scholarly journals Amino acid analysis at the picomole level. Application to the C-terminal sequence analysis of polypeptides

1981 ◽  
Vol 199 (3) ◽  
pp. 547-555 ◽  
Author(s):  
J Y Chang ◽  
R Knecht ◽  
D G Braun
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Analysis ◽  
Protein Hydrolysate ◽  
Visible Region ◽  
Reversed Phase ◽  
Terminal Sequence ◽  
Naturally Occurring ◽  
Ion Exchange Separation ◽  
Buffer Mixture

Amino acids labelled with dimethylaminoazobenzenesulphonyl chloride can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). All 19 naturally occurring amino acids can be separated on a Zorbax ODS column by employing two different gradient systems consisting of an acetonitrile/aqueous buffer mixture. As little as 2--5 pmol of an individual dimethylaminoazobenzenesulphonyl-amino acid can be quantitatively analysed with reliability, and only 10--30 ng of the dimethylaminoazobenzenesulphonylated protein hydrolysate is needed for each complete amino acid analysis. This new technique is as sensitive as any of the current amino acid analysis methods involving ion-exchange separation plus fluorescence detection, and is technically much simpler. By the combination of this sensitive amino acid-analysing technique with carboxypeptidase, we have been able to determine the C-terminal sequence of polypeptides at the picomole level.

Ion-Exchange Separation of Amino Acids with Postcolumn Orthophthalaldehyde Detection

1987 ◽  
Vol 70 (2) ◽  
pp. 248-252 ◽  
Author(s):  
Raymond B Ashworth
Keyword(s):  
Amino Acids ◽  
Liquid Chromatography ◽  
Amino Acid ◽  
Ion Exchange ◽  
Amino Acid Analysis ◽  
Corrosion Damage ◽  
Essential Amino Acids ◽  
Detection Sensitivity ◽  
Poultry Products ◽  
Ion Exchange Separation

Abstract Moore's and Stein's classical ion-exchange separation of amino acids remains the standard by which all methods are judged. The adaptation of liquid chromatography (LC) equipment to amino acid analysis was inevitable because microprocessor control of gradients allowed almost infinite variation in gradient shape, producing superior resolution with only 2 buffers. The versatility of LC equipment allowed the instruments to be used for other assays. Adaptation of orthophthalaldehyde (OPA) to amino acid analysis increased detection sensitivity to the picomole range. A method for essential amino acids analysis in mechanically separated red meat and poultry products has been adapted to liquid chromatography using postcolumn hypochlorite oxidation, OPA derivatization, and fluorescence detection. Separation is achieved with 2 sequential concave exponential gradients combining ionic strength and pH increases with halidecontaining buffers. Hydroxyproline and proline are detected with increasing sensitivity through the use of 3-mercaptopropionic acid in a stabilized OPA reagent. Sample preparation is a critical part of the method. A defatting procedure removes fat and other nonprotein nitrogenous substances. The hydrolysis procedure is designed to protect tryptophan which can be routinely assayed in hydrolysates with a modified flow program. Corrosion damage to the equipment by halide buffers has brought about a search for alternative methodology.

scholarly journals Year-Long Validation Study and Reference Values for Urinary Amino Acids Using a Reversed-Phase HPLC Method

2001 ◽  
Vol 47 (3) ◽  
pp. 575-583 ◽  
Author(s):  
Rafael Venta
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Reference Values ◽  
Amino Acid Analysis ◽  
Reference Intervals ◽  
Reversed Phase ◽  
Hplc Method ◽  
Rp Hplc ◽  
Age Related

Abstract Background: Reversed-phase HPLC (RP-HPLC) has become an alternative to ion-exchange chromatography for amino acid analysis in biological fluids. However, validation studies for its urine application are limited, and the corresponding reference values have not been reported extensively. We studied the long-term performance of a commercial HPLC method for urine amino acid analysis and established specific age-related reference values for urine amino acid excretion. Methods: Method performance was continuously assessed by recovery and precision studies with urine samples and controls, respectively. Healthy individuals were prospectively analyzed throughout a 5-year period. Excretion of individual amino acids, expressed as mmol/mol of creatinine, was included in six age-related groups for random urine samples (0–1 month, 1–12 months, 1–3 years, 3–8 years, 8–16 years, and >16 years) and in two groups for 24-h urine collections (8–16 years and >16 years). Results: Over a 1-year period, CVs for retention times were <0.5% and 3.3% for within- and between-run imprecision, respectively. For amino acid concentrations, within-run CVs were 2.9–17% and between-run CVs were 7.1–46% for the same period. Amino acid recoveries were 78–122%. Reference intervals for 35 amino acids were calculated and compared with the concentrations observed in patients diagnosed with specific pathologies. A few statistically significant differences were found between the reference intervals derived using random and 24-h urine collections. Conclusions: Long-term reliability of the RP-HPLC method for urine amino acid analysis has been demonstrated. Representative age-related reference intervals for the RP-HPLC method in both random urine and 24-h urine collections have been established, and their feasibility for diagnosis of aminoaciduria has been shown. These intervals could serve as a guide for laboratories changing to HPLC methods.

scholarly journals Pre-column dervatization of amino acids from nigella sativa L seed hydrolysates by reversed phase HPLC

2008 ◽  
Vol 5 (1) ◽  
pp. 125-130
Author(s):  
Baghdad Science Journal
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Seed Protein ◽  
Protein Hydrolysate ◽  
Nigella Sativa ◽  
Reversed Phase ◽  
Amino Acid Derivatives ◽  
Derivatizing Agent ◽  
Primary Amino ◽  
The Common

A rapid and sensitive method for analysis of amino acid hydrolysates of nigella sativa L seed has been developed using O-phthaldialehyde(OPA ) as a pre-column derivatizing agent. OPA reagents in the presence of mercaptoethanol react rapidly with primary amino acids ( less than 60 sec.) to form isindole derivatives which easily separated with good selectivity on ODS column. Resolution of amino acid derivatives is carried out with a methanol gradient in 0.01 maqueous sodium acetate. pH 7.1 . The quantitation of amino acid derivatives is reproducible within an average relative deviation of + 1.4% the linearity for most amino acids were more than 0.9993 with detection limit of 0.2 ppm. 15 amino acid were detected in the analysis of the seed protein hydrolysate. The presence of glutamic acid, alanine, leucine, cystine phenylalanine, aspartic acid in large quantities. The common separated amino acids were detected by U.V at 338 nm within 21 minutes.

scholarly journals Regulation of rat magnocellular neurosecretory system by D-aspartate: evidence for biological role(s) of a naturally occurring free D-amino acid in mammals

2000 ◽  
Vol 167 (2) ◽  
pp. 247-252 ◽  
Author(s):  
H Wang ◽  
H Wolosker ◽  
J Pevsner ◽  
SH Snyder ◽  
DJ Selkoe
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Amino Acid ◽  
Physiological Function ◽  
Neurosecretory System ◽  
Milk Ejection ◽  
Magnocellular Neurons ◽  
Rat Hypothalamus ◽  
Naturally Occurring

Little evidence is available for the physiological function of D-amino acids in species other than bacteria. Here we demonstrate that naturally occurring freed -aspartate (D-Asp) is present in all magnocellular neurons of rat hypothalamus. The levels of this naturally occurring D-amino acid were elevated during lactation and returned to normal thereafter in the magnocellular neurosecretory system, which produces oxytocin, a hormone responsible for milk ejection during lactation. Intraperitoneal injections of D-Asp reproducibly increased oxytocin gene expression and decreased the concentration of circulating oxytocin in vivo. Similar changes were observed in the vasopressin system. These results provide evidence for the role(s) of naturally occurring free D-Asp in mammalian physiology. The findings argue against the conventional concept that only L-stereoisomers of amino acids are functional in higher species.

The C-terminal sequence of amino acid residues of κ-casein isolated from buffalo's milk

1967 ◽  
Vol 34 (1) ◽  
pp. 85-88 ◽  
Author(s):  
M. H. Abd El-Salam ◽  
W. Manson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Peptide Chain ◽  
Amino Acid Residues ◽  
Terminal Sequence ◽  
Carboxypeptidase A ◽  
Phosphorous Content ◽  
Single Peptide

SummaryWhen κ-casein from buffalo's milk was treated with carboxypeptidase A (EC 3. 4. 2. 1),4 amino acids, valine, threonine, serine and alanine were released from the protein in a manner consistent with the view that they originate in the C-terminal sequence of a single peptide chain. The amounts produced suggest a minimum molecular weight for buffalo κ-casein of approximately 17000, in agreement with the value calculated from the phosphorous content on the basis of the presence of 2 phosphorus atoms/molecule. A comparison is made with the C-terminal sequence reported for bovine κ-casein.

Circular dichroism spectra of amino acid complexes. II. Chelated amino acid complexes with the CoN5O chromophore

1970 ◽  
Vol 23 (9) ◽  
pp. 1735 ◽  
Author(s):  
CJ Hawkins ◽  
PJ Lawson
Keyword(s):  
Amino Acids ◽  
Circular Dichroism ◽  
Amino Acid ◽  
Visible Region ◽  
Optically Active ◽  
Circular Dichroism Spectra ◽  
Amino Acid Complexes ◽  
Similar Series ◽  
Cotton Effects ◽  
Circular Dichroïsm

The circular dichroism spectra of a series of optically active (α-aminocarboxylato)tetraamminecobalt(111) complexes have been measured in aqueous solution, and in the presence of salts of polarizable anions. The observed spectra in the visible region have been analysed to determine the signs of the Cotton effects of the three components of the 1A1g ↔ 1T1g cobalt(111) transition. For L-amino acids, the transition with A2g(D4h) parentage is negative, and the two transitions with Eg(D4h) parentage have opposite signs. Published circular dichroism spectra of complexes of the type [Co(en)2(L-am)]2+ were similarly interpreted in terms of a perturbed tetragonal chromophore, and it was shown that the vicinal effect of the L-amino acids imposed the same signs onto the component transitions as for the tetraammines and for a similar series of pentaamminecobalt(111) complexes.

Effects of some Amino-acid and Purine Antagonists on Chick Embryos

Development ◽  
1958 ◽  
Vol 6 (2) ◽  
pp. 365-372
Author(s):  
C. H. Waddington ◽  
Margaret Perry
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Effective Concentration ◽  
Metabolic Inhibitors ◽  
Chick Embryos ◽  
Purine Analogue ◽  
Know How ◽  
Naturally Occurring ◽  
Different Types ◽  
Strong Action

Several authors have studied the effects on developing embryos of substances which are analogues of naturally occurring amino-acids and purines, and known to act, in other systems, as metabolic inhibitors. It was emphasized by Waddington, Feldman, & Perry (1955) that any particular substance may exhibit very different effects in embryos of different types. They found, for instance, that the purine analogue 8-azaguanine has a very strong action in the chick and a much lesser one in the newt embryo. It is therefore necessary to consider the various classes of embryos separately. In this communication we shall be concerned only with chick embryos. Substances under test can be administered to such embryos by injection through the shell, as was done in the paper cited above With this technique it is impossible to know how much diffusion takes place of the substance injected, and one cannot therefore be certain of the effective concentration which actually reaches the embryo.

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