On-line HPLC analysis of PTH-amino acids derived from Edman degradation of proteins and peptides: Optical sensor controlled sample injection

1989 ◽  
Vol 54 (4) ◽  
pp. 940-944 ◽  
Author(s):  
Manfred Pavlík ◽  
Jiří Jehnička ◽  
Vladimír Kostka
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Hplc Analysis ◽  
Edman Degradation ◽  
Isocratic Elution ◽  
Sample Injection ◽  
Optoelectronic Sensor ◽  
On Line ◽  
The Moment ◽  
Proteins And Peptides

A new principle in the system of identification and quantitation of amino acid phenylthiohydantoins derived from automated Edman degradation of proteins and peptides is described. The beginning of the on-line HPLC analysis of PTH's, the actuation of the valve and the sample injection are controlled by an optoelectronic sensor placed at the sample loop outlet. At the moment the loop has been loaded the sensor actuates the injection valve thus starting the chromatography run. This arrangement eliminates the possibility of incomplete (irreproducible) loading of the loop or of void injection. The chromatographic separation of the PTH's is carried out by isocratic elution.

scholarly journals Determination of amino acids in human biological fluids by high-performance liquid chromatography: critical review

Amino Acids ◽  
2021 ◽  
Author(s):  
Grażyna Gałęzowska ◽  
Joanna Ratajczyk ◽  
Lidia Wolska
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
High Performance ◽  
Hplc Analysis ◽  
Limit Of Detection ◽  
Biological Fluids ◽  
Analytical Techniques ◽  
Epidemiological Studies ◽  
Preparation Conditions

AbstractThe quantitation and qualification of amino acids are most commonly used in clinical and epidemiological studies, and provide an excellent way of monitoring compounds in human fluids which have not been monitored previously, to prevent some diseases. Because of this, it is not surprising that scientific interest in evaluating these compounds has resurfaced in recent years and has precipitated the development of a multitude of new analytical techniques. This review considers recent developments in HPLC analytics on the basis of publications from the last few years. It helps to update and systematize knowledge in this area. Particular attention is paid to the progress of analytical methods, pointing out the advantages and drawbacks of the various techniques used for the preparation, separation and determination of amino acids. Depending on the type of sample, the preparation conditions for HPLC analysis change. For this reason, the review has focused on three types of samples, namely urine, blood and cerebrospinal fluid. Despite time-consuming sample preparation before HPLC analysis, an additional derivatization technique should be used, depending on the detection technique used. There are proposals for columns that are specially modified for amino acid separation without derivatization, but the limit of detection of the substance is less beneficial. In view of the fact that amino acid analyses have been performed for years and new solutions may generate increased costs, it may turn out that older proposals are much more advantageous.

Isolation of a Crosslinked Peptide from Hitman Fibrin and Development of a Radioimmunoassay

1979 ◽  
Author(s):  
R. Canfield ◽  
B. Lahiri ◽  
R. D’Alisa ◽  
V. Butler ◽  
H. Nossel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Edman Degradation ◽  
Cnbr Cleavage ◽  
Normal Hemostasis

Factor XIIIa introduces up to six crossllnklng bonds per molecule of fibrin; the bonds between the γ chains on adjacent fibrin molecules form most rapidly. Since cross linking is essential for normal hemostasis and is likely to be important in tests to detect thrombosis, we have attempted to develop a radioimmunoassay that exhibits specificity for the γ chain crosslinks. The immunogen consisted of a 54 amino acid, crosslinked peptide, isolated from purified human γ-γ chains following CNBr cleavage, gel filtration on Sephadex G-50 and ion-exchange chromatography on SP-Sephadex. Amino acid analysis and Edman degradation through step 24 confirmed the sequence of Chen and Doolittle (Biochemistry 10: i486, 1971), and the two degradation steps that failed to liberate the expected PTH-amino acids matched the reported location of the Gin-Lys crosslinks. Antisera were obtained against this immunogen coupled either to bovine thyroglobulin or bovine serum albumin. All antisera elicited bound immunogen that was covalently coupled to ribonuclease radiolabeled with 125I as a tracer. The unlabeled γ-γ, crosslinked peptide effectively inhibited binding (0.03-0.08 picomoles for 50% inhibition), while with some antisera up to 500 times more of the 27 amino acid γ monomer peptide was required for the same degree of inhibition. Fibrinogen and fragment D also were poor Inhibitors. The results Indicate that it is possible by radioimmunoassay to distinguish the COOH-termlnal region of the γ-γ dlmer from that of uncrosslinked molecules.

scholarly journals Method to generate highly stable D-amino acid analogs of bioactive helical peptides using a mirror image of the entire PDB

2018 ◽  
Vol 115 (7) ◽  
pp. 1505-1510 ◽  
Author(s):  
Michael Garton ◽  
Satra Nim ◽  
Tracy A. Stone ◽  
Kyle Ethan Wang ◽  
Charles M. Deber ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Data Bank ◽  
Mirror Image ◽  
Proof Of Concept ◽  
Human Gut ◽  
Helical Peptides ◽  
Peptide Engineering ◽  
Small Molecule Drugs ◽  
Proteins And Peptides

Biologics are a rapidly growing class of therapeutics with many advantages over traditional small molecule drugs. A major obstacle to their development is that proteins and peptides are easily destroyed by proteases and, thus, typically have prohibitively short half-lives in human gut, plasma, and cells. One of the most effective ways to prevent degradation is to engineer analogs from dextrorotary (D)-amino acids, with up to 105-fold improvements in potency reported. We here propose a general peptide-engineering platform that overcomes limitations of previous methods. By creating a mirror image of every structure in the Protein Data Bank (PDB), we generate a database of ∼2.8 million D-peptides. To obtain a D-analog of a given peptide, we search the (D)-PDB for similar configurations of its critical—“hotspot”—residues. As a proof of concept, we apply our method to two peptides that are Food and Drug Administration approved as therapeutics for diabetes and osteoporosis, respectively. We obtain D-analogs that activate the GLP1 and PTH1 receptors with the same efficacy as their natural counterparts and show greatly increased half-life.

Nuclear Magnetic Resonance Studies of Phenylthiohydantoin Amino Acids

1975 ◽  
Vol 53 (9) ◽  
pp. 1005-1009 ◽  
Author(s):  
C. S. Tsai ◽  
N. L. Fraser ◽  
H. Avdovich ◽  
J. P. Farant
Keyword(s):  
Amino Acids ◽  
Nuclear Magnetic Resonance ◽  
Amino Acid ◽  
Magnetic Resonance ◽  
Diagnostic Purpose ◽  
Edman Degradation ◽  
Terminal Amino Acid ◽  
Magnetic Resonance Studies ◽  
Terminal Amino ◽  
Derivatives Of

Proton magnetic spectra of 3-phenyl-2-thiohydantoin derivatives of common amino acids in deuterated dimethyl sulfoxide were recorded. Spectral data pertaining to characteristic protons for diagnostic purpose were compiled. Their application to the N-terminal amino acid analysis of peptide by Edman degradation was examined.

scholarly journals Analisis Profil Protein dan Asam Amino Sarang Burung Walet (Collocalia fuchiphaga)Asal Painan

2014 ◽  
Vol 4 (1) ◽  
Author(s):  
Lina Elfita
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Aspartic Acid ◽  
High Performance ◽  
Hplc Analysis ◽  
Protein Profile ◽  
Essential Amino Acids ◽  
Bird Nest ◽  
Sds Page ◽  
West Sumatra

Abstrak Penelitian tentang profil protein sarang burung wallet masih terbatas, terutama sarang burung walet dari Indonesia. Oleh karena itu, penelitian ini bertujuan untuk menganalisa profil protein dan asam amino sarang burung walet yang berasal dari daerah Painan, Kabupaten Pesisir Selatan, Sumatera Barat. Analisis protein dilakukan dengan menggunakan SDS-PAGE, sedangkan analisis asam amino dilakukan dengan menggunakan kromatografi cair kinerja tinggi (KCKT). Analisa ekstrak air sarang burung walet dengan SDS-PAGE menunjukan bahwa sarang burung walet terdiri dari 6 protein. Keenam protein tersebut mempunyai bobot molekul masing-masing 147.2 kDa, 142.6 kDa, 133.4 kDa, 73.3 kDa, 66.2 kDa dan 37.7 kDa. Dari analisa asam amino burung walet dengan KCKT didapatkan 16 asam amino yang terkandung dalam sarang burung wallet, yang terdiri dari 7 jenis asam amino esensial yaitu Histidin (2.31%), Leusin (3.84%), Treonin (3.82%), Valin (3.93%), Metionin (0.48%), Isoleusin (1.80%), Fenilalanine (4.49%)  dan 9 asam amino non esensial yaitu Asam Serin (4.56%), Aspartat (4.48%), Arginin (3.93%), Lisin (2.34 %), Prolin (3.64%),  Asam glutamate (3.65%), Glisin (1.87%), Alanin (1.31%), Tirosin (3.92%). Serin merupakan asam amino dengan kadar tertinggi (4.56%), diikuti dengan Fenil alanine (4.49%) dan Asam aspartate (4.48%). Kandungan asam amino ini sedikit berbeda dengan kandungan asam amino sarang burung walet dari daerah dan negara lain. Kata kunci: sarang burung walet, protein, asam amino Abstract Study on protein profile of bird nest is still limited particularly protein profile of bird nest from Indonesia has not been reported. Therefore, this study was aimed to analyze protein profile and amino acid composition of bird nest from Painan, Pesisir Selatan Distric, West Sumatra. Protein analysis was performed by SDS-PAGE, and high performance liquid chromatography (HPLC) was used for amino acid analysis. SDS-PAGE analysis showed  six bands, which molecular weigh of 147.2 kDa, 142.6 kDa, 133.4 kDa, 73.3 kDa, 66.2 kDa and 37.7 kDa, respectively. On the other hand, HPLC analysis demonstrated that bird nest was composed of 16 amino acids. Seven of them were essential amino acids; histidine (2.31%), leucine (3.84%), threonine (3.82%),  valine (3.93%), methionine (0.48%), isoleucine (1.80%), phenylalanine (4.49%), and nine of them were non-essential amino acids; serine (4.56%), aspartic acid (4.48%), arginine (3.93%), lysine (2.34%), proline (3.64%), glutamic acid (3.65%), glycine (1.87%), alanine (1.31%), tyrosine (3.92%). Serine was the highest percentage of amino acid in the bird nest (4.56%), followed by phenylalanine (4.49%) and aspartic acid (4.48%). Composition of amino acid in this bird nest was slightly different with composition of amino acid in bird nest from other area. Keywords : bird nest, protein profile, amino acids

scholarly journals Complete amino acid analysis of peptides and proteins after hydrolysis by a mixture of Sepharose-bound peptidases

1972 ◽  
Vol 129 (3) ◽  
pp. 695-701 ◽  
Author(s):  
H. P. J. Bennett ◽  
D. F. Elliott ◽  
B. E. Evans ◽  
P. J. Lowry ◽  
C. McMartin
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Standard Deviation ◽  
Quantitative Determination ◽  
Edman Degradation ◽  
Amino Acid Residues ◽  
Peptide Fragments ◽  
Enzyme Mixture ◽  
Acid Labile

Incubation with a mixture of Sepharose-bound peptidases was shown to result in the quantitative release of amino acids from certain peptides and S-aminoethylated proteins. Subtraction of the low background values of amino acids generated by the enzymes enables amino acid ratios of corticotrophin-(1–24)-tetracosapeptide to be determined with a standard deviation on repeat digestions of 3–5%. Good values were obtained for amino acids that are completely or partially destroyed on acid hydrolysis, i.e. tryptophan, tyrosine, serine, asparagine and glutamine. Experiments with peptides containing d-amino acids showed that the enzyme mixture is stereospecific and could therefore be used to detect the presence of d-residues in peptides. The enzyme mixture completely hydrolyses peptide fragments obtained after Edman degradation and should therefore be useful for determining sequences of peptides containing acid-labile amino acid residues. The activities of the bound enzymes were unaltered over a period of 7 months and they provide a simple, reproducible procedure for the quantitative determination of amino acids in peptides and proteins containing l-amino acids.

scholarly journals Amino Acids, Peptides, and Proteins: Implications for Nanotechnological Applications in Biosensing and Drug/Gene Delivery

Nanomaterials ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 3002
Author(s):  
Simge Er ◽  
Ushna Laraib ◽  
Rabia Arshad ◽  
Saman Sargazi ◽  
Abbas Rahdar ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Amino Acids ◽  
Amino Acid ◽  
Gene Delivery ◽  
Cost Effective ◽  
Delivery Systems ◽  
Drug Molecules ◽  
Or Gene ◽  
Gold Nanoislands ◽  
Proteins And Peptides

Over various scientific fields in biochemistry, amino acids have been highlighted in research works. Protein, peptide- and amino acid-based drug delivery systems have proficiently transformed nanotechnology via immense flexibility in their features for attaching various drug molecules and biodegradable polymers. In this regard, novel nanostructures including carbon nanotubes, electrospun carbon nanofibers, gold nanoislands, and metal-based nanoparticles have been introduced as nanosensors for accurate detection of these organic compounds. These nanostructures can bind the biological receptor to the sensor surface and increase the surface area of the working electrode, significantly enhancing the biosensor performance. Interestingly, protein-based nanocarriers have also emerged as useful drug and gene delivery platforms. This is important since, despite recent advancements, there are still biological barriers and other obstacles limiting gene and drug delivery efficacy. Currently available strategies for gene therapy are not cost-effective, and they do not deliver the genetic cargo effectively to target sites. With rapid advancements in nanotechnology, novel gene delivery systems are introduced as nonviral vectors such as protein, peptide, and amino acid-based nanostructures. These nano-based delivery platforms can be tailored into functional transformation using proteins and peptides ligands based nanocarriers, usually overexpressed in the specified diseases. The purpose of this review is to shed light on traditional and nanotechnology-based methods to detect amino acids, peptides, and proteins. Furthermore, new insights into the potential of amino protein-based nanoassemblies for targeted drug delivery or gene transfer are presented.

scholarly journals System-directed pairing of protein amino acids. Part I

2021 ◽  
Author(s):  
Miloje M. Rakočević
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Code ◽  
Chemical Reactions ◽  
Review Paper ◽  
Point Of View ◽  
Second Phase ◽  
Protein Amino Acids ◽  
The Moment ◽  
Current Science

The idea of this review paper is as follows. If it can be shown (and it can!) that the pairing of protein amino acids is system-directed (determined), then the hypothesis of a prebiotically determined genetic code (Rakočević, 2004a) gets its full meaning. The hypothesis is supported by the fact that all these pairings come to the fore primarily through classes and subclasses of amino acid molecules. What is, however, unexpected and even unbelievable from the point of view of current science is the fact that the quantities, i.e. number of atoms (in a direct or indirect relation to the number of nucleons), in these classes and subclasses, are determined by Gauss’ number 51 (51 = 3 x 17), or Gauss’ sequence: 51 ± 10, 51 ± 20, 51 ± 30, 51 ± 40 and 51 ± 50; also by Dürer’s number 34 (34 = 2 x 17), even more either by its double value, 68; or by Dürer’s sequences: 34 ± 10, 34 ± 5 and 68 ± 10, 68 ± 5. Since the hypothesis refers to constituents – amino acids and nucleotides – it follows that in terms of the type and number of constituents, it makes no sense to talk about evolution of GC, but only about its generating. [Generated, not degenerated code!] It makes sense to talk about the evolution of the genetic code only from the "moment" when the resulting peptide and nucleotide chains enter into chemical reactions and interactions; although even then it can be said that this is just a second phase of GC generation.

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