Determination of biogenic amines using heterocyclic cation and aromatic anion elution

Heterocyclic cation and aromatic anion are used in the chromatographic buffers for the analysis of monoamines and diamines on a sulfonated cation-exchange column using an amino acid analyzer. All elution buffers employed for these analyses had a pH of 5.0 to maximize the ninhydrin color reaction. These methods have been successfully used for biological samples. Using a two-column (0.8 × 12 cm) system, with a buffer flow rate of 50 ml/h, analysis can be carried out in 330 min for monoamine and diamine mixtures on 0.5 to 10 nmol of each amine.     

An improved method for the determination of galactosaminitol,glucosaminitol, glucosamine,and galactosamine on an amino acid analyzer

Existing methods for the separation and quantitation of galactosaminitol, glucosaminitol, glucosamine, and galactosamine with an amino acid analyzer have been improved substantially by (1) addition of 15% ($\text{v}\text{v}$) 1-propanol to the citrate-borate buffer, (2) utilization of a two-buffer system, (3) doubling the length of the ninhydrin reaction coil, and (4) inclusion of an internal standard, ε-amino-n-eaproic acid, in each sample. These modifications decrease the time for the separation of these compounds to 110 min and increase the sensitivity for hexosaminitols to 3–4 nmol, and that for hexosamines, to 2 nmol.     

Effect of manganese on ninhydrin color development by amino acids

Addition of microgram quantities (5–10 μg per 2 ml) of Mn²⁺, Fe²⁺, and Mo²⁺ increases the intensity of color developed by amino acids two- to three-fold when reacting with ninhydrin. The lowest limit detected by the increased sensitivity of the reaction is 3 nmol of an amino acid. Cd²⁺, Zn²⁺, and Al³⁺ depress the color formation due to reaction between amino acids and ninhydrin. When chromatograms of amino acids are first sprayed with aqueous manganese chloride and later with ninhydrin or with ninhydrin solution containing manganese, not only is the sensitivity increased, but the stained spots retain their color for longer periods.     

A precise method for the quantitation of proteins taking into account their amino acid composition.

A method for the quantitation of protein in biological material is described which gives the same response for all proteins irrespective of their amino acid composition. The method is based on the ninhydrin reaction of amino acids released after total acid hydrolysis of 5- to 20-μl solutions containing 1 to 100 μg of protein. The ammonia is released from the hydrolysate by diffusion and the amino acids are quantitated without fractionation using the continuous-flow system of an amino acid analyzer. Calibration is obtained with solutions of known amino acid content. The protein of a sample is calculated by multiplying the nanomoles of total amino acids found by a conversion factor F. F is the weight in micrograms of 1 nmol of the specific mixture of amino acid residues that the protein of the sample is composed of F has to be determined once for all further quantitations of the same material by quantitative amino acid analysis following standard procedures. By this method as little as 30 ng of protein per aliquot of hydrolysate analyzed can be determined.     

Single column analysis of amino acids in protein hydrolysates utilizing the fluorescamine reaction

A system is described for the separation of the amino acids commonly found in protein hydrolysates at the picomole level using a single ion exchange column and for their quantitation by the fluorescamine (4-phenylspiro[furan-2 (3H),1′-phthalan]-3,3′-dione) reaction. Three sodium citrate buffers were required for the separation of the amino acids with an analysis time of approximately 3 hr. The amino acids in 1 μg of hydrolyzed bovine serum albumin were separated using a single ion exchange column and were detected in the effluent from the column by the fluorescamine assay. The results were compared with those obtained using a commercial amino acid analyzer and 150 μg of hydrolyzed bovine serum albumin. The chromatogram produced by the more sensitive analyzer utilizing the fluorescamine reaction to detect the amino acids compared favorably with the chromatogram produced by the commercial analyzer utilizing the ninhydrin reaction with the exception that the proline peak was missing. Proline and hydroxyproline fail to yield fluorescence on reaction with fluorescamine unless converted from imines to primary amines.     

Measurement of aspartylglucosamine in physiological fluids with an amino acid analyzer: Fused peak analysis with dual photometers

Methods for the quantitation of aspartylglucosamine using an amino acid analyzer equipped with a physiological column and dual photometers were developed. Aspartylglucosamine and urea coelute, but their ninhydrin derivatives have different color factors at 440 and 590 nm. The concentration of aspartylglucosamine may be determined in a urea mixture by amino acid chromatography, absolute peak area measurement, and the 590:440 peak area ratio. A second method was also developed in which urea was completely digested with urease before chromatography, permitting the direct quantitation of aspartylglucosamine.     

Identification of a crown-gall tumor growth factor as GABA

A crown-gall tumor growth factor (TGF-II) isolated from bean leaves inoculated with Agrobacterium tumefaciens strain 13333 is shown to be γ-aminobutyric acid (GABA). This identification is based on the comparative behavior of purified TGF-II and authentic GABA with respect to elution from preparative ion exchange and molecular sieve columns, ninhydrin reaction, TLC, co-chromatography on an automated amino acid analyzer, MS analysis and biological activity. GABA is detected by bioassay only in bean leaves infected with the bacterium and is in growth limiting supply when only a few tumors are present per leaf. GABA promotes tumor growth when as little as 1 ng is applied per leaf.     

Analysis of adipimidate-crosslinked lysine

The chromatographic conditions for separation of N,N′-bislysyl(?-N)adipamidine and N-lysyl(?-N)adipamidinic acid, which were the products of acid hydrolysis of proteins treated with adipimidate esters, from other amino acids on an amino acid analyzer were established including their ninhydrin color values. Kinetics of decomposition of these lysine derivatives under the conditions of total acid hydrolysis of protein are also reported.     

Determination of amino acids in human plasma by liquid chromatography with postcolumn ninhydrin derivatization using a hydroxyapatite cartridge for precolumn deproteination

Amino acids in human plasma were determined by liquid chromatography with postcolumn ninhydrin derivatization using a hydroxyapatite cartridge for precolumn deproteination. S-Carboxymethyl-l-cysteine, d-phenylglycine and S-aminoethyl-l-cysteine were found to be suitable internal standards. The proposed method is simple, rapid (deproteination time less than 1 min) and reproducible [relative standard deviation below 3% except for low-level aspartic acid (n = 3)]. The average recovery of 25 amino acids was above 90%. The elution time of amino acids in human plasma was approximately 2 h. Protein binding of tryptophan was also determined by the proposed method. The analytical data for amino acids in human plasma deproteinated using the proposed and published methods (5-sulphosalicylic acid and ethanol) were compared.     

A chromatographic method for the quantitative analysis of the deprotection of dithiasuccinoyl (Dts) amino acids.

The dithiasuccinoyl (Dts)-protecting group for amino acids is revoved by thiols (2 equivalents) through the intermediacy of an open-chain carbamoyl disulfide. Starting materials, intermediates, and products can be separated from one another on the standard amino acid analyzer 0.9 × 54-cm column of sulfonated polystyrene resin with 0.2 n sodium citrate buffers. Compounds are detected with the standard ninhydrin-hydrindantin reagent because the hydrindantin acts as a reducing agent and the released amino acid reacts in situ with the ninhydrin to give a purple color. Elution times, integration constants, and the ratios of absorbances at 570 and 440 nm are tabulated. Quantitative conversion of dithiasuccinoyl amino acids to the parent amino acids can be achieved with 0.1 n sodium hydroxide, 0.01 m alcoholic sodium borohydride, 0.1 m triphenylphosphine or 2 m m tri-n-butylphosphine in dioxane-H₂O (9:1) or water, and with a variety of thiols under various conditions. The chromatographic methodology is applicable to the determination of rate constants of the pseudo-first-order reductive deprotection of dithiasuccinoyl amino acids.     

Ribonucleotide reductase activity during the cell cycle of Saccharomyces cerevisiae

A fluorometric amino acid analyzer using fluorescamine for the assay of the full array of natural amino acids including proline on a single column is reported. The proline determination was carried out by specific introduction of a solution of N-chlorosuccinimide into the flow system. Single column fluorometric amino acid analysis was carried out in a significantly shorter time and with a sensitivity almost two orders of magnitude greater than that obtained with a commerical colorimetric ninhydrin amino acid analyzer.     

Inclusion of nonproteinous amino acids in thermally prepared models for prebiotic protein

Sixteen nonproteinous amino acids (those not coded for in contemporary protein biosynthesis) were incorporated during the thermal formation of polyamino acids under postulated prebiotic conditions, although not all into a single polyamino acid. The copresence of proteinous or even α-amino acids was not required. (Norleucine color equivalents and elution times on a Beckman model 120C amino acid analyzer were determined for these nonproteinous amino acids). The results suggest that prebiotically available nonproteinous amino acids would have been constituents of prebiotic protein if the latter were formed thermally. Some differences in properties of the polyamino acids could be attributed to particular nonproteinous amino acid residues; however, the tested properties did not suggest a means for evolutionary selection against nonproteinous amino acids as a group. Selection against this class of amino acids in toto was likely a later, biotic, event.     

Lysinonorleucine cross-link formation in alpha amino heptenoic acid-substituted peptide derivatives

Studies on the cross-linking of a tripeptide (t-butyloxycarbonyl-L -alanyl-D ,L -2-amino-6-heptenoyl-L -alanine methyl ester) have shown that it is possible to form specific cross-links in good yields through Schiff base formation of the ε amino group of lysine. The heptenoic acid residue has been ozonized to an aldehyde and condensed with the ε amino of lysine in the compounds alpha-t-butyloxycarbonyl-L -alanyl-L -lysine methyl ester and alpha-t-butyloxycarbonyl-L -lysine methyl ester to form the cross-link, lysinonorleucine. This compound has been stabilized by reduction with sodium borohydride and quantitated on the amino acid analyzer. This technique converts from 60 to 98% of the available aldehyde to lysinonorleucine.     

An automatic analyzer for the specific determination of amino sugars

The techniques previously employed for the extraction and determination of amino acids from different matrices are not necessarily optimal for the determination of the amino sugars. An analytical system is described which is a hybrid between the conventional amino acid analyzer and the liquid chromatographic system for the detection of reducing sugars. The major, naturally occurring amino sugars are separated in about 40 min, with sensitivites lying under the nanomole range, without interference from other co-extracted compounds such as amino acids and sugars. The reagent employed is noncorrosive and stable over long periods of time. The amino sugar analyzer can be readily constructed by simple modification of a conventional phenylketonuria or amino acid analyzer.     

Nitrotyrosine internal standard for amino acid analysis.

3-Nitrotyrosine is shown to be suitable as an internal standard for amino acid analysis. It is stable to acid hydrolysis, gives a color yield of 1.01 with ninhydrin which obeys Beer's Law, elutes in a position which is resolved from other amino acids on both Spinco and Durrum amino acid analyzers, and is commercially available. A method is also described for the preparation of a stable, pure, standardized stock solution of 3-nitrotyrosine.     

Sodium borohydride as a reducing agent for preparing ninhydrin reagent for amino acid analysis

Sodium borohydride, in place of stannous chloride or titanous chloride, is effective in the preparation of the ninhydrin reagent for automated amino acid analysis. The reagent, coupled with dimethyl sulfoxide as a solvent, a very stable ninhydrin solution which did not form precipitates in the flow lines of the analyzer.     

High-performance liquid chromatography determination of pipecolic acid after precolumn ninhydrin derivatization using domestic microwave

A novel procedure to specifically quantify low amounts of pipecolic acid and structurally related compounds in several types of biological materials has been characterized. From crude extracts of various types of biological material, the first step was to clear all low-molecular-weight compounds containing primary amino groups by a treatment of nitrous acid. Using a microwave-assisted reaction, the remaining substances containing secondary amino groups were then derivatized with ninhydrin and made soluble in glacial acetic acid. The derivatives produced were resolved by reverse-phase HPLC and detected by spectrophotometry at 570nm. This procedure allowed more rapid determination of pipecolic acid since microwave heating shortened the time needed for derivatization compared with heating at 95 degrees C in a water bath. The complete analysis of the chromogens for pipecolic acid and related substances was achieved in 20min. Under such conditions, the detection threshold for pipecolic acid was about 20pmol. The suitability of the technique was assessed in various biological matrices known to contain significant amounts of this amino acid. The data obtained are in accordance with those available in the literature. To our knowledge, this is the first method using the ninhydrin reaction in a precolumn, microwave-assisted derivatization procedure for detection and determination of heterocyclic alpha-amino acids.     

Lysosomal peptidase measurement by sensitive fluorometric amino acid analysis

A procedure has been developed for the measurement of lysosmal peptidase activity that is based upon highly sensitive fluorometric amino acid analysis. The fluorochrome results from the reaction of phthalaldehyde and mercaptoethanol with amino acids or similar compounds at pH 9.5. The fluorometric method is 10–100 times more sensitive than the colorimetric ninhydrin procedure for the measurement of lysosomal peptidases. The method lends itself to the measurement of any peptidase or protease that yields free amino groups in the product. The method has been applied to the measurement of cathepsin A and lysosomal dipeptidase activity.     

Quantitative aspects of the Ninhydrin-Schiff reaction on amino cellulose films and tissue sections

Summary In the ninhydrin-Schiff reaction primary amino groups are converted by oxidation with ninhydrin to aldehyde groups which are subsequently stained with pararosanilin. Amino cellulose films, used as a model, and sections of muscle tissue were submitted to this reaction. The amino groups were stained before and after the ninhydrin reaction with dinitrofluorobenzene and the generated aldehyde groups were stained with dinitrophenyl-hydrazine. The molar extinction coefficients used for the calculation of the molar amounts of chromophores from extinction values, and the conditions for maximal staining intensity were determined on the amino cellulose model. With these data the yields of the different steps in the reaction sequence could be calculated in molar amounts from the extinction measurements. The results showed that from the amino groups originally present in the tissue sections less than 40% were converted by ninhydrin. About 90% of the converted amino groups were found as aldehyde groups and from these only 7% reacted with pararosanilin. On amino cellulose similar data were obtained. Attempts were made by modification of the conditions in the ninhydrin oxidation step to increase the overall yield of the reaction. These were only partially successful, but indicate that further quantitative study of other reaction conditions and different aldehyde generating agents could be promising.     

A photoactivable amino acid based on a novel functional coumarin-6-yl-alanine

A novel fluorescent amino acid, l-4-chloromethylcoumarin-6-yl-alanine, was obtained from tyrosine by a Pechmann reaction. The assembly of the heterocyclic ring at the tyrosine side chain could be achieved before or after incorporation of tyrosine into a dipeptide, and amino acid and dipeptide ester conjugates were obtained by coupling to a model N-protected alanine. The behaviour of one of the fluorescent conjugates towards irradiation was studied in a photochemical reactor at different wavelengths (254, 300, 350 and 419?nm). The photoreaction course in methanol/HEPES buffer solution (80:20) was followed by HPLC/UV monitoring. It was found that the novel unnatural amino acid could act as a fluorescent label, due to its fluorescence properties, and, more importantly, as a photoactivable unit, due to the short irradiation times necessary to cleave the ester bond between the model amino acid and the coumarin-6-yl-alanine.