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.     

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.     

Ion-exchange chromatography of sulfur amino acids on a single-column amino acid analyzer.

Examination of the extent of production of the ninhydrin-colored derivative, Ruhemann's purple, under automated conditions of a single-column amino acid analyzer by several classes of sulfur-containing amino acids revealed a wide variation in the color factors relative to leucine. These ranged from 0.02 for the methyl ester of cysteine to 2.19 for D-homocystine. Color yields obtained by the manual ninhydrin reaction are generally lower than the corresponding values obtained on the amino acid analyzer. The elution positions ranged from 5.12 min for cysteic acid to 84.9 min for l-cystine dimethyl ester. The observed behavior of these compounds in the ninhydrin reaction is rationalized in terms of structural and electronic factors which they exhibit in reacting with ninhydrin to form the visible dye. Such an analysis should make it possible to predict ninhydrin color factors, and possibly also elution times, of structurally related compounds.     

Studies on the Decarboxylation of Amino Acids with Glyoxal

Decarboxylation of about twenty kinds of α, β and γ-amino acids in the reaction with glyoxal or ninhydrin was investigated. The decarboxylation rate of amino acids proved that steric and polar effects had important roles in the reaction.

From the data of pK₂ values and decarboxylation rates of amino acids, it can be concluded that under a similar steric environment, the decarboxylation rate depends on the anion concentration of amino acids.

Besides carbon dioxide, acetaldehyde, 2-propanone and propionaldehyde were respectively detected from the reaction of β-alanine, β and γ-amino-n-butyric acids with glyoxal or ninhydrin. The decarboxylation mechanism of these amino acids seemed to take place through the corresponding β- or γ-keto acid.

Oxygen absorption was also observed from the reaction of amino acids with dicarbonyl compounds.     

Synthesis of protein, nucleosides and other organic compounds from cyanamide and potassium nitrite under possible primitive earth conditions.

The mixing of cyanamide and KNO2 produced changes from white solids to yellow liquid and then to orange solid. The gases cyanogen and ammonia were formed. No external energy was used. The reactions were carried out with a small amount of O2. The presence of proteins in the reaction product formed 13 months after the mixing was indicated by the positive reactions of the cyanamide-KNO2 reaction product with ninhydrin, microbiuret, and Folin reagent; the ultraviolet absorption at about 280 nm; the yield of 24% of 15 amino acids; and molecular weight measurements of more than 160,000. The presence of nucleosides, nucleic acid bases, hydrocarbons, and organic esters in the reaction product formed 2 months after the mixing was indicated by ultraviolet absorption at about 260 nm, and the results of ligand-exchange chromatography, paper chromatography, infrared analysis, mass spectral analysis, and NMR spectroscopy. Possible cyanamide-mediated dehydration reactions and mechanisms are discussed.     

A step on the path in the discovery of new latent fingerprint development reagents: substituted Ruhemann’s purples and implications for the law

Energies corresponding to optimum geometries of ninhydrin, some of its analogs, the corresponding Ruhemann’s Purple analogs and some of the intermediates of the reaction between ninhydrin analogs and amino acids are calculated at Hartree–Fock/6-31G** level of theory. Such a study is significant from a forensic science point of view because of the strong interest in the forensic chemistry and law enforcement communities in developing alternatives to the current generation of ninhydrin-like chemicals for the detection and development of latent fingerprints. In examining our new predictions for the net energetics of the reactions in the formation of substituted Ruhemann’s Purples, we find that a fluorine-containing analog is the most thermodynamically feasible reaction. In light of this finding, we suggest further experimental studies to determine the kinetic feasibility of synthesizing the fluorine-containing analog, as well as other similar molecules and to determine their spectroscopic properties.     

Synthesis of protein,nucleosides and other organic compounds from cyanamide and potassium nitrite under possible primitive earth conditions

The mixing of cyanamide and KNO₂ produced changes from white solids to yellow liquid and then to orange solid. The gases cyanogen and ammonia were formed. No external energy was used. The reactions were carried out with a small amount of O₂. The presence of proteins in the reaction product formed 13 months after the mixing was indicated by the positive reactions of the cyanamide-KNO₂ reaction product with ninhydrin, microbiuret, and Folin reagent; the ultraviolet absorption at about 280 nm; the yield of 24% of 15 amino acids; and molecular weight measurements of more than 160 000. The presence of nucleosides, nucleic acid bases, hydrocarbons, and organic esters in the reaction product formed 2 months after the mixing was indicated by ultraviolet absorption at about 260 nm, and the results of ligand-exchange chromatography, paper chromatography, infrared analysis, mass spectral analysis, and NMR spectroscopy. Possible cyanamide-mediated dehydration reactions and mechanisms are discussed.     

Measurement of total protein in plant samples in the presence of tannins

A method for measuring total protein in situ in plant samples has been developed using the determination of amino acids released by acid hydrolysis of dried plant material. Standard proteins and plant samples were hydrolyzed with 3% sulfuric acid at 100 degrees C for 24 h and the amino acids released were measured with ninhydrin. Unhydrolyzed plant extracts were also analyzed for free amino acids with ninhydrin. Total amino acid equivalents (protein plus free amino acids) of a diverse set of plant samples was significantly correlated with total protein as estimated by elemental analysis (N X 6.25). The Lowry method as modified by precipitation of proteins with trichloroacetic acid was found to be unsatisfactory for dried plant samples due to the incomplete extractability of proteins. Although some alkaloids caused increased absorbance with ninhydrin, interference with quantification of protein is likely to be minimal. Tannins interfered with the Lowry and Bradford methods but not the ninhydrin method.     

Characterization of a new flavin metabolite from human urine

A new flavin metabolite comprising approximately 5% of the total flavin of human urine was isolated and characterized using absorption and fluorescence spectra, oxidation-reduction and hydrolysis data, and ninhydrin reactions. The flavin is a derivative associated with a peptide residue in ester linkage from an amino acid carboxyl to the ribityl chain of riboflavin, probably at the 5'-terminus.     

Isoelectric focusing of oligopeptides: detection by specific stains.

By using ultrathin (350 micrometers) polyacrylamide gels, which at the end of the fractionation are pasted to filter paper and dried in an oven at 110 degrees C, and after isoelectric focusing it has been possible to detect oligopeptides in the di- to tetradecapeptide range, which could not be detected by protein staining techniques. This is achieved by developing a series of specific stains for the following amino acids: Arg, Tyr, His, Trp, Met and Cys. Except for Met and Cys, the detection limits appear to be in the order of 0.2--2 micrograms of free amino acid loaded in the gel. The Pauli reaction for His and Tyr and the Sakaguchi stain for Arg can be developed sequentially in the same gel, thus allowing the detection of four different amino acids since, under these conditions, also Trp reacts. Unfortunately, more general reactions, such as the permanganate, the 'Lowry' and the ninhydrin stains, cannot be utilized since the carrier ampholytes react very strongly with all these reagents.     

Combined protein and DNA measurements by the ninhydrin-Schiff and Feulgen techniques

Summary Feulgen nuclear staining with pararosanilin-SO₂ was combined with the ninhydrin-Schiff technique. The aldehyde groups converted from primary amino groups are stained with an acriflavine-Schiff reaction. This results in a red nuclear fluorescence and a bright yellow cytoplasmic and nuclear fluorescence. The combined fluorescence staining facilitates cytofluorometric determination of total protein and DNA in the same cell.The ninhydrin-Schiff reaction is affected by the fixation procedure and the duration of the ninhydrin reaction. Investigations with a model system showed that proportionality beween the fluorescence intensity of acriflavine and the amount of protein stained by the procedure was obtained after fixation with a fixation mixture suggested by Böhm et al. (1968) and a reaction with ninhydrin at 37° C for 10 h.The ninhydrin-Schiff reaction has no effect on the fluorescence intensity of cells previously treated with pararosanilin-Feulgen staining and it is not affected itself by this previous procedure.Testing this double fluorescence staining on cytology specimens taken from patients with gastric carcinoma and uterine cervial carcinoma, cancer cells were shown to have markedly increased protein and DNA contents compared with those of normal cells.Partly supported by Deutsche Forschungsgemeinschaft (DFG), grant Nr. Bo 395/4     

A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids

1. An acid ninhydrin reagent was found to react specifically in forming a pink product (E(max.) 560mmu) with cysteine. 2. The method was highly sensitive for the determination of cysteine (in 28.0x10(3)). Homocysteine, glutathione, proline, ornithine and other naturally occurring amino acids tested did not give a similar reaction. 3. The reaction product was stable for at least 3-4hr. at room temperature and the extinction was proportional to the concentration in the range 0.05-0.5mumole of cysteine. 4. The acid ninhydrin reagent also gave yellow products (E(max.) 370-404mmu) with tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine and indol-3-ylacetic acid. 5. The method was applied for the determination of cysteine in perchloric acid extracts of rat brain, liver and blood.     

Use of fluorescamine in the chromatographic analysis of peptides from proteins

A routine procedure for the fluorometric analyses of peptides in the column chromatographic fraction has been described. Sensitivities of detection are 3–5 times higher in the direct fluorescamine method and 6–50 times higher in the method with alkaline hydrolysis than the conventional ninhydrin color method with hydrolysis (11).Reactivity of peptides with fluorescamine appears to depend mainly on the nature of amino acids occupying the amino termini; ?-amino groups of lysine residues in the peptides tested have been found not to contribute significantly in yielding fluorescence in the reaction.     

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.     

Ion-exchange chromatography in lunar organic analysis

Although ion exchange chromatography has been used in separating amino acids from mineral salts, quantitative recovery has not been possible for the basic amino acids or for subnanomole concentrations of amino acids.As an analytical tool for amino acid analysis, ion-exchange chromatography has made it possible to resolve a relatively complex mixture of amino acids in less than an hour with detection limits of less than 10^–12 moles of amino acids. Reasonable specificity for amino acids is achieved by multiple wavelength detection of the reaction product found with ninhydrin. Unequivocal specificity must be obtained in conjunction with other methods such as mass spectrometry.In the analysis of subnanomole levels of amino acids, it is necessary to carry both reagent blanks and low-level amino acid standards through the entire sample preparation step since both contamination and selective losses occur and must be monitored.     

华重楼内生菌抗菌肽的分离纯化及其特性

摘要：【目的】华重楼内生菌PCE45具有较强的抗菌活性,本文将对PCE45产生的抗菌物质进行分离纯化和性质分析报道。【方法】PCE45发酵液经硫酸铵盐析、丙酮沉淀、SephadexG75柱、DE52纤维素柱和SephadexG25凝胶柱纯化分离得到抗菌肽PCP-1。【结果】稳定性测试表明该抗菌肽对蛋白酶不敏感,对高温、强酸、强碱有较好的耐受性,可造成稻瘟病菌菌丝畸形并抑制孢子萌发。抑菌谱表明该抗菌肽对玉米弯胞病菌等真菌和大肠杆菌等细菌有较强的抑菌效果。质谱测得其分子量为1058.3D。氨基酸组成分析表明该     

Ultrastructural cytochemistry of hepatic lysosomes and their protein components is selectively revealed by the ninhydrin-dimethyl sulfoxide-thiocarbohydrazide-silver proteinate reaction

Summary Proteins in lysosomal membranes, lysosomes and within the transtubular network are readily accessible for electron microscopic analysis by a new three-step method. Oxidative deamination of tissue-bound amino acids by ninhydrin in a queous dimethyl sulfoxide and the concomitant formation of corresponding carbonyl groups comprise the first step. The addition reaction of thiocarbohydrazide to tissue-bound carbonyl groups comprises the second step, while the reduction of silver proteinate by tissue-bound thiocarbohydrazones is the final step of this sequential method. Glutaraldehyde-fixed and osmified ultrathin sections of rat liver embedded in LR White were oxidatively deaminated for 24 h by 1% w/v ninhydrin in aqueous 75% v/v dimethyl sulfoxide (DMSO). They were then incubated for 40 min in aqueous 1% w/v thiocarbohydrazide (TCH) and stained for 30 min at 50° C with silver proteinate (SP). The ninhydrin-dimethyl sulfoxide-thiocarbohydrazidesilver proteinate (N-DMSO-TCH-SP) reaction proved to be chemically specific and highly selective for ultrastructural resolution of the internal structure of lysosomes and their protein components. We conclude that the N-DMSO-TCH-SP reaction is the method of choice for cytochemical elucidation of the protein ultrastructure of lysosomes and their enzymatic aggregates.     

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.     

Identification of Products of the Uridinediphospho-N-acetyl-d-Glucosamine Oxidoreductase System from Citrobacter freundii ATCC 10053

Uridinediphospho-N-acetyl glucosamine is converted to one or more uridine-diphospho-4-keto-6-deoxy-2-acetamidohexose derivatives by an enzyme from Citrobacter freundii ATCC 10053. Borohydride reduction of reaction products followed by acid hydrolysis yielded several amino sugars. Two of these were identified as fucosamine and quinovosamine by chromatography and ninhydrin degradation.     

Protein precipitation assay for quantitation of tannins: determination of protein in tannin-protein complex

A protein precipitation method for the determination of tannins has been developed. The protein in the tannin-protein complexes was measured using the ninhydrin assay of amino acids released by alkaline hydrolysis of the complex. Standard protein and the complex were hydrolyzed with 13.5 N NaOH at 120 degrees C for 20 min and the amino acids released were measured with ninhydrin. Tannins did not interfere in the determination of protein by ninhydrin assay. The bovine serum albumin (BSA) precipitated (y; mg) increased linearly with increase in tannic acid (x) from 0.2 to 0.9 mg (y = 2.598x - 0.258). The protein precipitation capacities (mg BSA precipitated/g dry wt) measured by the method for young and mature leaves of oaks were Quercus incana (young, 42.21; mature, 79.51), Q. ilex (young, 1.86; mature, 1.86), and Q. semecarpifolia (young, 733.54; mature, 304.32). The method can provide valuable information on the mechanisms of protein-tannin interactions and nutritional and physiological significances of tannins.