End-group Determination Research Articles

Oxytocin biotransformation in the rat limbic brain: Chemical characterization of two oxytocin fragments and proposed pathway for oxytocin conversion

Two peptide fragments of oxytocin were isolated by high-pressure liquid chromatography from digests of oxytocin obtained after exposure to a SPM preparation of the rat limbic brain. The structures of these peptides, being Gln-Asn-Cys(O) x-Pro-Leu-GlyNH 2 and Gln-Asn-Cys(-S-S-Cys)-Pro-Leu-GlyNH 2, were assessed by quantitative amino acid analysis, combined with the determination of N-terminal end groups and cysteic acid residues after performic acid treatment. The fragments comprised the 4–9 and 1,4–9 sequences of oxytocin, respectively. The types of proteolytic enzymes involved in their formation are discussed and a pathway for the conversion of oxytocin by SPM is proposed.

Conversion of Des-tyrosine-γ-endorphin by brain synaptic membrane associated peptidases: Identification of generated peptide fragments

Des-tyrosine-γ-endorphin, a β-endorphin fragment with neuroleptic-like properties, was digested with a cSPM fraction of rat brain. A profile of metabolites and a time course of conversion were obtained by HPLC analysis of the digests. Quantitative amino acid analysis and a second HPLC fractionation step which was designed to separate and to identify very similar des-tyrosine-γ-endorphin fragments, combined with dansyl end group determination allowed the characterization of β-LPH 65–77, β-LPH 66–77 and β-LPH 62–73 as main conversion products. In the digests the C-terminal leucyl peptides β-LPH 67–77 and β-LPH 68–77 as well as the N-terminal glycyl peptides β-LPH 62–74 and β-LPH 62–76 were minor components. The data indicate the involvement of several types of peptidase activities in the conversion process. It is suggested that these peptidases have a role in mediating in vivo des-tyrosine-γ-endorphin effects. In addition, this study points to the capacity of the brain to gene-rate small peptides with neuroleptic-like properties.

Determination of the amino end groups in polyamide 6 and 6,6 with ninhydrin

AbstractThe amino end groups in polyamide 6 and 6,6 were reacted in a water/methylcellosolve/propionic acid/sodium propionate/pyridine/isopropanol system for 30 min at 100°C with ninhydrin to give a dye (λmax : 570 nm, ϵ570 : 18.6 · 103 cm2 mol−1), whose photometrically determinable concentration represented a measure of the amino end group content. Although the polymer remained undissolved during the analysis, the reaction proceeded quantitatively in all the samples studied (fibres with a maximum fineness of 44 dtex, corresponding to a diameter of 70 μm). ϵ‐Aminocaproic acid served as a low‐molecular calibration substance for the polymer.On account of its simplicity, rapidity, and good reproducibility (variation coefficient 3.4%), the ninhydrin method is suitable for the serial determination of the amino end groups in polyamide 6 and 6,6.

Determination of sulfate and similar end groups in polymers [poly(methyl methacrylate) and polystyrene] by an improved dye interaction method

AbstractIt has been observed that in a benzene medium the carbinol base of a cationic dye reacts with a polymer with SO3 Na and similar end groups that give rise to a reversible color formation of the dye cation. This reaction can be pushed to completion and consequent intensification of color by adding a benzene‐insoluble acid such as citric acid. This principle has been applied to develop an improved dye interaction method of determining such end groups. The end groups values thus obtained by this method for different poly(methyl methacrylate) and polystyrene samples were found to be in fairly good agreement with those obtained by the dye partition (extrapolation) method.

Determination of primary amine endgroups on polymers with fluorescamine in nonaqueous solvents

Determination of primary amine endgroups on polymers with fluorescamine in nonaqueous solvents

Photoaffinity labeling of insulin receptor proteins of liver plasma membrane preparations.

The photoreactive insulin derivatives N epsilon B29-(azidobenzoyl)insulin (MAB-insulin) and N alpha A1, N epsilon B29-di(azidobenzoyl)insulin (DAB-insulin) were synthesized by reacting bovine insulin with the N-hydroxysuccinimide ester of 4-azidobenzoic acid. These derivatives were purified by ion exchange chromatography on SP-Sephadex, and their identities were established by polyacrylamide gel electrophoresis, amino acid analysis, and end-group determination. Their biological activities were measured by receptor binding assay and fat cell assay. The photoreactivity of these two derivatives was demonstrated by spectral changes and by the formation of covalent polymers of high molecular weight when exposed to light. Radioactive MAB-insulin and DAB-insulin were prepared by iodination with [125I]iodine. These radioactive derivatives were characterized for their photoreactivity, immunoreactivity, and receptor binding to liver plasma membrane. Liver plasma membrane preparations of rat, mouse, and guinea pig were incubated with these radioactive insulin derivatives and irradiated with light. Sodium dodecyl sulfate gel electrophoresis of these plasma membrane preparations after solubilization and reduction showed that two proteins were specifically labeled. The molecular weights of the two radioactive bands were estimated to be about 130 000 and 90 000 in all three species of animals.

Bestimmung von Amino- und Carboxylendgruppen in Poly(paraphenylen-terephthalamid)

Two chemical methods are described for the quantitative determination of amino- and carboxyl end-groups in poly(paraphenylene terephthalamide). The amino end-groups are determined by heterogeneous reaction with 1-fluoro-2,4-dinitrobenzene. After washing out the excess of reagent, the modified polymer is dissolved in methanesulphonic acid and the introduced dinitrophenyl group is measured spectrophotometrically. The determination of carboxyl endgroups is based on a quantitative conversion to an amino group, nitrogen and carbon dioxide with sodium azide in 100 % sulphuric acid. The liberated carbon dioxide is determined by potentiometric titration. The standard deviations of the methods are 2.5 mmole COOH/kg and 0.7 mmole NH2/kg within the range of 5–200 mmole/kg.

Determination of Tert-Butoxy Endgroups in Polymers: The Mode of Reaction of Tert-Butoxy Radicals with Methyl Methacrylate and Styrene

Abstract A simple technique has been devised for the determination of tert-butoxy endgroups in poly(methyl methacrylate) and polystyrene. The method is based on cleavage of the tert-butyl ether with boron trichloride and analysis of the resulting tert-butyl chloride by gas-liquid chromatography. In polystyrene, tert-butoxy endgroups were also determined by dealkylation with trifluoroacetic acid, followed by reaction of the resulting hydroxy endgroups with o-sulfobenzoic anhydride and analysis for sulfonate by the dye-partition technique with methylene blue reagent. From these analyses it was possible to conclude that tert-butoxy radicals, derived from the decomposition of di-tert-butyl peroxalate (DBPOX), initiated styrene polymerization largely by direct addition, whereas in their reaction with methyl methacrylate (MMA) both direct addition and hydrogen abstraction from the monomer were important processes. The extent of hydrogen abstraction was determined from the yield of tert-butanol in the reaction o...

Purification and molecular characterization of two inhibitors of yeast proteinase B.

A rapid purification procedure for large scale preparations of yeast proteinase B inhibitors 1 and 2 (IB1 and IB2) is described. By disc gel electrophoresis, amino acid analysis, and end-group determinations, each of the inhibitors is homogeneous. Both inhibitors are polypeptides with molecular weights of 8,500, containing 74 residues. No components other than amino acids could be detected. There is no significant difference in the amino acid compositions of the two inhibitors as analyzed after acid hydrolysis. Both polypeptides are characterized by the total absence of arginine, tryptophan, and sulfur-containing amino acid residues. The proteinase B inhibitors of yeast, therefore, differ fundamentally from proteinase inhibitors of many other organisms, which generally contain a large number of disulfide bridges. Both proteinase B inhibitors have threonine as the NH2-terminal residue and -Val-His-Thr-Asn-COO- as the COOH-terminal sequence. Comparison of peptide maps after tryptic digestion reveals that the two inhibitors differ definitely in only a few tryptic peptides. The inhibitors are rapidly inactivated by digestion with carboxypeptidase A from bovine pancreas at pH 8.5. Inactivation occurs stoichiometrically with the release of threonine, the penultimate residue at the COOH-terminal end of both inhibitors.

Isolation and characterization of somatostatin from pigeon pancreas.

Most of the somatostatin-like activity from pigeon pancreas was found to correspond to small species with an apparent molecular weight of 1500--2500. This species was isolated under conditions minimizing intermolecular interactions and protease activities. The isolated product was characterized by two somatostatin radioimmunoassays, a bioassay, endgroup determination, and amino acid analysis. The structure of the isolated compound was determined to be H-Ala-Gly-cyclo-(Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys)-OH. Additionally, small amounts of des-Ala1-somatostatin, a possible degradation product of pancreatic somatostatin, and a large somatostatin-like species with an apparent molecular weight of 11,000--12,500 were detected. It is concluded that the main somatostatin-like polypeptide isolated from pigeon pancreas is identical to the mammalian hypothalamic tetradecapeptide somatostatin.

Isolation, purification and molecular properties of an acetylcholinesterase (E.C. 3.1.1.7) from the haemolymph of the sea mussel Mytilus edulis

1. The haemolymph of the sea mussel Mytilus edulis contains three cholinesterases (E.C. 3.1.1.8) and one acetylcholinesterase (E.C. 3.1.1.7). 2. Using fractional (NH 4) 2SO 4-precipitation, the acetylcholinesterase can be separated from other haemolymph proteins, e.g. agglutinins and the other cholinesterases. 3. Using gel filtration and ion-exchange chromatography, the desired enzyme could be obtained in a pure form with a specific activity of approximately 100 i.u. 4. Molecular weight, determined by gelfiltration, is about 240,000 with subunits of about 39,000 (determined by SDS-electrophoresis); the IP is 4.9 ± 0.1 and the end group determination reveals glycine as N-terminal amino acid. 5. The enzyme is active within a broad range; optimum pH is from 6.8–8.0 and optimum temperature from 28–40°C. Michaelis-Menten constants are given for some substrates, and K i values for inhibitor interactions with the enzyme.

An improved large scale procedure for the purification of porcine pancreatic lipase

A modified procedure for purifying porcine pancreatic lipase (triacylglycerol acyl-hydrolase, EC 3.1.1.3) is described. In comparison to the previous procedure reported by Verger, R., de Haas, G.H.; Sarda, L. and Desnuelle, P. (1969) Biochim. Biophys. Acta 188, 272–282) it is more rapid, more reproducible and results in a purer enzyme preparation. No colipase could be detected in the mixture of isoenzymes and, naturally, in the different separated lipases. In this process, butanol treatment is omitted. After pancreas powder extraction, a batch procedure was used for adsorption of DEAE-cellulose. Sephadex filtration (pH 8.0) was made in a large size column. Finally the isoenzymes were separated on CM-cellulose as in the Verger procedure, but under slightly modified conditions. Lipase L B was fully homogeneous as judged by end group determination, gel electrophoresis (in the presence or absence of sodium dodecyl sulfate) and sedimentation equilibrium.

Molecular weight–viscosity relationships for poly(1,4‐butylene terephthalate)

AbstractThe correlation between number‐average molecular weight and intrinsic viscosity in 60:40 phenol‐sym‐tetrachloroethane at 30°C for poly(1,4‐butylene terephthalate) was established from endgroup determinations as well as by gel permeation chromatography, eqs. (1) and (10a). The GPC data also yielded relationships between weight‐ and z‐average molecular weight and intrinsic viscosity, eqs. (10b) and (10c). Melt viscosities, corrected for the thermal history of the melt, were measured at shear stresses in the range of 0.02–0.55 MPa. Linear PBT melts were found to become non‐Newtonian at a shear stress of approximately 0.11 MPa, independent of molecular weight within the range studied. Correlations between melt viscosity at low shear stress versus intrinsic viscosity are presented, as well as the dependence of melt viscosity in the non‐Newtonian region on shear stress and low‐stress (Newtonian) melt viscosity.

Synthesis and characterization of functional diene oligomers in view of their practical applications

AbstractAn overview is given on the preparation and characterization of hydroxyl terminated liquid polybutadienes and polyisoprenes which are potential alternative backbones for polyurethanes. After stating the requirements for such precursors, selected routes to their production are discussed, taken from more recent literature and from own experience. Characterization encompasses new methods of end group determination, problems of functionality and fractionation. In spite of the progress achieved in various aspects, outstanding properties of e.g. carbon black‐filled polyurethanes have been reported rarely and on a larger scale commercial realization of such oligomers for high quality products seems somewhat uncertain at the moment.

Determination of hydroxyl end groups in poly(ethyiene terephthalate) and poly(butylene terephthalate) by reaction with dichloroacetic anhydride

A chemical method is described which provides a reproducible quantitative determination of hydroxyl end groups in poly(ethylene terephthalate) and poly (butylene terephthalate). It is based on esterification at 60° C of the hydroxyl end group with dichloroacetic anhydride as an acetylating reagent in dichloroacetic acid as a solvent. After removal of the excess of esterification reagent and solvent by precipitation techniques, the chlorine content of the modified polymer, which is a measure of the hydroxyl group content, is determined by X-ray fluorescence. The standard deviation of the method is 0.7 mmol OH/kg in the region of 10–200 mmol/kg.

End-group determination in DNA of mammalian cells after low doses of ionizing radiation: T. Coquerelle, C. Sexauer and U. Hagen, Kernforshungszentrum Karlsruhe, Institut für Strahlenbiologie, 75 Karlsruhe 1, Postfach 3640 (Germany)

End-group determination in DNA of mammalian cells after low doses of ionizing radiation: T. Coquerelle, C. Sexauer and U. Hagen, Kernforshungszentrum Karlsruhe, Institut für Strahlenbiologie, 75 Karlsruhe 1, Postfach 3640 (Germany)

Determination of unsaturated structures in poly(vinyl chloride) by means of fourier transform 1H‐NMR spectroscopy

AbstractA method based on the PFT‐NMR technique was developed for a direct determination of unsaturated end groups in PVC. By using NMR proton spectra it was found that the unsaturated groups in PVC contain allylic chlorine atoms. Three types of unsaturated end groups were identified, corresponding to the following models: 3‐chloro‐1‐pentene, 2,3‐dichloro‐1‐pentene, and 1,2‐dichloro‐2‐pentene. The last two types could also correspond to some unsaturated groups on the backbone bearing allylic chlorine atoms. The presence of such groups would partially explain the PVC degradation.

Grafting onto wool: 7. Determination of end groups of grafted polymer and homopolymer formed in methanol-benzoyl initiating system

A previous assessment of the end group incorporated in poly(methyl methacrylate) and polystyrene chains, based on the synthesis of the 2,4-dinitrophenyl (DNP) derivatives of hydroxyl and amino groups, has been extended. It has been found that in methanol-benzoyl peroxide initiating systems, hydroxyl, phenyl and benzoyloxy radicals initiate homopolymerization. The total number of end groups has been determined. From the results of the number of DNP-amino-acid end groups in the isolated polymer chain from the grafted fibres and from chemically modified fibres before and after grafting, it is postulated that two DNP-end groups are linked to the polymer chain, and one DNP—amino-acid is situated at each end of the polymer chain. No initiating mechanism visualized with respect to the homopolymerization is involved in the graft copolymerization.

Endonuclease Activities in Extracts ofMicrococcus Luteusthat Act on γ-irradiated DNA

Several protein fractions containing endonuclease activity against gemma-irradiated DNA (gamma-endonuclease) were isolated from M. luteus. The crude extract was eluted on a phosphocellulose column and chromatographed on TEAE cellulose and subsequently on hydroxyapatite. Five peaks of gamma-endonuclease were obtained from each preparation. Repeated experiments showed comparable chromatographic behavior of the fractions. There was no detectable activity of U.V.-endonuclease in the fractions with gamma-endonuclease but a small contamination of endonuclease against unirradiated DNA and against DNA with apurinic sites. The gamma-endonuclease is stimulated by, but is not dependent on, magnesium. Several tests for endonuclease activity have been used: the analysis of strand breaks in calf-thymus DNA or in PM2 DNA, and the determination of end-groups formed by endonuclease, either 3'OH end-groups or phosphomonoester end groups. From the results obtained it can be assumed that the strand breaks induced by the gamma-endonuclease carry 3'OH and 5' phosphate end groups.

The semisynthesis of portions of hen's-egg lysozyme by fragment condensation

1. We have evaluated several methods for coupling protected tryptic peptides and have selected one that is satisfactory in terms of efficiency and degree of racemization. 2. We report the use of the chosen method (which must be slightly modified for each application in the light of the result of micro-scale pilot experiments) for the preparation of five fragments of hen's-egg lysozyme ranging in length from 31 to 51 residues. 3. We report methods for the complete deprotection of these fragments. They have been characterized by end-group determination, amino acid analysis and tryptic digestion.