A new high-performance liquid chromatography assay for glutamine synthetase and glutamate synthase in plant tissues

Glutamic acid and glutamine, formed in plant tissue extracts by glutamate synthase and glutamine synthetase, respectively, were separated by derivatization with o-phthaldialdehyde followed by reverse-phase high-performance liquid chromatography on a μBondapak C₁₈ column. The derivatives were eluted by isocratic elution, and the mobile phase was a 20 mm sodium phosphate buffer (pH 6.8) with 36% methanol. The procedure is rapid, sensitive, and requires a minimum sample.     

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.     

The interaction of amino acids with o-phthaldialdehyde: A kinetic study and spectrophotometric assay of the reaction product

A detailed study has been made of the kinetics of interaction between amino acids and esters of amino acids and o-phthaldialdehyde in the presence of mercaptoethanol. The reaction products have been characterized. A spectrophotometric method for quantitative analysis of all amino acids, except proline and hydroxyproline, has been developed. The possibility of determination of amino acid esters in mixtures containing free amino acids has been demonstrated. It is noted that determination of glycine and histidine with the help of o-phthaldialdehyde has certain specificities associated with faster, compared to other amino acids, degradation of their derivatives. Optimal conditions for quantitative analysis of amino acids in solutions of higher than 10^?5m concentration are recommended. The reproducibility of the determination was ±2%.     

A method for hydrolyzing and determining the amino acid compositions of picomole quantities of proteins in less than 3 hours

A method is described for quantitatively hydrolyzing proteins in 45 min and for analyzing the hydrolysates by high-performance liquid chromatography in an additional 52 min. The α-amino acids were detected by the fluorescence of their o-phthaldialdehyde derivatives. Ten picomoles of each of the commonly occuring α-amino acids could be reliably determined. The method described yielded OPA-ethanethiolamino acid derivatives that were stable for $\text{1}\text{h}$ h and the HPLC method produced a better separation than previously published methods.     

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.     

Measurement of amino acid release from cultured cerebellar granule cells by an improved high performance liquid chromatography procedure

Endogenous amino acid release was examined in rat cerebellar primary cultures comprising more than 95% of glutamatergic granule cells. Eighteen amino acids were determined in the cell extracts and in the release fractions by high performance liquid chromatography, using precolumn derivatization witho-phthaldialdehyde and separation on a reverse-phase column using a multi-step gradient system of two solvents (0.1 M Na⁺acetate, pH 7.2/methanol: tetrahydrofuran, 97:3). The fluorimetric response was linear, at least in the range of 2–162 pmol, for all the amino acids analysed, with a detection limit of 1 pmole. We observed a good reproducibility in within-assay and between-assay coefficients of variation of the retention times and fluorescence yield. When cultured granule cells were exposed to the excitatory amino acid receptor agonist quisqualic acid (50 M), we observed a net increase in the release of glutamate (3 fold over the baseline) and a smaller increase in that of aspartate (2 fold) and taurine (1.6 fold). Other amino acids were not significantly affected. GABA levels were below detection limits, due to the minimal number of GABAergic neurons present in the cultures.     

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).     

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.     

Simultaneous analysis of the non-canonical amino acids norleucine and norvaline in biopharmaceutical-related fermentation processes by a new ultra-high performance liquid chromatography approach

In this study, a precise and reliable ultra-high performance liquid chromatography (UHPLC) method for the simultaneous determination of non-canonical (norvaline and norleucine) and standard amino acids (aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, tyrosine, methionine, valine, phenylalanine, isoleucine, leucine) in biopharmaceutical-related fermentation processes was established. After pre-column derivatization with ortho-phthaldialdehyde and 2-mercaptoethanol, the derivatives were separated on a sub-2 μm particle C₁₈ reverse-phase column. Identification and quantification of amino acids were carried out by fluorescence detection. To test method feasibility on standard HPLC instruments, the assay was properly transferred to a core–shell particle C₁₈ reverse-phase column. The limits of detection showed excellent sensitivity by values from 0.06 to 0.17 pmol per injection and limits of quantification between 0.19 and 0.89 pmol. In the present study, the newly established UHPLC method was applied to a recombinant antibody Escherichia coli fermentation process for the analysis of total free amino acids. We were able to specifically detect and quantify the unfavorable amino acids in such complex samples. Since we observed trace amounts of norvaline and norleucine during all fermentation phases, an obligatory process monitoring should be considered to improve quality of recombinant protein drugs in future.     

A comparative study of amino acid measurement in leaf extracts by gas chromatography-time of flight-mass spectrometry and high performance liquid chromatography with fluorescence detection

Gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS) has become a promising technique for simultaneous and rapid analysis of small metabolites in complex mixtures. The aim of this work was to establish the quantitative nature of the information generated by amino acid analysis of crude leaf extracts using GC-TOF-MS. Dried aliquots of methanol/water extracts of Arabidopsis leaves were analysed in parallel by GC-TOF-MS following trimethylsilylation or high performance liquid chromatography and fluorescence detection of o-phthaldialdehyde derivatives (OPA-HPLC). Twenty amino acids could be routinely detected in leaf extracts by both methods. Because of instability of some trimethylsilylated derivatives, all GC-TOF-MS analyses were performed within a window of 2 h 30 min following derivatization. Repeatability studies showed that relative standard deviations for multiple injections of a single extract were below 20% for both techniques, though significantly smaller for OPA-HPLC. Similar between-extract variability and condition-independent biological variation were detected by OPA-HPLC and GC-TOF-MS, and both techniques detected similar environmentally induced changes in four major amino acids. Recovery of standard compounds through the extraction procedure was between 80% and 120% for OPA-HPLC but more variable when analysed by GC-TOF-MS. When quantified on the basis of tissue fresh weight according to response factors of mixed standards, the two techniques gave consistent values for a number of amino acids but divergent values for others. Taken together, the results suggest GC-TOF-MS analysis of Arabidopsis leaves with the present protocol can be used for absolute quantification of 4–7 amino acids, accurate relative quantification of 8–11 amino acids, and more limited quantification for five compounds of this class.     

Biosynthesis of Cytokinin in Germinating Seeds of Zea mays

The embryos of germinating Zea mays seed were supplied with[¹⁴C]-adenine Following incubation, the tissue was extractedand extensively purified by non-exchange chromatography andthin layer chromatography. Radioactivity was found to be incorporatedinto zeatin nucleotide indicating that the embryo in the germinatingseed is capable of cytokinin biosynthesis. Key words: Zea mays cytokinin, zeatin nucleotide, biosynthesis, seed     

Evaluation of methods for the separation and analysis of proteins and free amino acids in phytoplankton samples

Due to their importance as not only major constituents in paniculatematter but also the metabolism of nitrogen in marine microorganisms,numerous methods have been employed to measure proteins andfree amino acids. However, two difficulties frequently complicatethese measurements. First, an initial separation of proteinsfrom free amino acids is helpful since most analytical methodsare somewhat sensitive to both compound types. Second, the choiceof detection techniques that minimize response differences betweenvarious proteins or amino acids is desirable since natural samplesof microorganisms consist of mixtures of many proteins and aminoacids. To address these problems, four protein detection techniques(modified Lowry et al., Dorsey et al., Bradford and fluorescamine)and two amino acid detection techniques (fluorescamine and o-phthaldialdehyde)were evaluated. Relative extraction efficiencies for proteinfrom phytoplankton samples were also evaluated with six homogenizationsolutions/protocols (TCA, NaOH, boiling NaOH, Triton X-100,NaOH plus Triton X-100 and distilled water). TCA homogenizationyielded the highest protein recoveries, and sufficient physicalseparations between proteins and free amino acids were obtainedwith TCA concentrations between 0.18 and 0.37 M. Results ofthese studies allowed for development of a method for extracting,separating and analyzing proteins and total free amino acidsfrom a common phytoplankton sample. The procedure involves initialhomogenization in a TCA solution, followed by centrifugationto separate protein and free amino acid fractions. Proteinsare then analyzed by a modification of the Lowry et al. procedure,and amino acids by a fluorescamine procedure. ²Present address: Science Applications International Corporation,4224Campus Point Court, San Diego, CA 92121, USA     

Reverse-phase liquid chromatographic analysis of amino acids after reaction with o-phthalaldehyde

Precolumn formation of o-phthalaldehyde (OPA) derivatives of amino acids, followed by reverse-phase separation and fluorescent detection, provides rapid, sensitive amino acid analysis. Eighteen OPA-amino acid derivatives are resolved on a Micropack MCH 5 column and can be measured at picomole levels. Ease of derivative preparation and separation makes liquid chromatographic analysis of OPA-amino acids a convenient and improved technique for measuring or confirming the presence of low levels of amino acids in aqueous solutions. Use of the method was demonstrated by measuring low concentrations of amino acids released from zooplankters stressed by contaminants.     

Effect of the Osmotic Environment on Assimilate Transport in Isolated Developing Embryos of Maize (Zea mays)

Embryos obtained from developing kernels of maize (Zea maysL.) were incubated in a bathing medium, to measure the effectof the osmotic environment on the balance between uptake andrelease of assimilates by the embryo. Net efflux of sucroseand amino acids from the embryo decreased with increasing mannitolconcentration in the bathing medium and net uptake of [¹⁴C]valine increased with increasing mannitol concentration. Therole of a high osmolality of the seed apoplast in seed developmentis discussed Zea mays, maize, embryo, seed development, assimilate transport, turgor-sensitive transport     

Alterations of Free Amide and Amino Acid Contents During the Development of Maize Plants, Zea mays L.

Concentrations of free amino acids and amides were measuredin organs of maize plants, Zea mays L. in the period from 14d before pollen liberation until complete seed maturation. Inthis time anthesis took place and only the ovaries of ear 9(numbered from below) developed into seeds. In mature leaf bladesNH₄ ion assimilation had ceased and asparagine and glutaminewere not found there. N redistribution induced the occurrenceof large amounts of aspartate, glutamate and alanine. The amountsof amides in leaf sheaths and stem parts depended on the neighbourhoodof generative parts. The generative plant parts can be distinguishedfrom adjacent vegetative plant parts in concentrations of freeproline or asparagine. Proline occurred in pollen but not inthe empty anthers. Ears had a small, early peak amount of prolinemostly before pollination. Only the ninth ear had a first maximumproline amount after the fifth day of pollen liberation. A secondproline peak in the ears coincided with the period of maximumincrease in d. wt. The occurrence of proline in the generativeorgans relative to metabolic processes inducing fertility orseed maturation is discussed. Zea mays L., amides, amino acids, amino-transferring components, asparagine, glutamine, proline     

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.     

Metabolism of [14C]Indole-3-Acetic Acid by Coleoptiles of Zea mays L.

Reverse-phase high-performance liquid chromatography was usedto analyse [¹⁴C]-labelled metabolites of indole-3-acetic acid(IAA) in coleoptile segments of Zeo mays seedlings. After incubationfor 2 h in 10^–2 mol m^–3 [2-¹⁴C]IAA, methanolic extractsof coleoptiles contained between six and ten radioactive compounds,one of which co-chromatographed with IAA. The metabolic productsin coleoptile extracts appeared to be similar to those in rootextracts, with an oxindole-3-acetic-acid-like component as theprincipal metabolite, but the rate of metabolism was slowerin coleoptile than in root segments. Decarboxylation did notappear to play a major role in the metabolism of exogenous IAAduring the short incubation periods. Moreover, external IAAconcentration had little effect on the pattern of metabolism.Coleoptile segments were also supplied with [¹⁴C]IAA from agardonor blocks placed at the apical ends, and agar receiver blockswere placed at the basal ends. After incubation for 4 h, theidentity of the single radioactive compound in the receiverblocks was shown to be IAA by both reverse-phase high-performanceliquid chromatography and gas chromatography-mass spectrometrytechniques. Key words: Zea mays, Coleoptile, High-performance liquid chromatography, Indole-3-acetic acid     

Root Growth Inhibitors from Root Cap and Root Meristem of Zea mays L.

A micro-assay based on the growth inhibition of root segmentsof the seminal roots of Zea mays has been used to investigatethe root-growth-inhibiting substances in root caps and meristemsrespectively of the roots of Zea mays. This micro-assay is sensitiveto 50 pg of IAA or less. Paper chromatography of the acid fractionof methanolic extracts shows the presence of one main inhibitorin root caps and a different main inhibitor in root meristems.Neither is IAA, whose presence in meristems is sometimes indicatedby small inhibitions (or stimulations) at the characteristicRf of IAA. A Commelina leaf-epidermis assay shows the presenceof one stomata-closing ABA-like substance in root caps and onein meristems, one corresponding in Rf to the main root-growthinhibitor from the root cap. The implications of these findingsfor the geotropic responses of roots is briefly discussed.     

A Comparative Study of the Structure-Function Relationship of Cross Veins in Leaves of Digitaria eriantha and Zea mays

Ultrastructural and functional differences between the crossveins of Digitaria eriantha and Zea mays were investigated.Cross veins of both genera possess similar conducting tissues,namely one tracheary element and one sieve cell. In Digitariaeriantha these conducting elements are associated with onlytwo parenchyma cells, and, those in Zea mays are completelysurrounded by chJorenchyma cells. The protein ribulose 1,5-bisphosphatecarboxylase/oxygenase (rubisco) was used as a probe for CO₂fixation sites by comparing its distribution in the varioustissue types in the leaves of the two genera. The protein wasfound to be equally and uniformly distributed in the stromalregions of the chlorenchyma sheath cell chloroplasts of longitudinalveins of both genera. The chlorenchyma sheath cells in crossveins of Zea mays show a similar distribution of the enzymeas the longitudinal bundles. However, this enzyme was shownto be absent in the cross vein parenchyma cells of Digitariaeriantha and in the mesophyll cells of both genera. Cross veins, immuno-gold labelling, ribulose 1,5-bisphosphate carboxylase/oxygenase, Digitaria eriantha, Zea mays     

Analysis of dansyl amino acids by reverse-phase high-performance liquid chromatography

All 24 dansyl amino acids were separated by reverse-phase high-performance liquid chromatography on Develosil C8-5, using a linear gradient made from Tris-HCl buffer (pH 7.75) and methanol. A linear relationship between the amount of sample and peak area was found over the range of 6 to 300 ng (0.02–1 nmol) of dansyl derivatives. An application of this method to the NH₂-terminal analysis of lysozyme is described.