The use of p-Iodophenyl[125I] isothiocyanate for determination of N-terminal amino acids in nanomole quantities of protein

1975 ◽  
Vol 28 (10) ◽  
pp. 2289 ◽  
Author(s):  
CJ Burrell ◽  
PD Cooper ◽  
JM Swan
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
High Specific Activity ◽  
Coupling Reaction ◽  
Molar Content ◽  
Terminal Residue ◽  
Terminal Amino

Identification of the N-terminal residue of 2.5-5 nmol of protein was possible by means of a modified Edman reaction and p-iodophenyl[125I] isothiocyanate of high specific activity. The second and third residues could also be identified. Yields of the terminal residue were 25-50%, allowing approximate quantitation of the molar content of protein. Use of the isotope allowed certain steps of the degradative cycle to be examined in some detail, and preparation of this relatively non- volatile labelled Edman reagent was safe, cheap and convenient. ��� The synthesis and properties of the p-iodophenylthiohydantoins of 19 amino acids, and some side products of the coupling reaction, are described.

scholarly journals The Determination of N-terminal Amino Acids during the Conversion of Fibrinogen to Fibrin.

1957 ◽  
Vol 11 ◽  
pp. 194-195 ◽  
Author(s):  
Birger Blombäck ◽  
Ikuo Yamashina ◽  
S. Ross ◽  
J. Schliack ◽  
L. Reio
Keyword(s):  
Amino Acids ◽  
Terminal Amino

scholarly journals The determination of oestradiol and oestrone in the plasma of the domestic fowl by a method involving the use of labelled derivatives

1968 ◽  
Vol 106 (1) ◽  
pp. 77-86 ◽  
Author(s):  
J. E. O'Grady
Keyword(s):  
Human Plasma ◽  
Paper Chromatography ◽  
Domestic Fowl ◽  
Specific Activity ◽  
High Specific Activity ◽  
Double Labelling ◽  
Plasma Samples ◽  
Labelling Technique ◽  
Preliminary Purification

1. A method involving the use of triple-labelled derivatives has been developed for the determination of total oestrone and oestradiol in the plasma of the domestic fowl. The double-labelling technique devised by Svendsen (1960) for the determination of free oestrogens in human plasma was modified to enable the total oestrogen recovery to be determined for each sample. 2. [6,7−3H2]Oestradiol-17β is added to the plasma samples (1–10ml.), which are hydrolysed with acid and the phenolic steroids then extracted and partially purified. The extract is esterified with iodobenzene-p[35S]-sulphonyl chloride of high specific activity. After addition of standard oestrogen [131I]iodobenzene-p-sulphonates the esters are finally purified by paper chromatography. 3. The oestrogens are determined by comparing the 3H/35S and 131I/35S ratios in the purified esters with similar ratios of appropriate standards. 4. With this procedure the recoveries of oestrone and oestradiol after hydrolysis were 70–85% and 72–84% respectively, and after hydrolysis and preliminary purification 38–53% and 39–51% respectively. With this procedure up to 500ng. of oestradiol can be determined. The sensitivity of the technique for oestrone is 3·0ng. and for oestradiol 2·1ng. 5. The ranges of oestradiol and oestrone concentrations found in six plasma samples were 8·3–21·4ng./ml. and 15·2–31·6ng./ml. respectively.

scholarly journals Quantitative determination of N-terminal amino acids of peptides and proteins with cobalt(III) chelates

1976 ◽  
Vol 153 (1) ◽  
pp. 137-138 ◽  
Author(s):  
K W Bentley
Keyword(s):  
Amino Acids ◽  
Quantitative Determination ◽  
Peptide Bond ◽  
Small Peptides ◽  
Acidic Hydrolysis ◽  
Terminal Amino ◽  
A Chain ◽  
Glucagon And Insulin

Quantitative N-terminal peptide-bond hydrolysis with the cis-beta-hydroxyaquo(triethylenetetramine) cobal (III) ion, i.e. β-[Co(trien)(OH)(OH2)]2+, is reported. The method has been demonstrated with 20 small peptides, a hexapeptide, bradykinin, insulin A chain (oxidized), glucagon and insulin. The procedure involves no acidic hydrolysis step and thus no destruction of labile amino acids.

Eine Methode zur qualitativen und quantitativen Bestimmung von drei Juvenilhormonen von Insekten

1974 ◽  
Vol 29 (3-4) ◽  
pp. 161-168 ◽  
Author(s):  
K. H. Trautmann ◽  
A. Schuler ◽  
M. Suchý ◽  
H.-K. Wipf
Keyword(s):  
Quantitative Determination ◽  
Specific Activity ◽  
High Specific Activity ◽  
Ether Extract ◽  
Qualitative And Quantitative ◽  
Work Up ◽  
First Time ◽  
Very High ◽  
Tenebrio Molitor Larvae

Abstract A method is presented permitting the qualitative and quantitative determination of all three presently known hormones (JH1-3). The determination is based on the method of radioactive isotope dilution, whereby a very small known amount of tritium-labelled JH-1 is added to the ether extract of the particular species. The addition of radioactive JH-1 permits the isolation of all three hormones, because of their similar behaviour during the chosen work up. The quantitative determination was carried out by gas chromatography and the identification was confirmed with the help of retention-times and GC-MS combination. The method was checked by using an extract of Hyalophora cecropia. For the first time methyl 10,11-epoxy-3,7,11-trimethyl-2-trans-6-trans-dodecadienoate (JH-3) could also be identified as the juvenile hormone of Melo­lontha melolontha. In Vanessa io larvae, Tenebrio molitor larvae and adults and in Musca domestica larvae none of the three known hormones could be detected. The preparation of JH-1 labelled with tritium in the methyl group of the ester was accomplished with very high specific activity (4.34 Ci/mmol) of the tritiated acid with diazomethane.

scholarly journals Determination of the N-Terminal Amino Acids in Proteins Using Fluoro-Nitropyridines

1969 ◽  
Vol 7 (3) ◽  
pp. 328-333 ◽  
Author(s):  
A. Signor ◽  
L. Biondi ◽  
A. M. Tamburro ◽  
E. Bordignon
Keyword(s):  
Amino Acids ◽  
Terminal Amino

An Improvement of the Hydrazine Method for Determination of C-Terminal Amino-acids

Nature ◽  
1956 ◽  
Vol 178 (4539) ◽  
pp. 912-913 ◽  
Author(s):  
J. H. BRADBURY
Keyword(s):  
Amino Acids ◽  
Terminal Amino

Improvements in the determination of the turnover rate of plasma thyroxine in avian species: application to the Japanese quail

1987 ◽  
Vol 114 (2) ◽  
pp. 191-198 ◽  
Author(s):  
E. J. Cookson ◽  
J. Glover
Keyword(s):  
Japanese Quail ◽  
Half Life ◽  
Turnover Rate ◽  
Specific Activity ◽  
Simple Procedure ◽  
High Specific Activity ◽  
Future Application ◽  
Sodium Thiocyanate ◽  
Plasma Samples

ABSTRACT The disappearance of the thyroid hormone thyroxine (T4) from plasma in fully grown male Japanese quail can be described as a first order process with a rate constant of 0·178 ± 0·013/h (mean±s.e.m., n = 8), which represents a half-life of 3·90 h. A small amount of [125I]T4 in relation to total circulating T4 was injected i.v. into Japanese quail and plasma samples were taken at appropriate time-intervals for the determination of residual plasma radioactivity. The rate of disappearance of [125I]T4 was subsequently equated to the turnover rate of the endogenous hormone. Previous methods were modified to overcome problems arising from possible disturbance of plasma T4 metabolism, recirculation of radiolabelled iodide, and to purify the [125I]T4 from the plasma samples. By using labelled T4 of very high specific activity, the amount of [125I]T4 administered was kept much smaller than has been used in previous studies on Japanese quail, thus limiting any interference with plasma T4 dynamics. To minimize any disturbance of plasma T4 metabolism, only four blood samples were taken, at three-hourly intervals after the injection of [125I]T4. The rapid turnover of T4 produced a large amount of labelled inorganic iodide, the re-entry of which into the plasma T4 pool was inhibited by s.c. administration of sodium thiocyanate 1 h before injection of[125I]T4. Assay of the true [125I]T4 turnover was significantly improved over that used in previous studies by purifying the [125I]T4 from the plasma samples chromatographically. The samples were applied to small Sephadex G-25 columns with sodium hydroxide (0·1 mol/l) as the eluant. This simple procedure clearly separated the [125I]T4 from the other radioiodinated plasma components such as free iodide, non-hormonal iodinated proteins and triiodothyronine (T3), thus enabling a more accurate assessment of the residual labelled T4 concentration in the plasma and hence the T4 half-life. The future application of this method to the study of plasma T4 turnover under various experimental conditions is discussed and the possible involvement of T4 turnover studies in the assessment of T4 to T3 conversion is considered. J. Endocr. (1987) 114, 191–198

scholarly journals A novel manual method for protein-sequence analysis

1976 ◽  
Vol 157 (1) ◽  
pp. 77-85 ◽  
Author(s):  
J Y Chang ◽  
E H Creaser
Keyword(s):  
Amino Acids ◽  
Sequence Analysis ◽  
Protein Sequence ◽  
Detection Sensitivity ◽  
Phenyl Isothiocyanate ◽  
Protein Sequence Analysis ◽  
Manual Method ◽  
Terminal Residue ◽  
A Chain

A novel manual method for protein-sequence analysis is described. Three peptides, the hexapeptide (Leu-TRP-Met-Arg-Phe-Ala), insulin A chain and glucagon were used to test this technique. Peptides (1 or 2 nmol) were hydrolysed with acid and their qualitative amino acid compositions were confirmed by reacting with 4-NN-dimethylaminoazobenzene-4'-sulphonylchloride and 4-NN-dimethylaminoazobenzene 4'-isothiocyanate. Sequence determination of 20-200 nmol of peptide was then performed by the combined use of phenyl isothiocyanate and 4-NN-dimethylaminoazobenzene 4'-isothiocyanate, a new procedure that is analogous to the dansyl-Edman method with the replacement of dansyl chloride by 4-NN-dimethylaminoazobenzene 4'-isothiocyanate as the N-terminal residue determination reagent. On t.l.c. this new N-terminal reagent gave brightly coloured 4-NN-dimethylaminoazobenzene-4-thiohydantoins of amino acids and showed the following advantages: (1) the detection sensitivity is in the pmol range; (2) u.v. observation is not required; (3) there is no destruction of acid-labile amino acids; (4) two-dimensional t.l.c. separation is adequate to identify 24 amino acids, except leucine and isoleucine (this pair of amino acids can be resolved by using 4-NN-dimethylaminoazobenzene-4'-sulphonyl chloride); (5) the determination of a new N-terminal residue (from coupling to t.l.c. identification) takes only 3 h; (6) the colour difference beteen isothiocyanate, thiocarbamoyl and thiohydantoin derivatives facilitates the identifications.

scholarly journals An Alkane-Responsive Expression System for the Production of Fine Chemicals

1999 ◽  
Vol 65 (6) ◽  
pp. 2324-2332 ◽  
Author(s):  
Sven Panke ◽  
Andreas Meyer ◽  
Caroline M. Huber ◽  
Bernard Witholt ◽  
Marcel G. Wubbolts
Keyword(s):  
Specific Activity ◽  
Styrene Oxide ◽  
Expression System ◽  
Regulatory System ◽  
Dry Weight ◽  
Pseudomonas Oleovorans ◽  
Structural Genes ◽  
Biotechnological Processes ◽  
Terminal Amino

ABSTRACT Membrane-located monooxygenase systems, such as thePseudomonas putida mt-2-derived xylene oxygenase, are attractive for challenging transformations of apolar compounds, including enantiospecific epoxidations, but are difficult to synthesize at levels that are useful for application to biotechnological processes. In order to construct efficient biocatalysis strains, we utilized the alkane-responsive regulatory system of the OCT plasmid-located alk genes of Pseudomonas oleovorans GPo1, a very attractive system for recombinant biotransformation processes. Determination of the nucleotide sequence of alkS, whose activated gene product positively regulates the transcription of the structural genes alkBFGHJKL, on a 3.7-kb SalI-HpaI OCT plasmid fragment was completed, and the N-terminal amino acid sequence of an AlkS-LacZ fusion protein was found to be consistent with the predicted DNA sequence. The alkS gene and the alkBp promoter were assembled into a convenient alkane-responsive genetic expression cassette which allowed expression of the xylene oxygenase genes in a recombinant Escherichia coli strain at a specific activity of 91 U per g (dry weight) of cells when styrene was the substrate. This biocatalyst was used to produce (S)-styrene oxide in two-liquid-phase cultures. Volumetric productivities of more than 2 g of styrene oxide per h per liter of aqueous phase were obtained; these values represented a fivefold improvement compared with previous results.

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