compared to determine which would produce the best experimental precision for analysis of thymalfasin (thymosin alpha-1). Four different methods were evaluated including elemental analysis (CHN), quantitative amino acid analysis (AAA), high-performance liquid chromatography (HPLC), and Kjeldahl. This study demonstrates that the AAA method is highly variable in one laboratory while quite precise in another laboratory. Similarly, HPLC results depended on the laboratory conducting the study with more precise values obtained under cGMP. On the contrary, the CHN method yielded highly precise [i.e. <2% coefficient of variation (CV)] values. As precise knowledge of protein content is fundamental for the compounding of final pharmaceutical product of a specific potency, the CHN analysis is recommended for peptide content determination of the drug substance thymalfasin.

Heme Binding to the Histidine-Rich Protein II from Plasmodium falciparum

The histidine-rich protein II (HRP II) from Plasmodium falciparum has been implicated in the formation of hemozoin, a detoxified, crystalline form of ferric protoporphyrin IX (Fe(3+)-PPIX) produced by the parasite. Fe(3+)-PPIX titrations coupled with quantitative amino acid analysis showed that HRP II binds 15 Fe(3+)-PPIX molecules per 30 kDa monomer. Circular dichroism spectroscopy was used to probe the secondary structure of HRP II with and without bound Fe(3+)-PPIX. These studies have revealed large changes in the secondary structure with Fe(3+)-PPIX binding, changing from a random coil in the absence of Fe(3+)-PPIX to a more ordered helical structure in the presence of Fe(3+)-PPIX. The Fe(3+)-PPIX-bound HRP II structure most closely resembles a 3(10)-helix. Coincident with this structural change caused by Fe(3+)-PPIX binding, the formation of an intermolecular disulfide bond occurs between HRP II monomers. In vitro pull-down assays show an interaction between monomers that is dependent on the presence of Fe(3+)-PPIX. One model that best fits with the data reported here requires formation of 15 intermolecular bishistidyl ligated Fe(3+)-PPIX molecules arranged in a head to head fashion, which would then allow for the formation of an intermolecular disulfide bond. The structure best able to accommodate these requirements is a 3(10)-helix.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based amino acid analysis

Amino acid analysis has been an integral part of analytical biochemistry for more than 50 years. However, its experimental design, which includes derivatization of amino acids followed by some kind of chromatographic separation, has not changed over the years. We have developed a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-based method for the quantitative analysis of amino acids. This method does not require any amino acid modification, derivatization, or chromatographic separation. The data acquisition time is decreased to several seconds for a single sample. No significant ion suppression effects were observed with the developed sample deposition technique, and the method was found to be reproducible. Linear responses between the amino acid concentration and the peak intensities ratio of corresponding amino acid to internal standard were observed for all amino acids analyzed in the range of concentrations from 20 to 300μM, and correlation coefficients were between 0.983 (for arginine) and 0.999 (for phenylalanine). Limits of quantitation were between 0.03μM (for arginine) and 3.7μM (for histidine and homocysteine). This method was applicable to the mixtures of free amino acids as well as to HCl hydrolysates of proteins. Furthermore, we have shown that this method can be applied to other biologically important low-molecular weight compounds such as glucose.

Metabolic Disorders Associated With Neonatal Hypoglycemia

After completing this article, readers should be able to: 1. Describe four sources of glucose available to the neonate in the first 24 postnatal hours. 2. Select the best routine diagnostic test to recognize fatty acid oxidation defects. 3. List three or more metabolic disorders that can be identified by newborn screening performed using tandem mass spectrometry. Hypoglycemia is a common problem in neonates that has many causes. This review focuses on metabolic disorders that may be associated with hypoglycemia in the neonatal period. During intrauterine life, the fetus derives fuel, as glucose, from the mother via the placenta. After birth, the energy demands on the former fetus increase dramatically. The baby now must maintain its own body temperature and must undertake the work of breathing and other activities. Further, the maintenance of blood glucose levels requires glycogenolysis and gluconeogenesis. Postnatally, there are four sources of glucose: dietary glucose; glucose derived from the cleavage of more complex sugars in the gut (eg, lactose to glucose and galactose); glucose released from glycogen stores (primarily in the liver); and gluconeogenesis, in which glucose is synthesized from carbon skeletons derived from certain amino acids using energy derived from catabolism of fatty acids. Most term infants have sufficient glycogen stores to maintain blood glucose levels for several hours before gluconeogenesis is required. Infants who are breastfed in the United States typically are offered only water as a supplement to human milk during the first few postnatal days, a period when the mother’s milk supply is not yet established. In contrast, formula-fed babies receive calories by mouth by the end of the first postnatal day. Consequently, the metabolic stress of prolonged fasting occurs more frequently in breastfed than in formula-fed babies. Although breastfed babies have higher levels of ketone bodies that appear to provide adequate energy during this …

Qualitative and quantitative analysis of amino acids by capillary electrophoresis-electrospray ionization-tandem mass spectrometry.

We describe a method to identify and quantify amino acids using capillary electrophoresis-electrospray ionization-triple-quadrupole tandem mass spectrometry (CE-ESI-MS/MS). Amino acids, including physiological amino acids, were first separated by CE under acidic pH conditions and then detected by MS/MS. To efficiently introduce the whole sample into the capillary, no electrical potential was applied to the electrospray probe until running electrophoresis. The position of the electrosprayer with respect to the MS capillary entrance drastically affected sensitivity and generation of cluster ions. MS/MS with multiple reaction monitoring (MRM) detection was performed to obtain sufficient selectivity and sensitivity. Under optimized CE-MS/MS conditions, the minimum detectable levels for 32 free amino acids normally found in proteins and other physiological amino acids were between 0.1 and 14 micromol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. For most amino acids, this constitutes a severalfold increase in sensitivity compared to CE-MS. The relative standard deviations (% RSD) for all amino acids were better than 0.4% for migration times and between 1.4% and 8.6% for peak areas (n = 10). Since amino acids exhibited characteristic MS/MS spectra, this approach is useful for the simultaneous, selective, quantitative, and reproducible analysis of amino acids in physiological and biological samples that contain various kinds of matrices. The power of the method was demonstrated by analyzing amino acids in human urine.

Planar Chromatography for Quantitative Analysis of Amino Acids in Fermentation Solutions of Industrial Producer Cultures

Proximate procedures for quantitative analysis of amino acids using high-performance thin-layer chromatography were developed.

Antifouling activity of brominated cyclopeptides from the marine sponge Geodia barretti.

In this work, we show the potent antifouling effects of two compounds, barettin (cyclo[(6-bromo-8-entryptophan)arginine]) (1), isolated as a Z/E mixture (87/13), and 8,9-dihydrobarettin (cyclo[(6-bromotryptophan)arginine]) (2), isolated from the marine sponge Geodia barretti. The compounds were isolated guided by their ability to inhibit the settlement of cyprid larvae of the barnacle Balanusimprovisus, and their structures were determined by means of mass spectrometry, NMR, and quantitative amino acid analysis. The activities of these brominated diketopiperazine-like cyclic dipeptides are in the range of antifouling agents in use today, as shown by their EC(50) values of 0.9 and 7.9 microM, respectively. However, contrary to today's antifouling agents, the effects of barettin and 8,9-dihydrobarettin are nontoxic and reversible. A small set of synthetic analogues, including l-arginine, l-tryptophan, 5-bromo-d,l-tryptophan, 6-bromo-d,l-tryptophan, and 6-fluoro-d,l-tryptophan, were tested for possible structure-activity relationships. None of these compounds showed any effect at a concentration of 10 microM. We hypothesize that the isolated compounds are part of the sponge's chemical defense to deter fouling organisms. This theory is supported by the fact that barettin is found in water exposed to living specimens of G. barretti in concentrations that completely inhibit barnacles from settling.

Quantitative and isotopic analysis of amino acids, allantoin, and allantoic acid in soybeans by LC-MS using the atmospheric pressure chemical ionization method

In this report, we determined whether liquid chromatography mass spectrometry (LC-MS) using the atmospheric pressure chemical ionization (APCI) method could be applied for isotopic analysis of the high polar compounds, allantoin, and allantoic acid, which are the major transport forms of N from soybean nodules. The peak areas of the ion chromatogram of authentic allantoin and allantoic acid obtained by selected ion monitoring (SIM) were proportional to the contents of the compounds. The data obtained from the ion intensity using the scan mode and the area of the ion chromatogram using the SIM mode were entirely consistent with the isotope ratio of the compounds. The allantoin and allantoic acid concentrations in the xylem sap of soybeans at various growth stages were measured by the following two methods using LC-MS; an absolute calibration method and an isotopelabeled internal standard method. The values estimated by the isotope-labeled internal standard method were more consistent with those obtained by the colorimetric method. Furthermore, amino acids could be detected under the same analytical conditions. The 15N ratio of asparagine and aspartic acid in the non-nodulating soybean xylem sap which had been supplied with 15N-labeled nitrate could be measured. These results indicate that LCMS (APCI) is a suitable method for isotopic analysis of high polar compounds, including allantoin and allantoic acid in plant xylem sap and tissues.

Revised Sections F7.5 (Quantitative Amino Acid Analysis) and F7.6 (Qualitative Amino Acid Analysis): American College of Medical Genetics Standards and Guidelines for Clinical Genetics Laboratories, 2003

Determination of plasma amino acid levels has become a key piece of information in the diagnosis and clinical management of a group of metabolic genetic disorders. Appropriate laboratory methodologies have been published for amino acid analysis, yet there is a need for direction for the laboratory in performing this testing. The following guidelines were generated by a working group of the American College of Medical Genetics Laboratory Quality Assurance Committee. Based upon a body of knowledge and professional experience, these guidelines and standards are to be the benchmark for performance of amino acid analysis for clinical interpretation.

Cytotoxic cyclotides from Viola tricolor.

A crude fraction of Viola tricolor rich in small lipophilic proteins was prepared and subjected to fractionation guided by bioactivity, using RP-HPLC and a fluorometric cytotoxicity assay. Two human cancer cell lines, U-937 GTB (lymphoma) and RPMI-8226/s (myeloma), were used in this study. The most potent compounds isolated, that is, the compounds showing the lowest IC(50) values, were shown to be three small proteins: vitri A (IC(50) = 0.6 microM and IC(50) = 1 microM, respectively), varv A (IC(50) = 6 microM and IC(50) = 3 microM, respectively), and varv E (IC(50) = 4 microM in both cell lines). Their sequences, determined by automated Edman degradation, quantitative amino acid analysis, and mass spectrometry, were cyclo-GESCVWIPCITSAIGCSCKSKVCYRNGIPC (vitri A), cyclo-GETCVGGTCNTPGCSCSWPVCTRNGLPVC (varv A), and cyclo-GETCVGGTCNTPGCSCSWPVCTRNGLPIC (varv E), of which vitri A is described for the first time. Each forms a head-to-tail cyclic backbone, with six cysteine residues being involved in three disulfide bonds, characteristic of the family of small proteins called the cyclotides. This is the first report on cyclotides from the species V. tricolor and the first report on the sequence of the cytotoxic cyclotide vitri A.

Primary and 3-D modelled structures of two cyclotides from Viola odorata

Two polypeptides named vodo M and vodo N, both of 29 amino acids, have been isolated from Viola odorata L. (Violaceae) using ion exchange chromatography and reversed phase HPLC. The sequences were determined by automated Edman degradation, quantitative amino acid analysis, and mass spectrometry (MS). Using MS, it was established that vodo M (cyclo-SWPVCTRNGAPICGESCFTGKCYTVQCSC) and vodo N (cyclo-SWPVCYRNGLPVCGETCTLGKCYTAGCSC) form a head-to-tail cyclic backbone and that six cysteine residues are involved in three disulphide bonds. Their origin, sequences, and cyclic nature suggest that these peptides belong to the family of cyclic plant peptides, called cyclotides. The three-dimensional structures of vodo M and vodo N were modelled by homology, using the experimentally determined structure of the cyclotide kalata B1 as the template. The images of vodo M and vodo N show amphipathic structures with considerable surface hydrophobicity for a protein modelled in a polar environment.

Cys/Gly-rich proteins with a putative single chitin-binding domain from oat ( Avena sativa) seeds

Through a reliable and repeatable procedure based on solid-phase extraction techniques, a protein fraction (P fraction) rich in Cys/Gly residues was extracted and captured from oat ( Avena sativa L.) seeds. Quantitative amino acid analysis and MS of the P fraction indicated that it contains a series of heterogeneous Cys/Gly-rich proteins with molecular masses of 3.6–4.0 kDa. Preliminary results from bioassays showed that these proteins possess weak to moderate antifungal properties to some fungal strains. From this fraction, a new polypeptide, designated avesin A, was purified and sequenced by Edman degradation. Avesin A consists of 37 amino-acid residues, with 10 glycine residues and eight cysteine residues forming disulfide bridges, and contains a single chitin-binding domain, which indicates that avesin A is a new member of the putative chitin-binding proteins. Avesin A is the first identified hevein-like small protein from cereal grains.

Quantitative amino acid analysis of bovine NADH:ubiquinone oxidoreductase (Complex I) and related enzymes. Consequences for the number of prosthetic groups

Bovine-heart NADH:ubiquinone oxidoreductase (EC 1.6.5.3; Complex I) is the first and most complicated enzyme in the mitochondrial respiratory chain. Biochemistry textbooks and virtually all literature on this enzyme state that it contains one FMN and at least four iron–sulfur clusters. We show here that this statement is incorrect as it is based on erroneous protein determinations. Quantitative amino acid analysis of the bovine Complex I, to our knowledge the first reported thus far, shows that the routine protein-determination methods used for the bovine Complex I overestimate its protein content by up to twofold. The FMN content of the preparations was determined to be at least 1.3–1.4 mol FMN/mol Complex I. The spin concentration of the electron paramagnetic resonance (EPR) signal ascribed to iron–sulfur cluster N2 was determined and accounted for 1.3–1.6 clusters per molecule of Complex I. These results experimentally confirm the hypothesis [FEBS Lett. 485 (2000) 1] that the bovine Complex I contains two FMN groups and two clusters N2. Also the protein content of preparations of the soluble NAD +-reducing [NiFe]-hydrogenase (EC 1.12.1.2) from Ralstonia eutropha, which shows clear evolutionary relationships with Complex I, scores too high by the colorimetric protein-determination methods. Determination of the FMN content and the spin concentration of the EPR signal of the [2Fe–2S] cluster shows that this hydrogenase also contains two FMN roups. A third enzyme (Ech), the membrane-bound [NiFe]-hydrogenase from Methanosarcina barkeri which shows an even stronger evolutionary relationship with Complex I, behaves rather normal in protein determinations and contains no detectable acid-extractable FMN in purified preparations.

Rapid diagnosis of phenylketonuria and other aminoacidemias by quantitative analysis of amino acids in neonatal blood spots by gas chromatography–mass spectrometry

A new method for quantifying specific amino acids in small volumes of plasma and whole blood has been developed. Volatile derivatives of amino acids are analyzed by gas chromatography–mass spectrometry. The method only takes a few minutes to perform and requires minimal sample preparation. The accurate assay of phenylalanine, tyrosine and other amino acids in dried blood spots could be used for neonatal screening for phenylketonuria and other aminoacidemias. Because of the low cost, this neonatal screening method is suited to application in developing countries such as China.