DNP-amino Acid Research Articles

Evaluation on the performance of flatten and flat-twisted tube multilayer coil in type-I counter-current chromatographic system

Recently, the multilayer coil was introduced in type-I CCC system, which showed the excellent peak resolution. Its performance was further improved by intermittently pressed tubing. In the present study, the whole plain tubing was pressed to make the flatten tubing. Then, it was twisted along its axis to form the flat-twisted tubing. The performance of these two tubing geometries was tested in type-I CCC system with three solvent systems of different polarities each with the suitable set of test samples of DNP-amino acids, dipeptides and proteins. The results showed that the retention of stationary phase was increased with the decreased flow rates and the increased polarity of solvent systems. In DNP-amino acid separation, flatten tubing produced the best peak resolution among all tubing geometries, where the resolution of three peaks was 2.17 and 2.30, respectively. In dipeptide separation, the result of flat-twisted tubing was good, but slightly worse than that of intermittently pressed tubing due to the lower retention of stationary phase. In protein separation, the resolution of intermittently pressed tubing was the best. The present study provided more options and ideas for type-I CCC application and development.

Novel Design for Centrifugal Counter-Current Chromatography: VI. Ellipsoid Column

A novel ellipsoid column was designed for centrifugal counter-current chromatography. Performance of the ellipsoid column with a capacity of 3.4 mL was examined with three different solvent systems composed of 1-butanol–acetic acid–water (4:1:5, v/v/v) (BAW), hexane–ethyl acetate–methanol-0.1 M HCl (1:1:1:1, v/v/v/v) (HEMH), and 12.5% (w/w) PEG1000 and 12.5% (w/w) dibasic potassium phosphate in water (PEG-DPP) each with suitable test samples. In dipeptide separation with BAW system, both stationary phase retention (Sf) and peak resolution (Rs) of the ellipsoid column were much higher at 0° column angle (column axis parallel to the centrifugal force) than at 90° column angle (column axis perpendicular to the centrifugal force), where elution with the lower phase at a low flow rate produced the best separation yielding Rs at 2.02 with 27.8% Sf at a flow rate of 0.07 mL/min. In the DNP-amino acid separation with HEMW system, the best results were obtained at a flow rate of 0.05 ml/min with 31.6% Sf yielding high Rs values at 2.16 between DNP-DL-glu and DNP-β-ala peaks and 1.81 between DNP-β-ala and DNP-L-ala peaks. In protein separation with PEG-DPP system, lysozyme and myolobin were resolved at Rs of 1.08 at a flow rate of 0.03 mL/min with 38.9% Sf. Most of those Rs values exceed those obtained from the figure-8 column under similar experimental conditions previously reported.

ΔG(CH2) in Biphasic Systems of Water and Bis(trifluoromethylsulfonyl)Imide-Based Ionic Liquids

Partition coefficients for a series of four dinitrophenylated (DNP) amino acids were experimentally determined in four biphasic systems, composed of hydrophobic ionic liquids (ILs) and water. The ILs were 1-alkyl-3-methylimidazolium bis(trifluoromethyl-sulfonyl)imide, [Cnmim][NTf2] (n = 2, 4, 6, and 8). DNP-amino acids distribute preferentially to the water-rich (top) phase. The experimental partition coefficients decrease as the size of the alkyl side chain of both the DNP-amino acid and the IL increase. The experimental data allowed the calculation of the free energy of transfer of a methylene group (CH2) between the two equilibrium phases, ΔG(CH2). This thermodynamic function is regarded to be a good measure of the relative hydrophobicity of the phases. This property was found to be independent of the size of the alkyl side chain in the ILs. This fact indicates that in this family of ILs the anion contributes much more significantly to the hydrophobicity of the compound than the alkyl chain in the cation.

Evaluation of the effect of column orientation in type-I high-speed counter-current chromatography

The performance of type-I high-speed counter-current chromatography was evaluated by changing the column inclination against the rotating centrifugal force field. The separations were performed with two different solvent systems composed of 1-butanol-acetic acid-water (4.75:0.25:5, v/v) (BAW) and hexane-ethyl acetate-methanol-0.1 M HCl (1:1:1:1, v/v) (HEMW) using dipeptides and DNP-amino acid as test samples, respectively. A set of short coiled columns connected in series is mounted around the holder hub in two different ways: in the parallel orientation, all column units are arranged in parallel to each other and mounted on the holder at various angles against the horizontal plane. In the zigzag configuration, the neighboring units of the same column are mounted symmetrically forming various angles apart. In the parallel configuration, for both the BAW and HEMW systems, Sf (the retention of stationary phase) first increased as the column angle decreased from 90 degrees to 60 degrees and then decreased, as the column angle further decreased from 60 degrees to 0 degrees, while Rs (peak resolution) continually declined over the entire column angle range from 90 degrees to 0 degrees. But, for both solvent systems, with the zigzag configuration, retention of stationary phase and resolution both decreased as the column angle decreased from 90 degrees to 0 degrees. In general, Sf and Rs for separation of dipeptides in the BAW system, from 90 degrees to 15 degrees, is better for the parallel orientation than for the zigzag configuration. However, at 0 degrees, Sf and Rs are better for the zigzag orientation. In the DNP-amino acid separation with the HEMW system, retention of the stationary phase and Rs for the parallel orientation is better than that for the zigzag orientation from 90 degrees to 30 degrees, whereas from 30 degrees to 0 degrees the results are opposite. Over all results of our studies revealed that the formally used column orientation at 90 degrees inclination yields the highest peak resolution in both solvent systems.

Mathematical model of computer-programmed intermittent dual countercurrent chromatography applied to hydrostatic and hydrodynamic equilibrium systems

Dual high-speed countercurrent chromatography (dual CCC) literally permits countercurrent flow of two immiscible solvent phases continuously through the coiled column for separation of solutes according to their partition coefficients. Application of this technique has been successfully demonstrated by separation of analytes by gas–liquid and liquid–liquid two-phase systems. However, the method cannot be directly applied to the system with a set of coiled columns connected in series, since the countercurrent process is interrupted at the junction between the columns. However, this problem can be solved by intermittent dual CCC by eluting each phase alternately through the opposite ends of the separation column. This mode of application has an advantage over the conventional dual CCC in that the method can be applied to all types of CCC systems including hydrostatic equilibrium systems such as toroidal coil CCC and centrifugal partition chromatography. Recently, the application of this method to high-speed CCC (hydrodynamic system) has been demonstrated for separation of natural products by Hewitson et al. using a set of conventional multilayer coil separation columns connected in series. Here, we have developed a mathematical model for this intermittent dual CCC system to predict retention time of the analytes, and using a simplified model system the validity of the model is justified by a series of basic studies on both hydrodynamic and hydrostatic CCC systems with a computer-programmed single sliding valve. The present method has been successfully applied to spiral tube assembly high-speed CCC (hydrodynamic system) and toroidal coil CCC (hydrostatic system) for separation of DNP-amino acid samples with two biphasic solvent systems composed of hexane–ethyl acetate–methanol–0.1 M hydrochloric acid (1:1:1:1 and 4:5:4:5, v/v).

Capillary electrophoresis investigation on the structure-enantioselectivity relationship in synthetic cyclopeptides as chiral selectors.

We recently reported the use of a deconvolution strategy to identify the best chiral selectors for Nalpha-2,4-dinitrophenyl (Dnp) amino acid racemates from a combinatorial library composed of thousands of homodetic cyclohexapeptides. Selection was based on the capillary electrophoresis (CE) enantioresolution for a set of Dnp-amino acids. The groups involved in the chiral discrimination were assessed by nuclear magnetic resonance (NMR) spectroscopy, which revealed a strong involvement of one of the aromatic rings of the cyclopeptide in the binding with the analyte. In order to better understand the recognition mechanism, and thus extend the applicability of the analytical system, modifications on both analyte and selector structure were introduced. The effects on separation were evaluated in terms of resolution values and mobility variation.

Enhanced capillary zone electrophoretic separation of dinitrophenyl-amino acid derivatives through control of electroosmotic flow by the buffer cation

The effect of Mg2+, Cd2+ and Zn2+ are evaluated as buffer additives to reduce electroosmotic flow (EOF) to facilitate the capillary zone electrophoretic (CZE) separation of 2,4-dinitrophenyl (DNP) derivatives of amino acids. As the divalent cation concentration increases EOF decreases and reduction of EOF follows the order Zn2+>Cd2+>Mg2+. The electrophoretic mobility of the DNP-amino acid derivatives remains constant for a given buffer and pH over a change in divalent cation concentration. The effect of the divalent cation buffer additive is consistent with cation-exchange at the silanol sites on the fused-silica wall. Higher buffer pH favors greater cation-exchange and reduced EOF. Separation of multicomponent mixtures of DNP-l-amino acid derivatives in the presence of Mg2+, Cd2+ and Zn2+ are compared at pH 7.00. Derivative migration time, resolution and peak shape are enhanced and are best for Mg2+ as the buffer additive although Mg2+ must be used at a higher concentration than either Cd2+ or Zn2+. Raising the buffer pH to 9.25 in the presence of Mg2+ increases migration time and resolution but analysis time is also increased.

Chiral interaction of a polyclonal anti-dinitrophenyl antibody with dinitrophenyl-amino acids determined by an enantioselective enzyme-linked immunosorbent assay

The enantioselectivity of the interaction of a polyclonal anti-DNP (dinitrophenyl) antiserum with a variety of DNP-amino acids has been determined by an indirect noncompetitive ELISA and the results obtained have been compared with those of an indirect competitive ELISA. The competitive ELISA offers an advantage to determine the I 50 values representing the relative affinities of the investigated DNP-amino acid enantiomers. In all cases, the I 50 values obtained were different for the individual DNP-amino acid enantiomers. While the l-enantiomers of ϵ-DNP-lysine, δ-DNP-ornithine, DNP-alanine and DNP-proline show a higher relative affinity for the antibody combining site than the d-enantiomers, an inverse chiral recognition was found for DNP-phenylalanine. On account of the high sensitivity of the ELISA system, the determination of small differences in the enantiomer recognition by the anti-DNP antibody is possible.

Mechanism of allergic cross-reactions—II. Cross-stimulation, by chemically unrelated ligands, of rat basophilic leukemia cells sensitized with an anti-DNP IgE antibody

Serotonin release from rat basophilic leukemia (RBL) cells, sensitized with a DNP-binding monoclonal IgE, was stimulated with solid surface (polystyrene)-bound DNP-amino acids. The stimulatory potency of DNP-amino acids was dependent on the structure of amino acid attached to DNP. Generally, DNP-amino acids with high affinities to the sensitizing IgE (I(50) < 10 μM) were stimulatory in polystyrene-bound form; DNP-amino acids with lower affinities (Pro, Cys, Trp), and aliphatic/aromatic DNP-amino acid derivatives were inactive. In addition to structural analogues of DNP, lymecycline, that is chemically unrelated to DNP but was found to have high affinity to IgE(aDNP), was also stimulatory in this system. This drug, and various quinones (e.g. acenaphthene-quinone) in BSA-conjugated forms also stimulated serotonin release from RBL cells sensitized with IgE(aDNP). These studies suggest that (1) There is a threshold of intrinsic ligand binding affinities at approximately I(50) = 10–100 μM; ligands with lower affinities do not stimulate mediator release even if they are presented in multivalent forms; (2) The above affinity threshold for mediator cell stimulation is valid for various ligands, irrespective of their chemical similarity to the immunogen; (3) Multispecific stimulation of mediator release may contribute to the frequently observed allergic cross-reactions, false positive tests for allergies, and anaphylactic reactions to drugs upon first exposure.

Inhibition of cryoprecipitation of murine IgG3 anti‐dinitrophenyl (DNP) monoclonal antibodies by anionic DNP‐amino acid conjugates

Previously we have demonstrated that eight out of nine IgG3 monoclonal antibodies (mAb) obtained from autoimmune MRL-lpr/lpr mice were able to self-associate and to precipitate in the cold (Gyotoku et al., J. Immunol. 1987. 138:3785). To determine whether the cryoprecipitation of IgG3 mAb is enhanced or inhibited in the presence of specific ligand, we have established eight IgG3 mAb reactive with 2,4-dinitrophenol (DNP) hapten: four mAb were obtained from fusion of spleen cells of C57BL/6 mice immunized with 2,4,6-trinitrophenylated keyhole limpet hemocyanin, three from 129/Sv and one from BALB/c immunized with DNP-lipopolysaccharide. Five of them induced cryoglobulins composed exclusively of the IgG3 mAb. The binding of negatively charged monomeric DNP-amino acid conjugates completely inhibited the cryoprecipitation of all the five cryoprecipitating anti-DNP IgG3 mAb, while the incubation with positively charged or neutral DNP-amino acid conjugates had variable effects: increase, inhibition or no change of the cryoprecipitation. In addition, positively charged DNP-amino acid conjugates were able to induce the cryoprecipitation of one of the non-cryoprecipitating anti-DNP IgG3 mAb. Our data showed that (a) IgG3 mAb derived from non-autoimmune strains of mice, similar to IgG3 mAb derived from an autoimmune MRL-lpr/lpr strain, possessed the unique property to self-associate and were able to form cryoglobulins in most cases; (b) although the Fc-Fc interactions of IgG3 mAb play a decisive role in IgG3 cold solubility, IgG3 cryoprecipitation was markedly influenced after interacting with their specific ligand, depending on the charge of the hapten-amino acid conjugate. This suggested that even minor interferences with the electrostatic equilibrium of the IgG3 by the binding of charged hapten molecules induced dramatic changes in the solubility of the IgG3 mAb at low temperature.

L-lactate-2-monooxygenase. Sequence of peptides containing residues modified by 1-fluoro-2,4-dinitrobenzene.

L-Lactate-2-monooxygenase (EC 1.13.12.4) inactivated by 1-fluoro-2,4-dinitrobenzene essentially as described previously (Choong, Y. S., Shepherd, M. G., and Sullivan, P. A. (1978) Biochem. J. 173, 255-262) incorporated 2.8 mol of the dinitrophenyl (DNP) moiety per mole of flavin. The inhibitors 2-methyl lactate or sulfite decreased the incorporation to 0.9 mol of DNP per mole of flavin. Peptide mapping by high performance liquid chromatography of radioactively labeled protein digested with trypsin showed three peaks of radioactivity. DNP-amino acid analysis and peptide sequencing showed that 2 distinct cysteine residues and a histidine residue had been modified. Both cysteine peptides were protected from modification by either of the inhibitors, whereas the histidine was only partially protected. The sum of the 2 cysteine peptides accounted for nearly 1 mole of label per mole of monomer. Since both of the cysteines are protected by inhibitors, they both must be in or near the substrate-binding site of the enzyme and appear to be modified in a mutually exclusive fashion. The histidine, on the other hand, does not lie directly in the substrate-binding site. It is possible that this histidine is the positively charged residue that is postulated to be near the N-1 position of the flavin.

Determination of chemical properties of individual histidine and tyrosine residues of concanavalin A by competitive labeling with 1-fluoro-2,4-dinitrobenzene.

A selective peptide-mapping procedure was devised to purify peptides containing histidine or tyrosine residues from proteolytic digests of concanavalin A (Con A). The protein was modified with maleic anhydride followed by 1-fluoro-2,4-dinitrobenzene (Dnp-F) and then digested with thermolysin. The resulting labeled peptides were separated by high-performance liquid chromatography, and the Dnp-histidine and Dnp-tyrosine peptides were identified by their spectral characteristics. From their amino acid compositions, the labeled peptides could all be assigned within the known sequence. Peptides representing five of the six histidines and all seven tyrosines were obtained. With the same peptide-mapping procedure, the chemical properties (pK and reactivity) of these residues were determined. Samples of concanavalin A at various pH values were labeled with trace amounts of [3H]Dnp-F, in the presence of Gln-Gly as an internal standard. To each sample was added an aliquot of a mixture of [14C]Dnp-Gln-Gly and [14C]Dnp-maleyl-Con A. Portions of each sample were removed, [14C]Dnp-Ala-Ala and epsilon-[14C]Dnp-lysine were added, and the mixtures were hydrolyzed. The various Dnp amino acid derivatives were purified by HPLC. The remainder of each [3H]Dnp sample was maleylated, dinitrophenylated, and digested with thermolysin and separated by HPLC as above. From the 3H/14C ratios of the Dnp amino acid derivatives and the Dnp peptides relative to the ratio of the internal standard, pK and reactivity data were obtained for (a) the average behavior of the lysine, histidine, and tyrosine residues and (b) the individual behavior of the N-terminal alanine residue and the five histidine and seven tyrosine residues in the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Hapten-specific prophylaxis of murine contact sensitivity by DNP-amino acid derivatives.

In mice, the elicitation of DNFB contact skin reaction was suppressed by the intravenous injection of relatively small amounts of DNP-amino acid conjugates when given at the same time of challenge test. Maximal suppression achieved was around 70% when optimal doses of DNP-amino acid were administered. The suppressive activity of 4 different amino acids, i.e., DNP-L-lysine, DNP-S-cysteine, DNP-alanine, and DNP-glycine, was nearly the same, respectively. This inhibitory effect was hapten-specific, and it failed to suppress the strong skin reaction elicited in cyclophosphamide-pretreated mice. Using the lymphocyte culture system, low doses of DNP-amino acid also suppressed the antigen-specific proliferation of sensitized lymph node cells (T prlf). Preincubation of sensitized cells with DNP-amino acid eliminated the transfer activity of T-DH cells as demonstrated previously. The mixture of lymph node cells and spleen cells from DNP-L-lysine-treated mice in vivo also suppressed the elicitation of skin reaction when transferred to recipient mice sensitized 5 days previously. Transfer studies demonstrated that this suppression was mediated by Lyt 1.2 (+), Thy 1.2 positive cells in vivo.

Recognition of DNP-amino acid determinants by primed guinea pig T cells

Guinea pigs were immunized with DNP-mycobacteria prepared by reaction of the bacteria with dinitrofluorobenzene. T cells from these animals responded as well against DNPazo-proteins and proteins haptenated by reaction with DNP-sulfonic acid as against proteins haptenated by the same method as the immunogen. These cells responded also to free DNP- O-tyrosine, to the DNPazo derivatives of N-acetyltyrosine and of aminocaproic acid, and to α- N-DNP- l-asparagine. The experiments suggest that at least part of the DNP-responsive lymphocyte population found in animals primed with DNP-mycobacteria recognized a determinant no larger than the hapten with a single amino acid side chain, and that there was no requirement for the side chain to be aromatic (such as tyrosine or histidine) in order for recognition to occur. Determinants of this size were capable, in free form, of stimulating primed cells to mount an in vitro proliferative response. DNP-amino acids were not effective, however, as primary immunogens.

Nature of Adenosine Triphosphatase Accelerating Peptide from Hydrolysate of Fur Seal Muscle

Ultrafiltered fur seal muscle hydrolysate was divided into eleven fractions by gel filtration on Sephadex G-15. One of the fractions (Fraction G9) accelerated the ATPase activity of carp myosin B to a rate about two-fold faster than that of the control. Fraction G9 showed a single ninhydrin spot in its silica gel thin layer chromatograph, and gave a positive test for tryptophan by the p-dimethylaminobenzaldehyde method, while tests for tyrosine, and for arginine were negative. The ion exchange amino acid analysis of its acid hydrolysate showed a predominant content of lysine, nearly equivalent to the amount of tryptophan determined from its UV absorbancy and the p-dimethylaminobenzaldehyde method. The N-terminal amino acid analysis gave di-DNP-Lys as the sole DNP-amino acid. The structure of the ATPase accelerating peptide fraction, Fraction G9, was deduced to be Lys-Trp.

Countercurrent chromatography with the flow-through centrifuge without rotating seals

The seal-free centrifuge device is applied to helix countercurrent chromatography. Capability of the scheme is demonstrated on DNP-amino acid separation on a CHCl 3/CH 3COOH/0.1 n HCl (2/2/1) phase system.

Horizontal flow-through coll planet centrifuge without rotating seals

This new scheme for countercurrent chromatography uses a horizontal helical column that rotates about its own axis and simultaneously revolves around the central axis of the apparatus at the same angular velocity to prevent twisting the flow tubes. This planetary motion allows both gravity and centrifugal force to perform high efficiency analytical and preparative scale separations on a broad spectrum of two-phase systems as demonstrated on separation of DNP-amino acid with CHCl 3:CH 3COOH:0.1 n HCl (2:2:1).

Gas chromatographic analysis of amino acids in food

1. The methylester derivative of DNP-amino acid was examined by gas-chromatography and amino acid shown in fig. 1 was detected. 2. The gaschromatography was more sensitive than the ion-exchange chromatography, but detectable peaks were few, and it must be further studied. 3. The measurements of five amino acids in food were almost coincident with those obtained by the ion-exchange method in literatures.

Isolation and Some Physico-chemical Properties of the Acidic Subunits of Soybean 11S Globulin

Four kinds of acidic subunits and three kinds of basic subunits of 11S globulin were separated by polyacryl amide gel electrophoresis in the urea system. The four acidic subunits designated as A1, A2, A3 and A4 (Rm=0.35, 0.40, 0.46 and 0.56 respectively) were isolated by stepwise elution followed by repeating gradient elution with DEAE-Sephadex A-50 in the presence of 6 m urea at 5°C. Subsequently, some physico-chemical properties of the subunits were determined. For example, N-terminal amino acids were determined as phenylalanine for both A1 and A2 and as leucine (or isoleucine) for both A3 and A4 by the DNP-amino acid method. The molecular weights of A1, A2 and A3 were shown as 37,000 and 45,000 for A4 by SDS-gel electrophoresis. The amino acid compositions of the acidic subunits were roughly similar to each other, but some remarkable differences were observed in the content of basic amino acids (lysine, histidine and arginine), serine and proline.

A method for concurrent measurement of picomole quantities of acetylcholine, choline, dopamine, norepinephrine, serotonin, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, tryptophan, tyrosine, glycine, aspartate, glutamate, alanine, and gamma-aminobutyric acid in single tissue samples from different areas of rat central nervous system

A rapid and sensitive method for separation and concurrent assay of 14 compounds at the picomole level in individual rat brain parts is described. The putative amino acid neurotransmitters (aspartate, γ-aminobutyric acid, glutamate, and glycine) are extracted from a 20–30 mg portion of the tissue with 5% TCA and assayed as their respective DNP-amino acid methyl ester derivatives by glc. Four other putative neurotransmitters (acetylcholine, dopamine, norepinephrine, and serotonin) and some of their precursors and metabolites (choline, tryptophan, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, and tyrosine) are extracted in 1 n formic acid-acetone (v/v:15/85) from the remaining tissue. The lipids are removed with a heptane-chloroform (v/v:8/1) wash and the aqueous phase is dried at 37°C under N 2. The dried extract is dissolved in water (pH 4). With one portion of this solution, acetylcholine and choline are assayed using a radioenzymatic method whereas with the rest, dopamine, norepinephrine, serotonin, 5-hydroxyindoleacetic acid, 5-hydroxytryptophan, tryptophan, and tyrosine are separated with three ion exchange resins arranged in tandem. These compounds are assayed fluorometrically with modified microadaptations of previously reported methods.