Separation of unmodified α-amino acid enantiomers by reverse phase HPLC

1980 ◽  
Vol 13 (11) ◽  
pp. 677-685 ◽  
Author(s):  
V. A. Davankov ◽  
A. S. Bochkov ◽  
A. A. Kurganov ◽  
P. Roumeliotis ◽  
K. K. Unger
Keyword(s):  
Amino Acid ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Amino Acid Enantiomers

scholarly journals Comparison of Organosulfur and Amino Acid Composition between Triploid Onion Allium cornutum Clementi ex Visiani, 1842, and Common Onion Allium cepa L., and Evidences for Antiproliferative Activity of Their Extracts

Plants ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 98 ◽  
Author(s):  
Željana Fredotović ◽  
Barbara Soldo ◽  
Matilda Šprung ◽  
Zvonimir Marijanović ◽  
Igor Jerković ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Hela Cells ◽  
Antiproliferative Activity ◽  
Dapi Staining ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Allium Species ◽  
Allium Cepa L ◽  
Common Onion

Species that belong to the genus Allium have been widely used for human food and traditional medicine. Their beneficial health effects, as well as the specific aroma, are associated with their bioactive chemical compounds, such as sulfur compounds and flavonoids. Gas chromatography and mass spectrometry (GC–MS) and reverse-phase high-performance liquid chromatography (reverse-phase HPLC) were used to identify organosulfur and amino acid content of triploid hybrid onion, Allium cornutum Clement ex Visiani, 1842, and common onion, Allium cepa L. Allium extracts were tested for their antiproliferative activity in three human cancer cell lines (HeLa, HCT116, and U2OS). DNA fragmentation and DAPI staining analysis were performed on HeLa cells to evaluate the effect of extracts on DNA damage and cell morphology. The mRNA expression of p53, Bax, and Caspase-3 genes involved in apoptosis were analyzed by real-time PCR. Using GC–MS, 27 compounds were found in two Allium species headspaces. Differences were noted among the main compound abundance in the headspace (although the major thiols and disulfides were qualitatively identic in both Allium species) and dipropyl disulfide, diisopropyl trisulfide, and (Z)-prop-1-enyl propyl trisulfide were predominant sulfides. Identification of amino acids and their quantities were determined by reverse-phase HPLC. Most abundant amino acids in both onions were arginine (Arg) and glutamic acid (Glu). The results of cytotoxicity testing confirmed antiproliferative effects of both species. The DNA fragmentation assay, DAPI staining and real time PCR analysis confirmed that A. cornutum and A. cepa extracts induced apoptosis in HeLa cells. This study presents the evidence for possible therapeutic use of A. cornutum and A. cepa extracts against human cervical carcinoma cell line.

Molecular Characterization and Pharmacology of Epoetin Delta − The Only Erythropoietin Produced in a Human Cell Line.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1299-1299
Author(s):  
Raymond D. Pratt ◽  
J. Dowell ◽  
Z. Shahrokh ◽  
S. Flatman ◽  
M. Davies ◽  
...  
Keyword(s):  
Amino Acid ◽  
Adverse Events ◽  
Human Cell Line ◽  
Pharmacokinetic Parameters ◽  
Epoetin Alfa ◽  
Healthy Individuals ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Dose Dependent ◽  
Serum Erythropoietin

Abstract Recombinant erythropoietins are used extensively in the management of anemia associated with chronic kidney disease (CKD). All the currently available erythropoietins are synthesized in Chinese hamster ovary (CHO) cell lines resulting in differences in glycosylation compared with human serum erythropoietin. We report the molecular characterization and pharmacokinetic properties of epoetin delta (Dynepo®, Shire plc), the only erythropoietin produced in a human cell line. A variety of techniques have been used to characterize epoetin delta, including amino acid sequencing, peptide mapping with reverse-phase HPLC/mass spectrometry, oligosaccharide profiling and MALDI-TOF of released glycans. Sialic acid and N-glycolylneuraminic acid (Neu5Gc) residues were quantified, following labeling of the released glycans, by reverse-phase HPLC analysis with fluorescence detection. Epoetin delta was produced as a highly pure and stable protein with the full human primary amino acid sequence. Neu5Gc residues were not detectable in epoetin delta within the validated limits of the assay but became readily detectable in CHO derived epoetins. Following characterization of the molecule early studies were done to determine the pharmacokinetic and pharmacodynamic properties of epoetin delta. Two studies investigated responses to epoetin delta in healthy individuals. The first study involved randomization of 21 men to either intravenous (i.v.) epoetin delta (15, 40 or 100 IU/kg) or placebo. The second was an open-label, crossover study, involving 32 volunteers randomized to receive single doses of epoetin delta 75 IU/kg given i.v. or subcutaneous (s.c.) by injection. Two further studies were carried out with CKD patients requiring hemodialysis who had previously received epoetin alfa. The first involved 40 patients randomized to epoetin delta or epoetin alfa (50 or 100 IU/kg), three-times per week for 4 weeks. The second was a single-dose study in 28 patients comparing epoetin delta 150 and 300 IU/kg given i.v. or s.c. Pharmacokinetic parameters were calculated from serum erythropoietin concentrations (determined by ELISA) by validated non-compartmental techniques using WinNonlin Professional v3.0A. Intravenous epoetin delta in healthy individuals displayed non-linear and dose-dependent pharmacokinetics, with a dose-dependent effect on serum hemoglobin. The bioavailability of s.c. epoetin delta is around 30%, and concentrations peak later and decline more slowly than with i.v. injection. In hemodialysis patients pharmacokinetic parameters were similar to those in healthy individuals, although AUC and half-life were increased. Compared with the 50 IU/kg dose, treatment with epoetin delta 100 IU/kg was associated with a trend to increased hemoglobin and hematocrit levels. Epoetin delta was well tolerated in all participants with the frequency of adverse events occurring in dialysis patients as expected for that population. Adverse events were similar with epoetin alfa and epoetin delta. No neutralizing anti-erythropoietin antibodies were detected in any patient. Epoetin delta was characterized at the molecular level and levels of Neu5Gc residues were undetectable within the validated limits of the assay. The pharmacokinetic profile of epoetin delta and effects on hemoglobin and hematocrit levels were suitable for the treatment of anemia in CKD patients by either s.c or i.v. administration.

scholarly journals Isolation and Characterisation of a Reserve Protein from the Seeds of Cereus jamacaru (Cactaceae)

2001 ◽  
Vol 44 (4) ◽  
pp. 331-335 ◽  
Author(s):  
Itayguara Ribeiro da Costa ◽  
Petrônio Augusto Simão de Souza ◽  
Carlos Bloch Jr. ◽  
Romulo Marino Llamoca-Zárate ◽  
Francisco A. P. Campos
Keyword(s):  
Amino Acid ◽  
Molecular Marker ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Storage Protein ◽  
Gel Filtration ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
2S Albumin ◽  
Reserve Protein

We describe here the isolation and characterisation of a major reserve protein from the seeds of Cereus jamacaru. (Cactaceae). This protein has a molecular mass of 5319 kDa and was isolated by a combination of gel filtration chromatography and reverse phase HPLC. The amino acid composition of the protein was determined and it was shown to have similarities with the amino acid composition of several proteins from the 2S albumin storage protein family. The usefulness of this protein as a molecular marker in the Cactaceae is also discussed.

scholarly journals Purification to homogeneity and amino acid sequence analysis of two anionic species of human interleukin 1.

1986 ◽  
Vol 164 (1) ◽  
pp. 237-250 ◽  
Author(s):  
P M Cameron ◽  
G A Limjuco ◽  
J Chin ◽  
L Silberstein ◽  
J A Schmidt
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Interleukin 1 ◽  
Amino Acid Residues ◽  
Amino Acid Sequence Analysis ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Molecular Weights ◽  
Maximal Stimulation ◽  
Anionic Species

Two anionic species of human IL-1 have been purified to homogeneity. These molecules were characterized as having pI of 5.4 and 5.2 and molecular weights identical to IL-1/6.8 (17,500). The specific activities of IL-1/5.4 and IL-1/5.2, as measured in the mouse thymocyte co-mitogenic assay, were identical to that of IL-1/6.8, namely 1.2 X 10(7) U/mg, with half-maximal stimulation observed at 2 X 10(-11) M. IL-1/5.4 and IL-1/5.2 were found to be antigenically distinct from IL-1/6.8 in an ELISA. IL-1/5.4 was structurally distinct from IL-1/6.8 based on reverse-phase HPLC or CNBr peptides. Intact IL-1/5.2 and three intact CNBr peptides of IL-1/5.4 were sequenced, with the identification of 74 amino acid residues. These sequences were found to correspond exactly with the amino acid sequence deduced from the IL-1-alpha cDNA reported by March et al.

Application of statistical design for the optimization of amino acid separation by reverse-phase HPLC

2008 ◽  
Vol 383 (1) ◽  
pp. 93-102 ◽  
Author(s):  
R. Gheshlaghi ◽  
J.M. Scharer ◽  
M. Moo-Young ◽  
P.L. Douglas
Keyword(s):  
Amino Acid ◽  
Statistical Design ◽  
Reverse Phase Hplc ◽  
Reverse Phase

TWO ABNORMAL FIBRINOGENS DESIGNATED AS OSAKA II AND MORIOKA WITH A HITHERTO UNIDENTIFIED AMINO ACID SUBSTITUTION; γARG-275 BY CYS

1987 ◽  
Author(s):  
S Terukina ◽  
M Matsuda ◽  
N Yoshida ◽  
K Yamazumi ◽  
Y Takeda ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Sequence Data ◽  
Total Amino Acid ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Functional Studies ◽  
Sds Page ◽  
Two Populations ◽  
Fibrin Monomers

A hitherto unidentified amino acid substitution of γ Arg-275 by Cys has been found in two abnormal fibrinogens, Osaka II and Morioka. The propositi are both asymptomatic heterozygotes for the abnormality characterized by altered polymerization of fibrin monomers. Reducing SDS-PAGE revealed that fibrinogens derived from thé propositi both consist of two populations; one with a normal and the other with an abnormal longer γ-chain by 0.5 Kd.The γ-γ cross-linking took place nearly normally, however. Analyzing plasmic digests of fibrinogen by SDS-PAGE, we located the abnormality residing in the γ-chain remnant of fragment D. Chromatofocusing of D1 obtained by plasmic digestion in 5 mM Ca++ of purified fibrinogen separated the variant D1 (vD1) from the normal one (nD1) distinctly, as confirmed by SDS-PAGE and functional studies. As anticipated, vD1 failed to interfere with normal fibrin polymerization and thrombin clotting of normal fibrinogen, whereas nD1 inhibited these reactions significantly. After reduction and pyridylethylation, vD1 and nD1 were individually digested with lysylendopeptidase (lysEP). Analyzing the digests by reverse phase HPLC, we noted a single peak present in the digests of vD1 but missing in those of nD1, and vice versa. Analysis of N-terminal five cycles of these peptides suggested that both of them corresponded to the peptide with residues 274302 based on the known sequence data. Primary sequence and total amino acid analyses revealed that γ Arg-275 has been substituted by Cys in both of these abnormal fibrinogens. Analysis of the lysEP-digests of the isolated γ-chain also gave the same result. Since no free SH has been identified at the γ Cys-275 substitute, the variant γ-chain may be endowed with some additive by an S-S linkage. Even if so, elucidation of an apparent elongation by SDS-PAGE of the γ-chain variant must await further investigation. In any case, however, the substitution of γ Arg-275 by Cys may have induced critical alterations in the γ-chain-dependent polymerization site in the D domain in these two abnormal fibrinogens.

scholarly journals Antiproteolytic activity of goose pancreas: purification, inhibitory properties and amino-acid sequence of a Kazal type trypsin inhibitor.

1996 ◽  
Vol 43 (3) ◽  
pp. 489-496 ◽  
Author(s):  
A Wilimowska-Pelc ◽  
D Stachowiak ◽  
M Gładysz ◽  
Z Olichwier ◽  
A Polanowski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Trypsin Inhibitor ◽  
Association Constant ◽  
Amino Acid Residues ◽  
High Homology ◽  
Reactive Site ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Antiproteolytic Activity

A trypsin inhibitor of Kazal type has been isolated from goose pancreas by affinity chromatography on immobilized anhydrotrypsin, anion exchange and reverse phase HPLC. It inhibits bovine beta-trypsin with the association constant (Ka) of 5.99 x 10(8) M-1. The complete amino-acid sequence was determined following CNBr treatment. The protein comprised a total of 69 amino-acid residues, corresponding to a molecular mass of 7.7 kDa. The P1-P'1 reactive site bond of the inhibitor was localized at position Lys25-Met26. The amino-acid sequence of GPTI shows extremely high homology to that of other inhibitors isolated from pancreas of birds.

Sign in / Sign up

Export Citation Format

Share Document