scholarly journals Chemical decarboxylation kinetics and identification of amino acid standards by benchtop NMR spectroscopy

2021 ◽  
Author(s):  
Angelika Czajkowska ◽  
Defne Ilayda Dayi ◽  
Helga Weinschrott ◽  
Hans-Peter Deigner ◽  
Magnus S. Schmidt
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Biochemical Analysis ◽  
Pyroglutamic Acid ◽  
Acidic Ph ◽  
Kinetic Measurements ◽  
All Solutions ◽  
Ph Conditions ◽  
The Stability ◽  
The Given

AbstractIn this paper, we examined the competence of amino acids as standards for instrumental biochemical analysis. The chosen amino acids were first dissolved in various aquatic solutions and then measured in a benchtop NMR spectrometer, which is not a common choice in such analytical investigations. Analysis by mass spectrometry was used in addition. As part of these investigations, we examined and determined the stability of the amino acids ornithine, glutamic acid, alanine, glycine, proline, pyroglutamic acid, phenylalanine and trans-4-hydroxy-D-proline under critical basic and acidic pH conditions and under various other conditions. We observed that not all solutions of the amino acid standards remain stable under the given conditions and a chemical transformation takes place. Given our findings by mass spectroscopy, additional kinetic measurements were carried out with the benchtop NMR spectrometer. We discovered that pyroglutamic acid becomes unstable under basic conditions and decarboxylates to pyrrolidone.

scholarly journals Produksi, isolasi dan karakterisasi superoksida dismutase dari Spirulina platensis yang dibiakkan dalam serum lateks Production, isolation, and characterization of superoxyde dismutase from Spirulina platensis cultured on latex serum

2016 ◽  
Vol 77 (1) ◽  
Author(s):  
. TRI-PANJI ◽  
. SUHARYANTO ◽  
Marini WIJAYANTI
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Spray Drying ◽  
Spirulina Platensis ◽  
Active Agent ◽  
Pilot Scale ◽  
Isolation And Characterization ◽  
The Stability ◽  
Major Amino Acid

AbstractSpirulina platensis is a blue-green microalgawhich is frequently used for food and feedsupplements and cosmetic active agent. Thismicroalga also produces a strong antioxidantnamely superoxide dismutase (SOD) used ascosmetic active agent for anti aging and anti freeradicals. SOD was isolated from S. platensis cellbiomass from local isolate grown on latex serumon semipilot (3.5 m 3 ) and pilot scale (40 m 3 )then dried with spray drying or sun drying andcharacterized. SOD was purified with sequentialtwo-stage sedimentation using ammoniumsulphate and fractionated in chromatographiccolumn containing Sephadex G 200. Thefractions were analysed to determine the activity,cofactor metal and amino acid composition of theantioxidant. The results showed thatsedimentation of SOD extract with 80%ammonium sulphate produced SOD with higheractivity compared to that of SOD fromcommercial S. platensis biomass. This SOD wassuccessfully isolated and purified. MetaloenzymeSOD was composed of subunits with molecularweight of 77.78; 71.74; and 19.2 kDa, whichcontained nine types of amino acids with tyrosineand lysine as the major amino acid components.Zn was the most predominant metal on SOD, thenfollowed by Fe and Mn. The main subunitcofactors consisted of Zn 72%, Fe 25%, Mn 2%,and Cu 1%, which were different from thesmall subunit that contained of Zn 55%, Mn 31%,Fe 14%, and Cu 4%. The stability of SOD wasachieved on pH 7.5 and temperature below 25 o C.AbstrakSpirulina platensis adalah mikroalga hijaubiru yang banyak digunakan sebagai suplemenpangan, pakan, dan bahan aktif kosmetika.Mikroalga ini juga menghasilkan antioksidankuat yaitu superoksida dismutase (SOD), yangmerupakan bahan aktif kosmetika anti penuaandini dan pencegah efek radikal bebas. SODdiisolasi dari biomassa sel S. platensis isolat lokalyang dibiakkan dalam serum lateks skalasemipilot (3,5 m 3 ) dan pilot (40 m 3 ) sertadikeringkan dengan cara pengeringan kabut(spray drying) atau penjemuran untuk kemudiandikarakterisasi. SOD dimurnikan dengan peng-endapan bertingkat menggunakan ammoniumsulfat dan dipisahkan dengan kolom kromatografiberisi Sephadex G 200. Hasil pemisahankemudian dianalisis untuk menentukan aktivitas,logam kofaktor serta komposisi asam amino antioksidan tersebut. Hasil penelitian menunjukkanbahwa pengendapan ekstrak SOD denganSOD lebih tinggi dari SOD asal biomassaS. platensis komersial. SOD tersebut telahberhasil diisolasi dan dimurnikan. MetaloenzimSOD tersusun atas subunit dengan BM 77,78;71,74; dan 19,2 kDa, yang mengandungsembilan jenis asam amino dengan tirosin danlisin sebagai komponen asam amino utama.Logam yang dominan pada SOD adalah Zn,disusul kemudian Fe dan Mn. Kofaktor sub unitbesar terdiri dari Zn 72%, Fe 25%, Mn 2%, danCu 1%, berbeda dengan sub unit kecil yangmengandung Zn 55%, Mn 31%, Fe 14%, dan Cu4%. Stabilitas SOD S. platensis dicapai pada pH7,5 dan suhu di bawah 25 o Cammonium sulfat 80% menghasilkan aktivitas

scholarly journals Quantitative Analysis of Branched-chain Amino Acids in Five Nutritional Supplements Using a Leucine Dehydrogenase Assay (P23-004-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Brendan Eley
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Nutritional Supplements ◽  
Sport Nutrition ◽  
Free Form ◽  
Leucine Dehydrogenase ◽  
Funding Sources ◽  
Branched Chain Amino Acid ◽  
Branched Chain ◽  
The Given

Abstract Objectives The objective of this experiment was to determine the branched-chain amino acid (BCAA) content in five different sport nutrition supplements compared to the amount claimed on the label. Methods To measure the BCAA content of five nutritional supplements, a leucine dehydrogenase enzyme assay was used. This enzyme catalyzes the reaction of turning the given BCAA (L-leucine, L-valine, and L-isoleucine), water, and NAD+ into their respective metabolite, NH3, NADH, and H+. Ultraviolet-visible light spectrophotometry (UV/Vis) was used at 340 nm to create a standard curve. This curve uses the Beer-Lambert Law to measure NADH concentration from absorbance. NADH is in a 1:1 ratio with each BCAA molecule thus relaying the content of the given sample. The assay is specific to the three BCAAs in their free form. Other amino acids, as well as BCAAs in oligopeptides, do not interfere with this experiment. Products including oligo- and polypeptides were not included for testing. The assay was performed for each product and ran against a known standard (≥98% L-leucine) for validation. Due to different supplements having different BCAA amounts per serving, % content of the claimed amount was measured. Results Compared to the amount provided by the labels of each supplement, BCAA content was on average only 61% of the manufacturer claims when compared to ≥98% L-leucine. This shows that these BCAA supplements do not meet label claims for BCAA content (P < 0.01). Conclusions The five tested nutritional supplements contain significantly less branched-chain amino acid content than claimed on the label. This experiment can be expanded on in the future to test content of other BCAA containing supplements to determine how common underdosing is in the industry as a whole. Funding Sources The author claims no funding sources.

scholarly journals The combination of proteins, amino-acids, etc., with acids and alkalis, and their combining weights as determined by physico-chemical measurements.—Preliminary paper

1925 ◽  
Vol 97 (684) ◽  
pp. 364-386 ◽  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Hydrogen Ion ◽  
Acid Mixture ◽  
Modus Operandi ◽  
End Points ◽  
Basic Group ◽  
Chemical Measurements ◽  
Physico Chemical ◽  
The Given

In previous communications (1) the writer described a system for estimating and characterising the amino-acids which depends on the k a and k b values of these bodies. The modus operandi of the technique is to determine the amounts of acid or alkali required to titrate the amino-acid- (or dipeptide-, etc.) solutions over definite ranges of p H or to definite p H end-points; it rests on the knowledge that the weaker an acidic (or a basic) group the greater will be the degree of alkalinity (or acidity) developed during its titration with strong alkali (or acid). After the addition of each increment of acid or alkali the p H is determined. One plots p H against, not the actual amount of alkali (or acid) added, but against this amount corrected for the “hydrogen ion error.” The correction is made by subtracting from the amount of alkali (or acid) added, the amount of alkali (or acid) which would have to be added to a hypothetical blank consisting of water only in order to produce the same total volume of liquid having the same p H as the amino-acid alkali (or acid) mixture. The corrected value represents the amount of alkali (or acid) required to bring the amino-acid itself to the given p H value, while the correction is the amount required by the water present.

How to fragment a polypeptide? An ab initio computational study of pair interactions between amino acids and ligand-amino acids in proteins

2011 ◽  
Vol 76 (5) ◽  
pp. 605-618
Author(s):  
Vojtěch Klusák ◽  
Petr Dobeš ◽  
Jiří Černý ◽  
Jiří Vondrášek
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ab Initio ◽  
Building Blocks ◽  
Basis Set ◽  
Single Amino Acid ◽  
Amino Acid Side Chain ◽  
Protein Chain ◽  
The Stability ◽  
Dispersion Term

To determine reasonably which amino acid side chain contributes significantly to the stability of a protein or to the stability of a protein–ligand complex is not a straightforward task. We suggest a partial but systematic solution of the problem by a specific fragmentation of a protein chain into blocks of single amino acid side chains with their corresponding backbone part. For such systems of building blocks, we have calculated the stabilisation/interaction energies by means of correlated ab initio calculations. We have shown that a reasonable way to treat an amino-acid residue composing the protein is to break the homonuclear C–C bond between the Cα atom and the C(O) carboxyl carbon. The reference data obtained by the RI-MP2 method with the cc-pVDZ basis set were compared with RIDFT, RIDFT augmented by the dispersion term, SCC-DFTB-D and Hartree–Fock calculations. The results clearly show the failure of those methods lacking an appropriate treatment of the correlation energy. The DFT methods augmented by the empirical dispersion term on the other hand describe the interaction in good agreement with the reference method.

scholarly journals CELLULAR MECHANISMS OF PROTEIN METABOLISM IN THE NEPHRON

1954 ◽  
Vol 99 (6) ◽  
pp. 621-628 ◽  
Author(s):  
Yin Chen Lee
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Enzymatic Activity ◽  
Protein Metabolism ◽  
Biochemical Analysis ◽  
High Concentration ◽  
Cellular Mechanisms ◽  
Theoretical Assumption ◽  
High Positive Correlation ◽  
Proximal Convolution

1. The cells of the proximal convolutions of the rat can be filled with fine droplets by the administration of certain amino acids in non-toxic dosage. 2. These minute droplets resemble in their Gram positivity and in their phospholipid and PNA content on biochemical analysis (3) the larger droplets that form in the cells of the proximal convolution during the absorption of certain proteins. 3. The positive or negative reaction to 3 specific histochemical procedures of the fine droplets forming after administration of 7 amino acids gave such a high positive correlation with theoretical assumption as to warrant the following inferences: (a) that the methods are specific under the conditions of the experiment; (b) that the droplets contain a higher concentration of the administered amino acid than the general cytoplasm of the cells which contain them. 4. The mitochondrial rodlets of epithelium of the proximal convolution contain sulfhydryl in high concentration, a finding which correlates well with their enzymatic activity.

Stability of Disodium Sulfobenzylpenicillin in Large Volume Parenteral Solutions

1978 ◽  
Vol 12 (7) ◽  
pp. 418-423
Author(s):  
Hiroshi Fukuchi ◽  
Minoru Yoshida ◽  
Michio Kumagai ◽  
Teruaki Kitaura
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Thin Layer ◽  
Large Volume ◽  
Degradation Product ◽  
Degradation Products ◽  
Residual Rate ◽  
The Stability ◽  
Layer Chromatography ◽  
Over Time

The stability of disodium sulfobenzylpenicillin (SB-PC) in several large-volume parenteral solutions was studied. A study was also made of the influences of other additive drugs upon the stability of SB-PC in SB-PC IV fluid admixtures. Quantitative determinations were made by iodometry of the residual rate over time of SB-PC. The residual rate of SB-PC in 5-fluorouracil (5-FU injection)-10 percent glucose admixture was 67 percent after 24 hours at 25 °C, (77 °F) and in aminophylline (aminophylline injection J.P.)-10 percent glucose admixture it was 54 percent after 24 hours. It was found that the decomposition of SB-PC was due to amines which were added to these injections. In an admixture of amino acid and SB-PC, the residual rate of SB-PC decreased linearly with the increase in amino acid concentration. In the 12 kinds of amino acids used in this study, the greatest decrease was observed in the admixture with lysine, the residual rate being 76.7 percent. For the study of degradation products of SB-PC, thin layer chromatography was employed and a spot of the degradation product was detected at an Rf value of 0.2.

scholarly journals Consensus Finder web tool to predict stabilizing substitutions in proteins

2020 ◽  
Author(s):  
Bryan J. Jones ◽  
Chi Nok Enoch Kan ◽  
Christine Luo ◽  
Romas J. Kazlauskas
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Consensus Sequence ◽  
Fine Tuning ◽  
Protein Fold ◽  
Web Tool ◽  
Multiple Sequence ◽  
Homologous Sequences ◽  
The Stability ◽  
Conserved Amino Acids

AbstractThe consensus sequence approach to predicting stabilizing substitutions in proteins rests on the notion that conserved amino acids are more likely to contribute to the stability of a protein fold than non-conserved amino acids. To implement a prediction for a target protein sequence, one finds homologous sequences and aligns them in a multiple sequence alignment. The sequence of the most frequently occurring amino acid at each position is the consensus sequence. Replacement of a rarely occurring amino acid in the target with a frequently occurring amino acid is predicted to be stabilizing. Consensus Finder is an open-source web tool that automates this prediction. This chapter reviews the rationale for the consensus sequence approach and explains the options for fine-tuning this approach using Staphylococcus nuclease A as an example.

Assembly properties of altered beta-tubulin polypeptides containing disrupted autoregulatory domains

1989 ◽  
Vol 9 (8) ◽  
pp. 3418-3428
Author(s):  
W Gu ◽  
N J Cowan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Tubulin Isotype ◽  
The Stability ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

beta-Tubulin synthesis in eucaryotic cells is subject to control by an autoregulatory posttranscriptional mechanism in which the first four amino acids of the beta-tubulin polypeptide act either directly or indirectly to control the stability of beta-tubulin mRNA. To investigate the contribution of this amino-terminal domain to microtubule assembly and dynamics, we introduced a series of deletions encompassing amino acids 2 to 5 of a single mammalian beta-tubulin isotype, M beta 1. Constructs carrying such deletions were inserted into an expression vector, and the ability of the altered polypeptide to coassemble into microtubules was tested by using an anti-M beta 1-specific antibody. We show that the M beta 1 beta-tubulin polypeptide was competent for coassembly into microtubules in transient transfection experiments and in stably transfected cell lines when it lacked either amino acid 2 or amino acids 2 and 3. The capacity of these mutant beta-tubulins to coassemble into polymerized microtubules was only slightly diminished relative to that of unaltered beta-tubulin, and their expression did not influence the viability or growth properties of cell lines carrying these deletions. However, more extensive amino-terminal deletions either severely compromised or abolished the capacity for coassembly. In analogous experiments in which alterations were introduced into the amino-terminal domain of a mammalian alpha-tubulin isotype, M alpha 4, deletion of amino acid 2 did not affect the ability of the altered polypeptide to coassemble, although removal of additional amino-terminal residues essentially abolished the capacity for competent coassembly. The stability of the altered assembly-competent alpha- and beta-tubulin polypeptides was measured in pulse-chase experiments and found to be indistinguishable from the stability of the corresponding unaltered polypeptides. An assembly-competent M alpha 4 polypeptide carrying a deletion encompassing the 12 carboxy-terminal amino acids also had a half-life indistinguishable from that of the wild-type alpha-tubulin molecule. These data suggest that the universally conserved amino terminus of beta-tubulin acts largely in a regulatory role and that the carboxy-terminal domain of alpha-tubulin is not essential for coassembly in mammalian cells in vivo.

scholarly journals Assembly properties of altered beta-tubulin polypeptides containing disrupted autoregulatory domains.

1989 ◽  
Vol 9 (8) ◽  
pp. 3418-3428 ◽  
Author(s):  
W Gu ◽  
N J Cowan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Tubulin Isotype ◽  
The Stability ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

beta-Tubulin synthesis in eucaryotic cells is subject to control by an autoregulatory posttranscriptional mechanism in which the first four amino acids of the beta-tubulin polypeptide act either directly or indirectly to control the stability of beta-tubulin mRNA. To investigate the contribution of this amino-terminal domain to microtubule assembly and dynamics, we introduced a series of deletions encompassing amino acids 2 to 5 of a single mammalian beta-tubulin isotype, M beta 1. Constructs carrying such deletions were inserted into an expression vector, and the ability of the altered polypeptide to coassemble into microtubules was tested by using an anti-M beta 1-specific antibody. We show that the M beta 1 beta-tubulin polypeptide was competent for coassembly into microtubules in transient transfection experiments and in stably transfected cell lines when it lacked either amino acid 2 or amino acids 2 and 3. The capacity of these mutant beta-tubulins to coassemble into polymerized microtubules was only slightly diminished relative to that of unaltered beta-tubulin, and their expression did not influence the viability or growth properties of cell lines carrying these deletions. However, more extensive amino-terminal deletions either severely compromised or abolished the capacity for coassembly. In analogous experiments in which alterations were introduced into the amino-terminal domain of a mammalian alpha-tubulin isotype, M alpha 4, deletion of amino acid 2 did not affect the ability of the altered polypeptide to coassemble, although removal of additional amino-terminal residues essentially abolished the capacity for competent coassembly. The stability of the altered assembly-competent alpha- and beta-tubulin polypeptides was measured in pulse-chase experiments and found to be indistinguishable from the stability of the corresponding unaltered polypeptides. An assembly-competent M alpha 4 polypeptide carrying a deletion encompassing the 12 carboxy-terminal amino acids also had a half-life indistinguishable from that of the wild-type alpha-tubulin molecule. These data suggest that the universally conserved amino terminus of beta-tubulin acts largely in a regulatory role and that the carboxy-terminal domain of alpha-tubulin is not essential for coassembly in mammalian cells in vivo.

The basic hydrolysis of amino acid esters

1966 ◽  
Vol 19 (7) ◽  
pp. 1197 ◽  
Author(s):  
RW Hay ◽  
LJ Porter ◽  
PJ Morris
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Steric Effects ◽  
Ionization Constants ◽  
Amino Acid Esters ◽  
Kinetic Measurements ◽  
Basic Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Acid Esters

The kinetics of alkaline hydrolyses of nine amino acid esters by the process H,2NCHRCO2R'+OH- + H2NCHRCO2-+R'OH have been studied at 25� and an ionic strength of 0.1M in water over a range of pH. The acid ionization constants of the various amino acid esters have been determined, and these are roughly 2 pK units lower than those of the corresponding amino acids. The kinetic measurements are interpretable on the basis of electrostatic and steric effects.

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