Polyamide Sheets Research Articles

New liquid chromatographic approaches for free amino acid analysis in plants and insects

A simple and rapid method of analysis for free amino acids in plants and insects is presented. The amino acids were determined as their 5-dimethylaminonaphthalen-1-sulfonyl (Dns)dervatives. Two-dimensional thin-layer chromatography on polyamide sheets and high-performance liquid chromatography on μBondapak C 18 columns were used to resolve the Dns dervivatives. R F values of 45 Dns derivatives are reported.

A rapid procedure for detection of bacterial amino acid decarboxylases

The detection of decarboxylases of arginine, glutamic acid, histidine, lysine, ornithine, phenylalanine, tryptophan, and tyrosine in bacteria by thin-layer chromatography on polyamide sheets is described. The bacteria were grown on agar medium plates supplemented with eight amino acids at pH 5.5 for induction of amino acid decarboxylases, then transferred to amine-production media. The decarboxylation products in the spent media (amines and/or γ-amino- n-butyric acid) were dansylated and the dansyl derivatives were separated by thin-layer chromatography on polyamide sheets. This method requires only two separate incubations of the decarboxylase-induced bacteria in amine-production media for 1 h at 37°C for simultaneous detection of eight bacterial amino acid decarboxylases using 0.4 μl of the spent media.

Conversion of lipotropic peptides by purified cathepsin D of human pituitary: Release of γ-endorphin by cleavage of the Leu 77-PHE 78 bond

Cathepsin D (EC 3.4.3.23) purified from human putuitary by affinity chromatography on pepstatin-Sepharose cleaved human β-endorphin (LPH 61–91) at the Leu 77-Phe 78 bond after incubation at pH 3.2 for 1–3. Incubation with smaller lipotropic fragments (enkephalin, α-endorphin, β-endorphin) did not lead to further degradation. Cleavage sites were identified following the separation of danyslated or iodinated peptides on polyamide sheets accompanied by N-group determination, or following slab-gel electrophoresis of the iodinated peptides. Cleavage at the above site was blocked using an analog containing D-Leu 77. Breakdown of porcine β-lipotropin (LPH 1–91) was slower and required an 18 h incubation. A product LPH 1–77 was tentatively identified based in part on its mobility on slab gels as compared to β-LPH and β-endophine and by N-group determination of cleavage products.

Solvent systems for thin-layer chromatography of Dns-amino acids on polyamide sheets

Solvent systems for thin-layer chromatography of Dns-amino acids on polyamide sheets

Separation of two types of diphenylindenone derivatives of amino acids (ITH- and DIS-amino acids) applicable to sequencing of proteins on polyamide sheets

Several chromatographic systems are proposed that provide the possibility of determining all amino acids commonly found in proteins as diphenylindenone thiohydantoins and diphenylindenone sulphonamides at the picomole level using two-dimensional thin/3-layer chromatography on polyamide sheets.

Identification of subnanomole amounts of PTH-amino acids by high performance thin-layer chromatography

High performance thin-layer chromatography (HPTLC) of PTH-amino acids on 5×5 cm silica gel plates precoated with a fluorescence marker gives 10–20 fold increase in sensitivity compared to ordinary silica gel plates. Separation of PTH-Leu from PTH-Ile is easily achieved in contrast to chromatography on polyamide sheets. Only two solvent systems are required and as many as 12 samples can be chromatographed on each plate. However, if the sample is contaminated with N-phenylthiourea a third solvent system is necessary.

Thin-layer chromatography of dansyl amino acids on polyamide.

Thin-Layer Chromatography of Dansyl Amino Acids on Polyamide A two-dimensional thin-layer chromatography system for the separation of the dansyl derivatives of the amino acids commonly found in proteins is described. Chromatography was performed on polyamide sheets using water-pyridine-formic acid (93∶3.5∶3.5 v/v) and benzene-acetic acid (4.5∶1 v/v) as the solvents.

High-sensitivity sequence analysis of peptides and proteins by 4-NN-dimethylaminoazobenzene 4′-isothiocyanate

A manual high-sensitivity sequencing method is described, in which 4-NN-dimethylaminoazobenzene 4'-isothiocyanate is used for the stepwise degradation of amino acid residues from the peptides. The 4-NN-dimethylaminoazobenzene 4'-thiazolinones of amino acids that were released, after conversion into their thiohydantoin derivatives, were identified by t.l.c. on polyamide sheets. This new method is simple and sensitive, and requires only 2-10nmol of peptides or proteins for extended sequence analysis. The method was tested on the sequence analysis of a hexapeptide (Leu-Trp-Met-Arg-Phe-Ala), bradykinin, glucagon and native lysozyme. Results show that the proposed procedure is a sensitive method for the sequence determination of short peptides as well as for the partial sequence determination of intact proteins.

A new approach for the solid phase sequence determination of proteins

The use of the modified Edman reagent, 4-N,Ndimethylaminoazobenzene 4’-isothiocyanate, in the stepwise degradation of peptides and proteins has provided a useful method for sequence determination [l-3] . Released N-terminal amino acids are identified as 4-N,N-dimethylaminoazobenzene 4’-thiohydantoins directly by thin-layer chromatography (t.1.c.) on polyamide sheets, the intense colour of the amino acid derivatives allowing detection in subnanomol quantities. Low repetitive yields due to extraction losses and time-consuming operations at each drying step have reduced both sensitivity and efficiency of the manual sequencing work. However, the solid phase degradation of Laursen [4,5] whereby peptides are attached covalently to an insoluble support, eliminates extraction losses and shortens greatly the time needed for the drying of residues after the extractions. A solid phase degradation procedure using 4-N,Ndimethylaminoazobenzene 4’-isothiocyanate has been developed. The N-terminal sequence of native lysozyme (first 19 residues) and glucagon (first 10 residues) attached to controlled porosity glass beads activated by p-phenylene diisothiocyanate have been obtained. Reduced Histidinol dehydrogenase (mol. wt 40 000) from Salmonella typhimwium, of hitherto unknown sequence has been attached via cysteine residues to an iodoacetamide derivative of controlled porosity glass beads and the first eight amino acid residues determined. This new solid phase sequenching method is sensitive and efficient, requiring practically, 210 nmol protein as the starting material. Each cycle of

Amino acids and biogenic amines in smooth muscles and hemolymphs of different molluscs

1. Microchromatography of dansyl compounds on 3 × 3 cm polyamide sheets was used to study the distribution of amino acids and biogenic amines in smooth muscles and hemolymps of the king crab and some molluscs. 2. Using liquid scintillation counting, the amounts of glycine, glutamic acid, γ-amino butyric acid and 5-hydroxytryptamine was quantitatively determined. 3. After stimulation of the penis retractor muscle (PRM), a smooth muscle in the mollusc Helix pomatia, variations of the concentrations of these putative neurotransmitters were quantitatively determined and compared with an untreated PRM. 4. Only in the marine mollusc, Mytilus edulis, large amounts of free amino acids and taurine were detected; all other tissues and hemolymphs of the other animals examined contained essentially less free amino acids. 5. Some rare compounds could be identified in a few tissues or hemolymphs: sarcosine, ethanolamine, putrescine and tryptamine. The occurrence of free choline has not been clearly indicated. 6. The significance of these experiments has been briefly discussed.

Determination of plasma amino acids in small samples with the use of Dansyl-chloride

A radiochemical method based on dansylation of plasma samples with Dansyl-chloride and thin alyer chromatography on polyamide sheets is presented for the quantitative individual determination of plasma amino acids in very small blood samples. The results are first corrected with a norvaline internal standard and secondly with specific recovery factors for each amino acid. The results agree slosely with other methods and with already published plasma normal amino acids values in healthy adult rats.

Low-fluorescence polyamide sheets for thin-layer chromatography

Low-fluorescence polyamide sheets for thin-layer chromatography

Identification of aqueous phase amino acid phenylthiohydantoins on polyamide sheets

The aqueous phase phenylthiohydantion (PTH) derivatives of arginine, histidine, and S-pyridylethylcysteine are successfully separated by thin-layer chromatography on polyamide sheets using 10% aqueous pyridine as solvent. Trials on fractions from sequenator runs show that this solvent also separates these PTHs from contaminants which are usually present in the aqueous phase. Development with a second-dimension solvent containing a fluorescent indicator provides a method for identifying these PTHs in subnanomole quantities.

An improved system for the separation and identification of phenylthiohydantoin derivatives of histidine and arginine by thin-layer chromatography

An improved technique for the separation and identification of phenylthiohydantoins of histidine and arginine by thin-layer chromatography of Cheng-Chin polyamide sheets has been described. The solvent system used was n-butyl acetate:methanol:glacial acetic acid (29:20:1; 300 mg of butyl-PBD/liter).

The application of 0.1 M quadrol to the microsequence of proteins and the sequence of tryptic peptides.

In an effort to extend automated Edman degradation to nanomole quantities of protein, the method of sequenator analysis described by Edman and Begg (Edman, P., and Begg, G. (1967), Eur. J. Biochem. 1, 80) was modified to permit long degradations in the absence of carrier proteins. By using an aqueous 0.1 M Quadrol program with limited, combined benezene-ethyl acetate solvent extractions, as well as a change in the delivery system for heptafluorobutyric acid, it was possible to recover and identify the first 30 amino acid residues from a sequenator run on 7 nmol of myoglobin. For 3 nmol of myoglobin, 20 steps could be identified. PTH-amino acids were identified by gas-liquid chromatography and thin-layer chromatography on polyamide sheets. Without using a carrier protein the cup to prevent mechanical losses (Niall, H. D., Jacobs, J. W., Van Rietshoten, J., and Tregear, G. W. (1974), FEBS Lett. 41, 62), the repetitive yield using this program was 93-96%. The same program has been applied successfully to peptides of 14 or more residues with or without modification by Braunitzer's reagent and to a number of larger peptides and proteins including a 216 residue segment of rabbit antibody heavy chain in which a sequence of 35 steps was accomplished on 25 nmol.

Application of the dansylation reaction to the characterization of low molecular weight peptides by dodecyl sulfate-polyacrylamide-gel electrophoresis

Polyacrylamide-gel electrophoresis of low molecular weight DNS-peptides was performed in the presence of 0.1% sodium dodecyl sulfate and 8 m urea. This procedure enabled an estimation of the molecular weight of peptides at the nanomole level without staining. A linear relationship between molecular weight and mobility was obtained over a molecular weight range of 1,000–12,000, although a few anomalous peptides (e.g., glucagon and insulin) and small peptides of molecular weight less than 1,000 deviated from linearity. The N-terminal amino acid of a peptide can be determined in combination with thin layer chromatography on polyamide sheets. The usefulness of this procedure for checking small amounts of contaminant in an oligopeptide sample was also noted.

Mikroquantitative bestimmung von aliphatischen aminen mit 7-chlor-4-nitrobenzo-2-oxa-1,3-diazol

Microquantitative determination of aliphatic amines with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole Fluorometric determination of microanalytical quantities of aliphatic amines with 7-chloro-4-nitrobenzo-2-oxa-1-3-diazole is described. Reaction conditions in a one-phase system, clean-up methods and column or thin-layer chromatographic separation procedures are investigated in order to know the potential of the reagent. For quantitative investigation, separation on polyamide sheets with subsequent in situ measurement by means of a Zeiss chromatogram spectrophotometer is particularly suitable. A linear calibration curve is obtained for quantities between 15 and 150 ng per spot. The high stability of the reaction products and the efficient reproducibility of the procedure justify recommendation of the reagent for the microanalytical determination of amines.

Thin-layer chromatography of sub-nanomole amounts of phenylthiohydantoin (PTH) amino acids on polyamide sheets

Thin-layer chromatography of PTH amino acids on polyamide sheets with a fluorescent indicator gave a 50–100-fold increase in sensitivity when compared to thin-layer polyamide plates developed by spray techniques. Resolution of the individual residues in a standard mixture remains comparable to that described previously. Using this technique, the two-dimensional chromatography of each residue from automated sequence analysis is quite feasible even when small amounts of material are available.