End-group Assay Research Articles

Bis(polypseudorotaxane)s Formed by Multiple Metallo-Bridged β-Cyclodextrins and the Thermodynamic Origin of Their Molecular Aggregation

Bis(polypseudorotaxane)s possessing the bridged bis(β-cyclodextrin)s with multicoordinated metal centers are prepared, in which β-cyclodextrin (β-CD) acts as the host structure, amino-terminated polypropylene glycol (PPG; MW = 2000) as the guest, and nickel(II) complexes as templating moieties. The assembly behavior is comprehensively studied by NMR spectroscopy, gel permeation chromatography (GPC), end group assay, static light scattering, thermogravimetric (TG), differential thermal analysis (DTA), and scanning tunneling microscopy (STM). The obtained results indicate that not only could the two PPG molecules be respectively threaded into metallo-bridged bis(β-CD)s forming the double-strand structure, but they also could play a stabilizing role in the molecular aggregation. The fluorescence titrations are performed in a mixture of acetonitrile/water to calculate the effective binding constants and thermodynamic parameters for the threading process of 2 onto PPG. The results reveal that the assembly proc...

The polycondensation of lithium 11-bromoundecanoate in aqueous media

To obtain a biodegradable plastic, polycondensation, the substitution of bromine by carboxylate was carried out on lithium 11–bromoundecanoate. Using water as a solvent, the formation of a double bond was detected between 70 and 100°C, yielding a good quantity of polymer. Water is recommended as the best solvent for the reaction. Thermodynamic investigations were carried out by using ethylene glycol and water. The reaction is believed to proceed according to the SN2 mechanism. The ΔH and ΔG values for condensation in ethylene glycol were slightly higher than for those in water, but the reaction rate in water at 70°C was 7 times greater than that in ethylene glycol. The enhancement of dissociation accounts for the advantageous condensation in aqueous media. Furthermore, ΔS shows negative values and ΔG shows positive values, meaning that the reaction should proceed exothermically and not spontaneously. Experiments to condense 11–bromoundecanoate in aqueous media were carried out for ten days in a water bath, kept at 70°C, using 25 mL of 0.05M 11–bromoundecanoate. The degree of polymerization for the resulting polymer was found to be 23 by the end group assay for both bromine and carboxylate. Neither a melting point nor glass transition point for the product were observed. The polymer formed decomposes at 300°C and instantly disperses. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 130–134, 2003

Hyaluronidase in ram semen. Quantitative determination, and isolation of multiple forms.

A study was made of hyaluronidase in ram semen. The end-group assay conditions used to determine activity quantitatively were chosen to ensure reliability as well as sensitivity [Gacesa, Savitsky, Dodgson & Olavesen (1981) Anal. Biochem. 118, 76-84]; they led to 1 W.H.O. Standard International Hyaluronidase Unit displaying 0.1263 EC munit (1 EC unit of activity releases 1 mumol equivalent of N-acetylglucosamine end groups/min at 37 degrees C). All the activity in the semen was shown to be sperm-derived, and intact spermatozoa were estimated to contain 1.23 EC units per 10(9) cells. In a low-ionic-strength medium, only some 20% of the hyaluronidase was extractable, although up to 80% of the activity could be extracted as the ionic strength was increased; further addition of detergent extracted the remainder. During purification of the enzyme, it was found that inclusion of poly(vinyl alcohol) in the media stabilized the activity; detergent inclusion also improved the yield, especially during early stages. As a consequence both of reliable quantitative determination and of stabilization, a number of forms of hyaluronidase could be isolated in high yield, by using anion-exchange chromatography, cation-exchange chromatography, affinity chromatography and gel filtration. The existence of all these forms was confirmed by electrophoresis and immunoblotting with the use of a monoclonal anti-(ram hyaluronidase) antibody, and their presence in very freshly prepared sperm extracts was demonstrated. The specific activity of the isolated major hyaluronidase form was 15.0 EC units/mg; this was equivalent to 119,000 W.H.O. units/mg, higher than any other previously reported values.

Synthetic analogues of polynucleotides—XIII: The resolution of dl-β-(thymin-1-yl)alanine and polymerisation of the β-(thymin-1-yl)alanines

dl-β-(Thymin-1-yl)alanine has been resolved into d(+) and l(−) forms. The pure d(+) form was obtained by fractional crystallisation of the (+)α-methylphenylethylamine salts of the α-N-formyl derivatives. The pure l(−) isomer was obtained on a small scale by chromatography of the same salts. The optically active amino acids and the dl-mixture were polymerised by the mixed anhydride procedure to give polymers which showed no evidence of base stacking or of interaction with polyadenylic acid. The molecular weights of the polymers were in the range 2–4 × 10 3. These were determined by end group assay which involved the synthesis of α- N-(2,4-dinitrophenyl)- dl-β-(thymin-1-yl)alanine as a standard.

Polysaccharide synthesis from mono- and oligo-saccharides by the action of phosphorus pentoxide in dimethyl sulphoxide

Phosphorus pentoxide-dimethyl sulphoxide (P 4O 10-DMSO) was found predominantly to catalyse the polymerization reaction of carbohydrates at below 35°C and the oxidation reaction at 60–65°C. A series of new synthetic polysaccharides were prepared from mono- and oligo-saccharides including 2-acetamido-2-deoxy- d-glucose and hexuronic acids in up to 48% yield by the action of P 4O 10-DMSO at 10–25°C. These synthetic polysaccharides showed s 20,w 0.68–1.34S, and the degree of polymerization fell in 15.3–4.7 monosaccharide units per polysaccharide chain on the basis of reducing end-group assay. A structural analysis by the methylation of the synthetic glucan (2) revealed α-1,4- and α-1,6-glucosidic linkages as main chains with various branchings. The synthetic polysaccharides contained 1.3–15.9% phosphorus, but the linkages are unknown.

Galactosamine polymer isolated from the cell wall of Neisseria sicca.

A D-galactosamine polymer having a molar ratio of galactosamine–acetyl–phosphorus (1.0:1.0:0.35) has been isolated from the cell wall of Neisseria sicca. Its homogeneity was established by free-boundary electrophoresis, ultracentrifugal sedimentation, and gel permeation chromatography. Molecular weight estimation by reducing end group assay gave a value of [Formula: see text] 18 500. Methylation analysis indicated that the main glycosidic linkages were (1 → 4) and (1 → 6) with a minimum of 10% branch points. The glycosidic bonds were indicated as being in the α configuration by the high positive optical rotation of the polysaccharide and also from infrared spectrum evidence. Both the acetyl and phosphate contents were relatively unchanged by the Hakamori methylation procedure. Although galactosamine polymers have been found in a number of fungi, the present instance appears to be the first report of their presence in bacteria.

PHYSICO-CHEMICAL PROPERTIES OF GLYCOGEN FROM THE RAT LIVER AND MUSCLE

As reported previously (6, 7, 8), the glycogen particles from the rat liver and muscle appeared as the specific forms of spheroidal branching bodies. The branching structures were more clearly demonstrated in the glycogen particles from the fasted animal and these appearances were different from the aggregated structures. The chemical and physico-chemical properties of glycogens from the rat liver and muscle are demonstrated and discussed in this paper.1. End group assay of the glycogens and β-amylase digestion degree suggested that the average chain length of the liver glycogen was longer than the muscle glycogen.2. The viscosity of glycogen, measured by varing the temperature from 10°C to 70°C, showed the non-aggregated structure of the glycogen. particles.3. The optical rotartory dispersion and circular dichroism spectra of the glycogen were measured using an addition of iodine solution, and only the muscle glycogen showed the Cotton effect.4. The sedimentation coefficient of the liver glycogen showed the larger s value than the muscle one, and the relationship between s value and concentration of the glycogen was discussed.

Chromosomal RNA of calf thymus chromatin

Calf thymus chromatin, the most widely studied deoxyribonucleoprotein, is shown to contain an associated chromosomal RNA as do the chromatins of other species. The chromosomal RNA of calf thymus chromatin is present in an amount corresponding to approx. 1% of that of DNA. The purified material eluted from DEAE-Sephadex column at a NaCl concentration of 0.56 M as do the chromosomal RNA's of other organisms. Its average chain length by end-group assay is approx. 40 nucleotides and it contains approx. 3–4 dihydrouridylic acid residues per chain. Calf thymus chromosomal RNA, like the chromosomal RNA's of other organisms, is associated with chromosomal protein in a form not dissociable by high salt concentration.

Linear polypeptides of a known repeating sequence of amino-acids. Synthesis of poly-(L-glutamyl-L-glutamyl-L-alanylglycyl)[1-14C]glycine ethyl ester

Selective removal of the o-nitrophenylsulphenyl protecting group from N-o-nitrophenylsulphenyl-γ-t-butyl-L-glutamyl-γ-t-butyl-L-glutamyl-L-alanylglycine pentachlorophenyl ester gave γ-t-butyl-L-glutamyl-γ-t-butyl-L-glutamyl-L-alanylglycine pentachlorophenyl ester hydrochloride. Polymerisation of this material on the partially blocked monomer [1-14C]glycine ethyl ester hydrochloride, at a relatively high dilution, gave poly-(γ-t-butyl-L-glutamyl-γ-t-butyl-L-glutamyl-L-alanylglycyl)[1-14C] glycine ethyl ester. The t-butyl ester protective groups were removed to give the title compound. The majority of the radioactivity was incorporated into the polypeptide. Comparison of the number average molecular weights obtained by end group assay and calculated from osmometry, 2·6 × 105 and 2·3 × 105 respectively, indicates that the polypeptide is linear.

The protein-polysaccharide complex of bovine nasal cartilage. Studies on the protein core.

1. Chondromucoprotein from bovine nasal cartilage was purified by cetylpyridinium chloride or by bismuth nitrate in acetone. 2. Amino acid compositions of crude and purified preparations were compared and few differences were found, in spite of the decrease in protein content on purification. 3. Amino acid analysis of bismuth-purified material revealed the existence of four groups of amino acids. Within each group, the amino acids were present in approximately equimolar concentrations. 4. Amino end-group assay on the same material showed six alpha-DNP derivatives. 5. A molecular weight of 6.3x10(5) for the protein-polysaccharide complex was calculated from the latter analysis.

真菌多糖類の研究 (第4報)

Mannan of Candida albicans was isolated by modified Vogel's method and purified by preparation of its copper complex. This material showed [α]D+53°, molecular weight of 4500 (Akiya-Barger method and from reducing end-group assay). Its infrared spectrum showed no β-configuration of mannopyranose.Periodate oxidation products of this mannan were identified with glyoxal (as p-nitrophenylosazone, m.p. 310°, and by infrared spectrum) and glyceraldehyde (as p-nitrophenylhydrazone, paper chromatography and electrophoresis). From these results, it was concluded that the mannan has been 1-2, 1-6, or 1-2-6 α-linkages, but not 1-3 or 1-4 linkages.

Studies on Fungal Polysaccharides. II. On the Componental Sugars and Partial Hydrolysis of the Capsular Polysaccharide from Cryptococcus neoformans.

Capsular polysaccharide of the Cryptococcus neoformans was found to be a xyloglucu-ronomannan, which consisted of D-glucuronic acid, D-xylose, and D-mannose (in 1 : 1 : 3 ratio), and its molecular weight was about 6600 by the Akiya-Barger's method and from results of end-group assay. D-Xylose and a part of D-glucuronic acid in this polysaccharide were liberated more easily than D-mannose.

525. α-1,4-Glucosans. Part XII. End-group assay of glycogens by oxidation with sodium metaperiodate

The first page of this article is displayed as the abstract.

Reduction of the products of periodate oxidation of carbohydrates. VII. The constitution of glycogen

Periodate oxidation of glycogen yields glycogen polyaldehyde which, upon reduction either with hydrogen and a palladium catalyst or with sodium borohydride, gives glycogen polyalcohol. Hydrolysis of the polyalcohol, obtained from glycogen samples of different origin, affords glycerol, erythritol, glycolic aldehyde, and glucose. Determination of the molar ratio of glycerol to erythritol which was approximately 1:10 provides a new route for the end-group assay of glycogen. The result by this new technique is in good agreement with those from periodate and methylation studies. The amount of glucose stable to periodate oxidation in various specimens of glycogen polyaldehyde and the corresponding polyalcohols was 0.6-0.7% of the parent glycogen. It is suggested that this glucose arises from d-glucose residues in the glycogen molecule that are linked so that they are immune to periodate cleavage. This observation appears to require a modification of the hitherto accepted structure of glycogen.

On the molecular weight determination of poly(ethylene terephthalate)

AbstractNumber average molecular weights were determined on a series of unfractionated poly(ethylene terephthalate) samples by carboxyl and hydroxyl end group assay methods.The relationship between intrinsic viscosity in phenol‐tetrachloroethane (50 : 50) solution and molecular weight was found to be

423. α-1 : 4-Glucosans. Part V. End-group assay of glycogens by periodate oxidation, and the oxidation of maltose by sodium metaperiodate

423. α-1 : 4-Glucosans. Part V. End-group assay of glycogens by periodate oxidation, and the oxidation of maltose by sodium metaperiodate

Reduction of the Products of Periodate Oxidation of Carbohydrates. II. A New Method for the End-group Assay of Amylopectin1

Reduction of the Products of Periodate Oxidation of Carbohydrates. II. A New Method for the End-group Assay of Amylopectin¹

Fragmentation of the myosin molecule

1. 1. Evidence is given for the existence of actin and tropomomysin impurities in “cyrstalline” myosin. 2. 2. The existence of subunits in myosin has been demonstrated by studying the depolymerisation of purified myosin in concentrated urea and guanidine hydrochloride solutions, dilute acid and alkali. Collateral data from end-group assay (Bailey 15) show that in all cases no peptide bonds are split except in alkali at pH 13. 3. 3. The fractionation of subunits of myosin has been effected in a medium of urea and ethanol or with ammonium sulphate after complete depolymerisation in urea; and the size and shape of the fragments studied with osmotic pressure, viscosity and fluorescence-polarisation techniques. 4. 4. Purified myosin appears to contain two types of subunits: about 92% of highly asymmetric units of average particle weight 165,000 which may be cyclic, and about 8% of units of average weight 16,000 and axial ratio ∼ 12 5. 5. The small fragments could only be detached from the myosin macromolecule by treatment with urea, but not by alkali or acid, while maximum depolymerisation of the myson “skeleton” seems to occur at pH 10.7. 6. 6. The depolymerisation of myosin with 5.7 M urea of 5.7 M guanidine hydrochloride is a very slow process. At 20°C, pH 6.5, apparent equilibrium was not reached until after several months treatment in urea. Guanidine hydrochloride appears to be less effective. 7. 7. There is no evidence as yet that tropomyosin forms a part of the myosin molecule, nor has it been possible to separate the adenosine triphosphatase activity.

End-group assay in some proteins of the keratin-myosin group.

End-group assay in some proteins of the keratin-myosin group.

Action of thrombin in the clotting of fibrinogen.

IN spite of much literature on the clotting of blood, the exact mechanism of the final event, the mode of transformation of soluble fibrinogen to insoluble fibrin by the action of thrombin, remains unknown. The concept of thrombin as a hydrolytic or proteolytic enzyme1,2 has fallen into disfavour3, and in recent years other plausible but unproved mechanisms have been proposed4–6. In a study7 of the proteins of the keratin–myosin–fibrinogen group by end-group assay with fluorodinitrobenzene8, only small amounts of terminal amino-groups could be detected in fibrinogen, although the amounts were not sufficiently small to conclude, as in the case of myosin and tropomyosin, that the molecule is constructed of cyclo-peptide units. The action of purified thrombin, however, results consistently in the appearance of amino-terminal residues of glycine, and whatever the ensuing mechanism, we believe that the fundamental enzymic step in the transformation of fibrinogen to fibrin is associated first with the appearance of these groups. This action of thrombin, which tentatively may be considered as a very specific proteolysis, was discovered independently in Cambridge and in Leeds, and for this reason the relevant observations are reported jointly.