End-group Determination Research Articles

Specific binding of a nonhistone chromosomal protein from lymphocyte to DNA.

The isolation of a nonhistone chromosomal protein, NH30 000, from bovine lymphocytes by affinity chromatography on a DNA-agarose column is described. The procedure starts with nonhistone fraction NH4 obtained by hydroxyapatite chromatography as described previously [Blüthmann, H., Mrozek, S. & Gierer, A. (1975) Eur. J. Biochem. 58, 315-326]. Protein NH30 000 migrates as a single band with a molecular weight of 30 000 on sodium dodecylsulfate polyacrylamide gels. It contains, on a molar basis 25.3% acidic amino acids and 18.3% basic amino acid residues, 10.3% being lysine. End-group determination confirms that this protein is composed of one polypeptide chain with the NH2-terminal amino acid arginine. The binding of protein NH30 000 to native DNA of different molecular weights has been investigated by the nitrocellulose filter assay. The results suggest that in the presence of 0.2 M NaCl it binds about 30% of the DNA with binding sites every 1700 nucleotide pairs. Competition experiments show that protein NH30 000 exhibits a higher binding affinity for lymphocyte DNA in comparison to Escherichia coli DNA.

The arrangement of 18-S and 28-S ribosomal ribonucleic acids within the 40-S precursor molecule of Xenopus laevis.

The arrangement of 18-S rRNA and 28-S rRNA within their 40-S common precursor molecule (pre-rRNA) of Xenopus laevis was investigated by electron microscopic analysis of secondary structure of nascent pre-rRNA chains of oocytes, and by 5'-end analysis of 18-S rRNA and 28-S rRNA hybridized to the EcoRI fragment of rDNA cloned as plasmid pCD42. Secondary structure mapping of phenol-extracted RNA from nucleolar cores revealed complete pre-rRNA chains or molecules at various stages of processing and pre-rRNA molecules apparently lacking one end. In this latter group, which was regarded as representing nascent chains, more than 90% of the molecules had no 28-S rRNA REGION. This shows that the 28-S rRNA sequence is transcribed after the 18-S rRNA region and hence must be located nearer to the 3' end of the pre-rRNA molecule. For 5' end-group determination [3H]uridine-labelled 18-S rRNA and 28-S rRNA were hybridized, as fragments of about 200 nucleotides, to the plasmid pCD42 containing coding sequences for four-fifths of the 18-S rRNA sequence, the external transcribed spacer, the non-transcribed spacer and a tenth of the 28-S rRNA sequence. The RNA was recovered from the hybrids and analyzed for uridine 3',5'-bisphosphate (pUp) after alkaline hydrolysis. The pUp content of the hybridized 18-S rRNA fragments was 20-fold higher than in those of 28-S rRNA, THUS DEMONSTRATING THAT THE 5' END OF THE 18-S rRNA is located next to the external spacer region. From these results it is concluded that the 18-S rRNA is located close to the 5' end of the 40-S pre-rRNA molecule.

The structure and stability of trypsin-resistant segments from rabbit tropomyosin

Tropomyosin was found to undergo only limited digestion by trypsin at 0 °C and the two segments that accumulated amounted to two-thirds of the original protein. They are referred to as segments A and B. These segments were not resistant to trypsin digestion at 20 °C and at the latter temperature no large fragments remained as judged by disc gel electrophoresis. Segments A and B were separated from each other on the basis of solubility differences and were found to have molecular weights of 24 600 and 21 900 respectively. Each of the segments appeared to retain about 70–75% of the helical conformation as judged by circular dichroism at 20 °C. However, the segments did not show any of the inhibitory activity of the parent tropomyosin molecule when mixed with troponin in the Mg 2+-actomyosin ATPase system. Amino acid analysis showed that the portion of tropomyosin that was digested by trypsin (EC 3.4.21.4) had a lower content of the helix stabilizing residues Glu and Leu and a higher content of the helix-destabilizing residues Arg and Lys. These differences indicate that the digested portion should be less stable in the helical conformation than the two trypsin-resistant segments. End group determinations along with the results of the amino acid analysis indicated that segment A was probably derived from the central one-third of tropomyosin and segment B from the C-terminal one-third. By the process of elimination the N-terminal third appears to have been the more labile region that was digested by trypsin. The segments A and B were shown to differ in their stability to denaturation by guanidine·HCl and elevated temperature. All of these observations indicate that tropomyosin is not a uniform structure and is composed of regions of different stability.

Studies on the pituitary "fettstoffwechselhormon". VII. Analytical studies on two lipolytically high active peptides from hog pituitary glands.

First results of analytical studies on the highly purified lipolytic active hog pituitary fractions P-LF II C and P-LF II D are reported. Electrophoresis and end-group determinations indicate homogeneity for both peptides. The molecular weights, amino acid compositions, and the N- and C-terminal sequences of these peptides have been established and are compared with the corresponding results of other laboratories. The relation between biological activity and the structure of lipolytic active peptides is discussed.

A rapid assay method for hyaluronidase

Hyaluronidase activity has been measured by a semimicro radiochemical method. 3H-Hyaluronic acid was prepared using hydrazine to remove some of the acetyl groups followed by reacetylation with tritiated acetic anhydride. Hydrolysis was carried out using an extract from rat liver lysosomes and a cruder extract from other tissues. At the completion of hydrolysis, cetylpyridinium chloride was added to precipitate unreacted substrate. Smaller molecular-weight oligosaccharides produced by hydrolysis remain in solution to be determined by liquid scintillation counting. This assay gives results that are similar to those obtained by the assay using N-acetylglucosamine end-group determination but, in the case of tissue extracts, in 1 hr instead of 18 hr.

Subunit structure of d-galactose dehydrogenase from Pseudomonas saccharophila

1. 1. d-Galactose dehydrogenase from Pseudomonas saccharophila (molecular weight 102 000) dissociates in 8 M urea into its subunits (molecular weight 25 000) which migrate in polyacrylamide gels, containing 8 M urea, as a single band. 2. 2. The N-terminal residue determination by the dansyl method revealed only serine. 3. 3. The C-terminal group determination with carboxypeptidase A and B indicated the sequence -Tyr-His-Leu. Leucine as the single C-terminal amino acid was confirmed by the tritiation method and by tritiation and subsequent degradation with carboxypeptidases. 4. 4. The fragmentation of d-galactose dehydrogenase (24 mol methionine per mol enzyme) by CNBr resulted in six peptides, as detected in disc electrophoresis and substantiated by end group determination, indicating the identity of the subunits. 5. 5. The treatment of d-galactose dehydrogenase (24 mol lysine and 52 mol arginine per mol enzyme) with trypsin and subsequent peptide mapping showed 21, perhaps 22 peptides, indicating a structure comprising four identical subunits.

Structural studies on the coat protein of alfalfa mosaic virus. Isolation and characterization of the tryptic peptides and the alignment of the cyanogen-bromide fragments.

The reduced and carboxymethylated coat protein of alfalfa mosaic virus (AMV 425) was fragmented by means of cyanogen-bromide cleavage. The tryptic peptides from the protein and its four cyanogen-bromide fragments were isolated on a preparative scale by combinations of column and paper separation techniques. The tryptic digest of the carboxymethylated protein contained 24 peptides and two free amino acids. All peptides have been characterized by amino acid analyses and end-group determinations. Together the tryptic peptides account for a total chain length of 228 amino acids. The data are in good agreement with previous reports from this laboratory.

Isolation and characterization of alpha-1-antitrypsin from the Cohn fraction IV-I of human plasma.

Abstract Cohn Fraction IV-I from pooled human plasma was used as a starting material in the large-scale purification of alpha-1-antitrypsin (alpha-1-AT). Following ion-exchange, blospecific affinity and gel exclusion chromatographic procedures, material of high biological activity was obtained in 307percnt; overall yield. Homogeneity was demonstrated by acrylamide gel electrophoresis, immunoelectrophoresis, ultracentrifugation, gel filtration and end-group determination. The present preparation should be applicable to large scale industrial processing of alpha-1-AT with the potential for use in protein replacement therapy.

Correlation between sedimentation coefficient and molecular weight of denatured DNA

The relation between the sedimentation coefficient s 0 20, w of denatured DNA and its weight average molecular weight, M w , was investigated in the range between 4 · 10 4 and 2 · 10 7. The molecular weight was determined by means of viscometry, by end group determination with exonuclease III or polynucleotide kinase or by equilibrium sedimentation. When denatured DNA is dissolved in 0.2 M NaCl−0.025 M phosphate buffer , pH 7.3-2% formaldehyde, the relation follows the equation: S° − 2.74 = 0.0055( M ̄ w ) 0.52 where the sedimentation coefficient S 0 is expressed in Svedberg units. The data allow us to discuss the denaturated DNA molecules in neutral 0.2 M NaCl-2% formaldehyde as worm-like chains, each with a statistical segment length of about 300 Å. In the course of the studies it was shown that polydisperse mixtures of small DNA molecules with a random distribution in size can be analysed by equilibrium sedimentation.

γ-Glutamylwillardiine and γ-glutamylphenylalanylwillardiine from seeds of Fagus silvatica

γ- l-Glutamyl- l-willardiine [γ- l-glutamyl-3-(1-uracil)- l- l-alanine] and γ-glutamylphenylalanylwillardiine have been isolated from seeds of Fagus silvatica. The structures have been established by spectroscopy, hydrolysis to give the constituent amino acids, and for the tripeptide end-group determination and partial hydrolysis to give the two constituent dipeptides.

Determination of carboxyl endgroups and comonomers in poly(ethylene terephthalate) with hydrazine

On complete hydrazinolysis of poly(ethylene terephthalate), terephthalomonohydrazide is formed from carboxyl-end terephthaloyl residues in a quantity equivalent to the content of carboxyl endgroups in the polymer. The compound is separated from the reaction mixture by ion exchange and determined photometrically [epsiv;240 in 0.1 N HCl = 16,700 (1000 cm2/mole)]. A COOH determination carried out in this way is endgroup specific and, unlike titration, is not subject to interference by ionogenic fiber additives. Aromatic comonomers with acidic substituents (e.g., 5-sulfoisophthalic acid) in chemically modified, cationically dyeable poly(ethylene terephthalate) are determined simultaneously with the carboxyl endgroups by the same analytical method. In this case, the terephthalamonohydrazide and 5-sulfoisophthalodihydrazide are separated by ion exchange, and the difference in their spectral behavior is used for quantitative determination with the aid of a two-component analysis: where c1c2 = concentration of terephthalomonohydrazide and 5-sulfoisophthalodihydrazide, respectively; and D240 D212 = optical density at 240 and 212 nm, respectively. The content of carboxyl endgroups in polyether esters poly(p-(2-ethyleneoxy)-benzoate), is determined on the basis of the p-(β-hydroxyethoxy)benzoic acid [epsiv;258 in 0.1 N HCl = 16,100 (1000 cm2/mole)] liberated from carboxyl-end monomer units by hydrazinolysis. For copolyether esters with p-(β-hydroxyethoxy)benzoic acid as a comonomer, the contents of carboxyl-end terephthalic acid and p-(β-hydroxyethoxy)benzoic acid are determined simultaneously with the acid of a spectrophotometric twocomponent analysis: where c2, c2 = concentration of terephthalomonohydrazide and p-(β-hydroxyethoxy)-benzoic acid, respectively; and D240, D258 = optical density at 240 and 258 nm, respectively.

The Impact of Natural Fiber Science on Manmade Fiber Technology1

Analytical methods are described for the chemical characterization of polyamide and polyester fibers. Their origin from natural fiber chemistry is elucidated. End-group data (amino, acylated amino, and carboxyl groups), number-average molecular weights and breaking stress values are given for commercial polyamide fibers produced by several manufacturers. Amino groups were deter mined with 1-fluoro-2,4-dinitrobenzene, using a new technique. Besides its specifity, this chemical method allows the differentiation of primary and secondary amino groups and their determination in the presence of each other. A related topic is the detection of heat damage in polyamides. Heat treatment leads to the formation of basic groups, which can be distinguished spectroscopically from amino groups after reaction with 1-fluoro-2,4-dinitrobenzene. As a practical application the elucidation of the damage of a nylon needle felt, used in a paper machine, is described. Hydrazinolysis was applied for the determination of carboxyl end groups. The use of this new analytical method for polyester studies is described, also its application on several commercial polyethylene terephthalate fibers. Furthermore, hydrazinolysis allows the determination of the amount of 5-sulfo-isophthalic acid in acidic modified fibers. Values ob tained are confirmed by determination of the uptake of a basic dyestuff (methylene blue). The importance of end- group analysis is stressed by its application to the analysis of hydrolytically damaged polyester fibers. The increase in carboxyl groups was determined for steam-treated modified and unmodified polyethylene terephthalate. Results concerning the analysis and generation of oligomers in polyester are added.

Cyanogen bromide splitting of human immunoglobulin M

Human Waldenström IgM (κ) (Dau), its polypeptide chains and its F(c) 5 μ and Fab μ fragments were split by cyanogen bromide (CNBr). The fragments formed by CNBr were fractionated by gel filtration, ion exchange chromatography and paper electrophoresis. They were characterized in terms of polyacrylamide disc electrophoresis, peptide maps, amino acid composition, end group determinations and limited primary structure determination. Two CNBr fragments were formed from the κ chain, consistent with the presence of one methionine residue. Five fragments were isolated from the partially S-sulfonated μ chain. Three additional fragments were released after destruction of all disulfide bonds. The present data are unclear as to whether there are eight or nine CNBr fragments released. The comparison of CNBr pieces from the IgM, μ chain, F(c) 5 μ and Fab μ affords a tentative arrangement of their order, as well as the relative location of the disulfide bonds within the molecule.

Purification and characterisation of different active forms of pork trypsin.

1 Pork trypsin has been separated into several active forms by chromatography on sulphoethyl-Sephadex. 2 Two of these forms have been characterised by end-group determinations as a single chain form (the β-form) and a form (the α-form) with a single internal split in its peptide chain between a lysine and a serine residue. 3 The two peptide chains of the α-form have been separated by gel electrophoresis in the presence of sodium dodecylsulphate under reducing conditions and their molecular weights appear to be approximately 11000 and 13000. 4 At a substrate concentration of 0.56 mM the β-form is 25% more active than the α-form against α-N-benzoyl-dl-arginine-p-nitroanilide.

Gas-chromatographic determination of methylester end groups in polyethylene terephthalate (PET) and ester-interchange products

A method for the determination of methylester end groups in polyester and ester-interchange product is described. The sample is decomposed by boiling with hydrazine hydrate. The methanol liberated is determined by gas chromatography using Tenax GC as column packing and ethanol as internal standard. The gas chromatographic determination has passed through a number of development stages, viz. backflush and run methods.

Fractionation of tyrosine-rich proteins from oxidized wool by ion-exchange chromatography and preparative electrophoresis.

On oxidizing wool by performic acid a soluble protein fraction is obtained containing high amounts of tyrosine, phenylalanine and glycine. This fraction, termed “δ‐keratose”, is extremely heterogeneous as shown by solubility characteristics, electrophoresis and end‐group determination. Anion‐exchange chromatography allows isolation of subfractions with different amino‐acid compositions. This process, however, involves a marked loss of tyrosine‐rich proteins absorbed irreversibly. Repeated preparative electrophoresis on polyvinylchloride layers permits isolation of many subfractions on a preparative scale without loss of aromatic amino acids. The amino‐acid composition of these subfractions is investigated. In spite of the wide differences among them some common features for all fractions can be established. It is shown that possible contamination by other proteins is not responsible for the heterogeneity of δ‐keratose but rather due to a wide variation in the amino‐acid composition among the demonstrated tyrosine‐rich proteins.

Fractionation of toxins from Hydrophis cyanocinctus venom and determination of amino acid composition and end groups of Hydrophitoxin a : C.-S. Liu, G. S. Huber, C.-S. Lin and R. Q. Blackwell, Toxicon, 1973, 11, 73. (Department of Biochemistry and Toxicology Section, Department of Clinical Investigation, U.S. Naval Medical Research Unit No. 2, Taipei, Taiwan, Republic of China and Publications Office,

Fractionation of toxins from Hydrophis cyanocinctus venom and determination of amino acid composition and end groups of Hydrophitoxin a : C.-S. Liu, G. S. Huber, C.-S. Lin and R. Q. Blackwell, Toxicon, 1973, 11, 73. (Department of Biochemistry and Toxicology Section, Department of Clinical Investigation, U.S. Naval Medical Research Unit No. 2, Taipei, Taiwan, Republic of China and Publications Office,

Fractionation of toxins from Hydrophis cyanocinctus venom and determination of amino acid composition and end groups of hydrophitoxin

Three toxic fractions were separated from crude venom of the sea snake Hydrophis cyanocinctus by column chromatography. The most abundant fraction, called Hydrophitoxin a, has been studied for amino acid composition and found to contain 19 of the common amino acids (including asparagine and glutamine); phenylalanine is absent. The total number of amino acid residues per molecule is estimated to be 59. Methionine occupies the N-terminal position and asparagine is at the C-terminal end. From these preliminary results Hydrophitoxin a appears to be similar in molecular size and structure to several other sea snake venoms previously reported.

Etching of crystalline poly(ethylene terephthalate) by hydrolysis

AbstractConditions have been found for the etching of poly(ethylene terephthalate) with water so that the crystalline portions alone remain. Initial sample and hydrolysis products are analyzed by extraction of low molecular weight products; density, viscosity molecular weight, and endgroup determination; heating‐rate‐dependent thermal analysis; low‐angle and wide‐angle x‐ray analysis; and electron microscopy. On hydrolysis of a 67% crystalline polymer at 180°C for about 300 min, almost fully crystalline extended‐chain oligomers can be obtained with about 65% yield. The morphology of melt‐crystallized poly(ethylene terephthalate) and the melting behavior of oligomer lamellae are discussed.

The hemoglobins of Myxine glutinosa L.—II. amino acid analyses, end group determinations and further investigations

1. 1. The amino acid composition of the three main hemoglobins, Hb I, Hb II and Hb III, of the hagfish ( Myxine glutinosa L.) has been determined. The composition differs so much from that of the lamprey ( Lampertra fluviatilis) hemoglobin that a diphyletic origin of the two species seems likely. 2. 2. The N-terminal residue of Hb III (hagfish) is proline as in also that of lamprey hemoglobin. The N-terminal group of Hb I and II seems to be blocked. 3. 3. Because the three main hagfish hemoglobins have different molecular weights the amino acid composition is not sufficient for a discussion of their phylogenetic interrelationship but consideration of the amino acid sequences would be necessary.