Extraction of fixed, stained protein bands from gels for micro amino acids analysis using o-phthaldialdehyde.

A method is presented for extraction of fixed, stained protein bands from polyacrylamide gels suitable for automated fluorescence analysis of amino acids using o-phthaldialdehyde. Bands, containing microgram quantities of protein and stained with Coomassie blue, are extracted from homogenized gel slices with sodium dodecyl sulfate. The Coomassie blue and sodium dodecyl sulfate do not interfere with the amino acid determination, and contamination by ammonia from the gels is low. The method has been applied to the analysis of human carbonic anhydrase C, and the amino acid composition is found to be similar to that obtained by other methods requiring larger amounts of protein.     

Separation of o-Phthaldialdehyde Derivatives of Free Amino Acids from Plant Tissues by Isocratic Reverse-phase High-performance Liquid Chromatography

A reverse-phase high-performance liquid chromatography procedurehas been developed for rapid separation and quantitation offree amino acids as o-phthaldialdehyde derivatives. A two stepisocratic solvent system was used which enabled an accurateanalysis at nanomole level. However, two major disadvantagesto this procedure were the lack of reaction of proline and theco-elutions of threonine/glycine and tryptophan/methionine.The free amino acids in Zea mays roots were separated by usingthe method described. Amino acids, liquid chromatography, o-phthaldialdehyde derivatives, reverse-phase chromatography, Zea mays L, maize, corn     

Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated pre-column derivatization with o-phthaldialdehyde

A sensitive and reproducible fully automated method for the determination of amino acids in plasma based on reversed-phase high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. A 5-μm Spherisorb ODS 2 column (125 × 3 mm I.D.) was selected for routine determination. Over 40 physiological amino acids could be determined within 49 min (injection to injection) and 48 samples could be processed unattended. The coefficients of variation for most amino acids in plasma were below 4%. We were also able to measure trace amounts of amino acids in plasma normally not detected in a routine analysis. The results obtained with the method described compared favourably with those of conventional amino acid analysis (r = 0.997) and were in excellent agreement with those of other laboratories (r = 0.999).     

Measurements of polyamines and their acetylated derivatives in cell extracts and physiological fluids by use of an amino acid analyzer

A fast and sensitive method for the determination of free polyamines and their acetylated derivatives is presented. The separation is carried out on a Durrum DC-6A cation-exchange resin with an automated amino acid analyzer. The determination is based on a stepwise elution with a sodium chloride—sodium citrate buffer system. Detection is done by fluorescence of the o-phthaldialdehyde—polyamine conjugates. The sensitivity is in the picomole range. No prior purification step is needed. The method has been applied to cell extracts and urine samples.     

Staining for protease activity on polyacrylamide gels

A high-performance liquid chromatographic procedure has been developed for the detailed analysis of amino acids and related compounds in 10-μl samples of perilymph from the guinea pig cochlea (inner ear). The procedure employs an Aminco amino acid analyzer and combines the use of a single chromatographic column, lithium citrate buffers for elution, a change of column temperature, and fluorometric detection of o-phthaldialdehyde/2-mercaptoethanol adducts of primary amines. Sensitivity is about 0.2 pmol referenced to leucine. Fifty-four primary amine components are detectable in perilymph collected in relative silence. Twentynine compounds have been identified, and six are putative amino acid neurotransmitters. The present method provides new information about the chemical composition of perilymph and is suitable for the analysis of physiological fluids available only in volumes of several microliters.     

Determination of d- and l-aspartate in amino acid mixtures by high-performance liquid chromatography after derivatization with a chiral adduct of o-phthaldialdehyde

A sensitive and convenient method for the simultaneous determination of d- and l-aspartic acid in amino acid mixtures is described. The method involves derivatization of the mixture with a chiral fluorogen, followed by high-performance liquid chromatography on a reverse-phase column. The fluorogen used is an adduct of o-phthaldialdehyde with an optically active thiol, N-acetyl-l-cysteine. The sensitivity and accuracy of this method is similar to that using adducts of o-pthaldialdehyde with the achiral thiol, 2-mercaptoethanol. Five picomoles of d-aspartate can be accurately detected in the presence of a 100-fold excess of l-aspartate with a total analysis time (including derivatization) of 10 min.     

Determination of d-amphetamine in biological samples using high-performance liquid chromatography after precolumn derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid

An HPLC method is described for the determination of amphetamine using fluorometric detection after derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid. This procedure is more sensitive (detection limit 370 fmol in microdialysate buffer standards, 1.5 pmol in extracted plasma and tissue samples) than most of the previous methods described for the determination of amphetamine with HPLC-fluorescence detection. Due to the stability of the derivative it is also suitable for autosampling after manual derivatization. Investigators currently using o-phthaldialdehyde derivatization and fluorometric detection for amino acid determination should be able to rapidly implement this method.     

Automated fluorometric amino acid analysis: the determination of proline and hydroxyproline.

A fluorometric method for the automated determination of the imino acids proline and hydroxyproline has been developed. The assay is based on the postcolumn reaction of the imino acids with alkaline sodium hypochlorite, which yields oxidation products amenable to detection with fluorogenic amine reagents. The method is simple and can be adapted readily to high-sensitivity amino acid analyzers which use o-phthalaldehyde for detection. As little as 10 pmol proline and 20 pmol hydroxyproline can be determined accurately. Thus the full array of natural imino and amino acids can now be determined on a high-sensitivity amino acid analyzer using o-phthalaldehyde.     

Quantitative gas chromatographic analysis of amino acids on a short glass capillary column

A study of the quantitative gas chromatographic analysis of protein amino acids as their N-heptafluorobutyric amino acid n-propyl esters on a glass capillary column has been made. The analysis is completed within 35 min with good separation of the common protein amino acids in a single-column run.Hydrolyzed peptides have been analyzed. The analyses were performed with a precision varying between 1 and 6% (mean relative standard deviation) depending on the number of amino acid residues in the peptide. The amount taken for analysis was 20–300 μg. The results agree with the known sequences of the peptides and with the analyses by ion-exchange chromatography except for cysteine. This amino acid can be analyzed after modification as S-methylated cysteine.     

Gas Chromatography-Mass Spectrometry of N- Heptafluorobutyryl Isobutyl Esters of Amino Acids in the Analysis of the Kinetics of [N]H(4) Assimilation in Lemna minor L

Rapid, sensitive, and selective methods for the determination of the ¹⁵N abundance of amino acids in isotopic tracer experiments with plant tissues are described and discussed. Methodology has been directly tested in an analysis of the kinetics of [¹⁵N]H₄⁺ assimilation in Lemna minor L. The techniques utilize gas chromatography-mass spectrometry selected ion monitoring of major fragments containing the N moiety of N-heptafluorobutyryl isobutyl esters of amino acids. The ratio of selected ion pairs at the characteristic retention time of each amino acid derivative can be used to calculate ¹⁵N abundance with an accuracy of ±1 atom% excess ¹⁵N using samples containing as little as 30 picomoles of individual amino acids. Up to 11 individual amino acid derivatives can be selectively monitored in a single chromatogram of 30 minutes. It is suggested that these techniques will be useful in situations where the small quantities of N available for analysis have hitherto hindered the use of ¹⁵N-labeled precursors.     

Fluorometric analysis of N alpha-methylamino acids using fluorescamine

The fluorometric amino acid analyzer based on fluorescamine has been utilized for quantitative determination of N^α-methylamino acids. N-Chlorosuccinimide (1 × 10^?3m in 0.05 m HCl) was continuously introduced into the column eluate to convert N^α-methylamino acids to fluorescamine-sensitive methylamine. As little as 100 pmoles of l-N-methylalanine was detectable with a linear fluorescence response up to 10.0 nmoles. Distinction of primary and secondary amino acids was achieved by carrying out duplicate analyses with and without the introduction of the N-chlorosuccinimide solution.     

Determination of free amino acid enantiomers in rat brain and serum by high-performance liquid chromatography after derivatization with N-tert.-butyloxycarbonyl- -cysteine and o-phthaldialdehyde

The concurrent determination of free amino acid enantiomers and non-chiral amino acids in rat brain and serum was accomplished by high-performance liquid chromatography with fluorimetric detection after derivatization with N-tert.-butyloxycarbonyl- -cysteine and o-phthaldialdehyde. The method revealed the presence of a large amount of free -serine (0.22 μmol/g of tissue; + RATIO = 0.25) in the brain whereas -aspartate and -alanine were established to be at trace levels. These results further support the presence of -serine in adult brain tissues as demonstrated by recent work using gas chromatography.     

Analysis of the amino acid 3-methylhistidine by gas-liquid chromatography

A method for the quantitative separation of 3-methylhistidine from other amino acids, by gas-liquid chromatography, has been developed. This method gives complete resolution of the N-heptafluorobutyryl isobutyl esters of 20 amino acids with the use of a single column packed with 3% SE-30 on $\text{100}\text{120}$ mesh Gas Chrom Q. Using this method the 3-methylhistidine content of urine and meat has been determined.     

Drought stress increases the production of 5-hydroxynorvaline in two C4 grasses

Plants produce various compounds in response to water deficit. Here, the presence and identification of a drought-inducible non-protein amino acid in the leaves of two C₄ grasses is first reported. The soluble amino acids extracted from the leaves of three different species were measured by high-performance liquid chromatography of derivatives formed with o-phthaldialdehyde and β-mercaptoethanol. One amino acid that increased in amount with drought stress had a retention time not corresponding to any common amino acid. Its identity was determined by metabolite profiling, using ¹H NMR and GC-MS. This unusual amino acid was present in the dehydrated leaves of Cynodon dactylon (L.) Pers. and Zoysia japonica Steudel, but was absent from Paspalum dilatatum Poir. Its identity as 2-amino-5-hydroxypentanoic acid (5-hydroxynorvaline, 5-HNV) was confirmed by synthesis and co-chromatography of synthetic and naturally occurring compounds. The amount of 5-HNV in leaves of the more drought tolerant C₄ grasses, C. dactylon and Z. japonica, increased with increasing water deficit; therefore, any benefits from this unusual non-protein amino acid for drought resistance should be further explored.     

Application of tritium high resolution NMR spectroscopy to analysis of tritium-labelled amino acids and peptides

Summary A method has been developed for the qualitative and quantitative analysis of complex isotopic mixtures of tritium-labelled amino acids and peptides by using high resolution³H NMR spectroscopy at 266.8 MHz. Determined were tritium distribution in alanine, glycine, tryptophan and 4-hydroxyproline amino acids, as well as in glycine and valine residues of peptides. Approaches have been worked out for the determination of spin coupling constants and isotope chemical shifts for the strongly coupled nonequivalent atoms of the methylene groups.     

A fast and sensitive method for measuring picomole levels of total free amino acids in very small amounts of biological tissues

Summary. In the present study we describe a simple and fast method to measure the concentration of total free amino acids in very small amounts of biological tissues. The procedure described here is based on the reaction of free amino acids with o-phthaldialdehyde (OPA) in the presence of a reducing agent, β-mercaptoethanol (MET), to give a complex which can be measured by fluorescence. It is a very rapid process and has the same reliability as the conventional ninhydrin method of Moore and Stein but is about 500 times more sensitive. The sensitivity of the new protocol is such to permit the determination with high reliability of very small amounts of free amino acids at picomole levels, either in a standard amino acid mixture or in biological tissues, without chromatographic separation of the amino acids. It is particularly useful when the amount of the sample is very low, e.g. on a single pituitary or pineal gland of small animals or on single cells, such as oocytes or eggs, as well as single ganglions or axons of marine invertebrates. Received September 22, 1999 Accepted July 5, 2000     

Micro-analysis of amino acids

An automated amino acid analyzer has been developed for the analysis of amino acids with the sensitivity at the 10–100 pmol level except for proline which requires >50 pmol. o-Phthalaldehyde, in the presence of 2-mercaptoethanol, is used for the fluorometric detection of amino groups (Roth, M. (1971) Anal. Chem. 43, 880–882). A post-column reaction of the amino acid with sodium hypochlorite (Bohlen, P. and Mellet, M. (1979) Anal. Biochem. 94, 313–321) gives oxidation products amenable to detection with o-phthalaldehyde. The instrument uses high-performance liquid chromatographic pumps capable of micro-flow rates with a minimum pulsation. The method is suitable for routine analyses of amino acids at picomole levels with reproducibility and accuracy comparable to the ninhydrin-based amino acid analysis.     

Changes in free amino acid composition in haemolymph of larvae of the wax moth,Galleria mellon ella L., during cold acclimation

• 1.1. Free amino acids were analysed in the haemolymph of Galleria mellonella larvae by HPLC chromatography with o-phthaldialdehyde (OPA)-l-thio-β-d-glucose as derivatization agent.
• 2.2. Fourteen primary amino acids were detected among which glutamine, alanine, γ-aminobutyric acid (GABA) and glycine predominated and constituted 67.7% of the amino acids found.
• 3.3. The concentration of GABA increased significantly with the age of larvae entering the wandering phase and reached a maximum during metamorphosis.
• 4.4. Analysis of cold-acclimated larvae revealed a net increase of free primary amino acids from 96 to 151.8 μmol/ml during consecutive acclimation to 0°C within 20 days and to 205.4μmol/ml during cold shock injury at 0°C (3 hr).
• 5.5. The bulk of this increase was accounted for by alanine, glycine, phenylalanine and lysine.

     

A simple quantitative method for the determination of 3-fluorotyrosine substitution in proteins

A rapid quantitative method is described for determining 3-fluorotyrosine incorporation into proteins. Derivatives of tyrosine and 3-fluorotyrosine with o-phthalaldehyde are well separated from one another by a reverse-phase high-performance liquid chromatography system used for routine analyses of o-phthalaldehyde-amino acid derivatives. Since both amino acids are well resolved from all other derivatized amino acids, the method is useful for amino acid analyses of proteins. Determination of the fluorotyrosine content of proteins by this method involves a single separation step, is reproducible, and requires no corrections for stability or yield. Further, the o-phthalaldehyde derivatives of 5-fluorotryptophan, 2-fluorophenylalanine, 3-fluorophenylalanine, and 4-fluorophenylalanine can also be resolved. The method may be generally applicable to fluorinated aromatic amino acid-labeled proteins that are studied structurally and dynamically by nuclear magnetic resonance.     

Fluorescence stopped-flow study of the o-phthaldialdehyde reaction

The fluorogenic reaction involving three species, namely, a primary amine, o-phthaldialdehyde (OPA), and a thiol compound was studied with the fluorescence stopped-flow technique. The results are consistent with the reaction of the amine with a 1:1 adduct of OPA and the thiol compound. The equilibrium constant for the formation of the adduct, OPAME, from OPA and mercaptoethanol (ME) was determined to be 164 m^?1. A survey of the rates of reaction of OPAME with various amino acids demonstrated that with OPA: ME:amine equal to 1:2.4:1 (total OPA concentration 0.5 to 3.0 × 10^?3m), the reaction followed second-order kinetics, with k = 150 to 450 m^?1s^?1 at pH 9.O. The differences in rates are discussed in relation to structural differences between the amines. The reaction, when conducted under conditions of excess OPAME yielded pseudo-first-order kinetics, with rates consistent with the second-order rate constants. The rate of reaction of OPAME with alanine was maximal at pH 10.5–11, and a great excess of ME resulted in a slower rate. Slower rates were also observed if ME was replaced by dithiothreitol or 1-propanethiol.