Stable thiobarbituric acid chromophore with dimethyl sulphoxide. Application to sialic acid assay in analytical de-<i>O</i>-acetylation

Skoza, L; Mohos, Steven C.

doi:10.1042/bj1590457

Cited by 268 publications

(116 citation statements)

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“…of 1 M Tris-HCI, pH 8·3. Neuraminidase was assayed with N-acetylneuraminyl-o-Iactose as substrate as described by Dickson and Messer (1978) except that dimethylsulfoxide was used instead of acid butanol in the thiobarbituric acid determination of sialic acid (Skoza and Mohos 1976).…”

Section: Methodsmentioning

confidence: 99%

Intestinal Glycosidase Activities in one Adult and two Suckling Echidnas: Absence of a Neutral Lactase (b-D-Galactosidase).

Stewart¹,

Messer²,

Walcott³

et al. 1983

Aust. Jnl. Of Bio. Sci.

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The activities of various glycosidases in homogenates of the small-intestinal mucosa of one adult and two suckling echidnas, Tachyglossus aculeatus, were investigated. The activities of lactase (!i-D-galactosidase), !i-N-acetylglucosaminidase, neuraminidase and ()(-L-fucosidase were higher in the sucklings than in the adult animal. Maltase and isomaltase activities were lower. Sucrase and cellobiase activities were absent or present in trace amounts only. The lactase activity had a pH optimum of 4· 0--4· 5, was predominantly in the soluble fraction following ultracentrifugation and was inhibited by p-chloromercuribenzene sulfonate, suggesting that it was due to a lysosomal acid !i-galactosidase and not a brush-border neutral lactase. The maltase activity of the sucklings also had the characteristics predominantly of a lysomal acid hydrolase. It is proposed that in suckling echidnas, the oligosaccharides (mainly neuraminyllactose and fucosyllactose) of the mother's milk are digested intracellularly by lysosomal enzymes, rather than at the brush border, of the epithelial cells of the small-intestinal mucosa.

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Section: Methodsmentioning

confidence: 99%

Intestinal Glycosidase Activities in one Adult and two Suckling Echidnas: Absence of a Neutral Lactase (b-D-Galactosidase).

Stewart¹,

Messer²,

Walcott³

et al. 1983

Aust. Jnl. Of Bio. Sci.

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“…The thiobarbituric acid method of Aminoff as modified by Skoza and Mohos [9] is also applicable for detection and quantification of 1. Considering relative sensitivities (zl/ .zNeuSAc) the method of Svennerholm [7] is the method of choice for characterizing the elution profile during isolation.…”

Section: Colorimetric Analysismentioning

confidence: 99%

4,8-Anhydro-N-acetylneuraminic acid. Isolation from edible bird's nest and structure determination

1987

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A new, sialic-acid-derived compound was isolated from the acid hydrolysate of edible bird's nest by ionexchange chromatography. Combined use of mass spectroscopy and 'H-and 13C-NMR spectroscopy established that it is the 4,g-anhydro derivative of N-acetylneuraminic acid and that in solutions it exists in two tautomeric forms.The formation of the new compound supports and earlier finding that in the glycoprotein of edible bird's nest at least a portion of N-acetylneuraminic acid is acetylated at HO-4.Recently we needed gram quantities of N-acetyl-D-neuraminic acid (Neu5Ac) for synthetic work aimed at reconstructing Neu5Ac-containing oligosaccharides of the capsular polysaccharides of group B streptococci [l]. Neu5AcCollocalia niucoid, the nest-cementing glycopratein substance of Chinese swift, which can be obtained [2] by aqueous extraction of edible bird's nest, a Chinese delicacy, is reported [2] to contain about 9% of Neu5Ac and is, therefore, a practical source of Neu5Ac. The isolation of Neu5Ac from this glycoprotein has been the subject of several publications, using hot water extraction [3, 41, acid hydrolysis [5] and combined enzymatic and acid hydrolysis [6]. Although in our hands hot water extraction of edible bird's nest gave no practical amounts of NeuSAc, mild acid hydrolysis, followed by ionexchange chromatographic isolation, as described [5], gave Neu5Ac in yields comparable to those published [3 -51. Also obtained was a second, previously uncharacterized compound (I). In the present paper we describe the isolation and structural investigation of this new compound. MATERIALS AND METHODSEdible bird's nest was obtained from a local Chinese grocery. N-Acetyh-neuraminic acid was obtained from Pfanstiehl Laboratories, Inc. (Waukegan, IL) and was used without purification. All other chemicals were analytical grade commercial reagents. Analytical methodsThin-layer chromatography was carried out on precoated, 0.25-mm silica gel plates (Kieselgel 60 F254 of E. Merck, Darmstadt, FRG; cat. no. 5765) with propanol/water ( 3 : 1, by vol.) (solvent A) and on pre-coated, 0.1-mm cellulose plates (pre-coated TLC Sheets, cellulose of E. Merck, Darmstadt, FRG; cat. no. 5537) with propanol/butanol/O.l M HCI (2:1:1, by vol.) [6] (solvent B). Detection was made by spraying with resorcinol/Cu2 + reagent [7] followed by heating at 110°C.Colorimetric tests, using resorcinol/Cu2 +, periodic acid/ resorcinol/Cu2 + and periodic acidlthiobarbituric acid were carried out as published [7 -91 using an LKB Ultrospec 4050 spectrophotometer.Optical rotation was measured with a Perkin-Elmer 243 automatic polarimeter.Combined gas-liquid chromatography/mass spectrometry of derivatives of lwas performed on a Hewlett-Packard 5985B instrument operated in the electron impact mode at an ionization potential of 70 eV, ion-source temperature 200 "C, using a 3% OV-17-coated fused-silica capillary column (25 m x 0.3 mm) at a temperature program at 200 -260 "C at 2 "C/ min .The 'H-and 13C-NMR spectra were recorded with a Bruker AM-500 spectromet...

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“…Amino acids were determined on the amino acid analyzer after hydrolysis of duplicate samples in constant-boiling HCI at 110°C for 24 h. Glycoproteins, glycopeptides or released oligosaccharides were analyzed further as follows : (a) hydrolysis in 2 M HCI at 100°C for 2.5 h and analysis of the neutral sugars using an automated borate ion-exchange chromatography system [13] with fluorescence detection [14]; (b) hydrolysis in 2 M HCI at 110°C for 4 h or 14 h (some samples after prior reduction with NaBH4 [15]) and determination of glucosamine and glucosaminitol with the amino acid analyzer [16]; and (c) hydrolysis in 0.05 M H2S04 at 80°C for 1 h and determination of neuraminic acid [17]. All hydrolyses were performed in evacuated, N2-flushed tubes.…”

Section: Compositional Analysesmentioning

confidence: 99%

Detection and quantification of peptide‐N⁴‐(N‐acetyl‐β‐glucosaminyl)asparagine amidases

Plummer

Phelan

Tarentino

1987

European Journal of Biochemistry

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A detailed study of the oligosaccharide specificity of the almond enzyme, peptide-N4-(N-acetyl-Pglucosaminy1)asparagine amidase A, was undertaken by comparing the rate of release of intact oligosaccharide chains from defined glycopeptides of all significant classes. The oligosaccharide of a trisialo-triantennary pentaglycopeptide from fetuin was released at the highest rate. A procedure was developed for the isolation of this glycopeptide in high yield from 5 g fetuin. Sequence analysis established the structure as Leu-Ala-Asn(CH0)-Cys-Ser. The Cys(Cm) and the Cys(Ae) derivatives of the glycopeptide were reacted with 4-(dimethylamino)-azobenzene-4-sulfonyl (dabsyl) chloride to yield a monosubstituted and a disubstituted glycopeptide respectively. This chromophore confers high sensitivity at 436 nm on a pentapeptide backbone having minimal bonds for protease cleavage. A procedure was developed wherein these dabsyl derivatives were used in a high-performance liquid chromatography assay. The dabsyl-pentapeptide was retarded significantly from the dabsyl-glycopeptide and provided a sensitive method (1 -2 nmol) of detection of peptide-N4-(N-acetylg1ucosaminyl)asparagine amidase activity. Enzyme was detected in crude extracts of all eight seed sources surveyed. The enzyme from Pisum sativum was partially purified and its properties were compared with the correponding enzyme from almonds.Takahashi and Nishibe [l, 21 first demonstrated the presence of a new amidase in almond emulsin that released intact oligosaccharides from defined glycopeptides of bromelain and ovalbumin. Unlike previously described endoglycosidases, such as endo H' [3], which hydrolyzed the di-N-acetylchitobiosyl core of N-linked glycans, this new type of enzyme hydrolyzed the p-aspartylglycosylamine linkage of asparagine-oligosaccharides in peptide linkage. Plummer and Tarentino [4] reported that this enzyme, peptide-N4-(N-acetyl-P-glucosaminyl)asparagine amidase (PNGase A), was much broader in specificity than all previously described endoglycosidases ; not only were high-mannose and hybrid-type oligosaccharide chains released, but also complex di-, tri-and tetraantennary chains [5, 61.Oligosaccharide-chain-cleaving enzymes with broad substrate specificity provide an attractive alternative to hydrazinolysis for isolating N-linked glycans from proteins, since yields are typically 90 -95% and there is no modification Abbreviations. endo, endo-P-N-acetylglucosaminidase; PNGase, peptide-N4-(N-acetyl-~-glucosaminyl)asparagine amidase (formerly known by the trivial name, peptide: N-glycosidase); dabsyl, 4-dimethylaminoazobenzene-4-sulfonyl; dns, 5-dimethylaminonaphthalene-1-sulfonyl; Ae, aminoethyl; Cm, carboxymethyl; SDS, sodium dodecylsulfate; CHO, intact oligosaccharide moiety.Enzymes. of the reducing end. Such enzymes continue to have great potential for investigating glycoprotein structure and function, and there is a need for specificities directed at other linkages on ohgosaccharide chains. Because of this we have developed a new method of detecti...

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Stable thiobarbituric acid chromophore with dimethyl sulphoxide. Application to sialic acid assay in analytical de-O-acetylation

Cited by 268 publications

References 16 publications

Intestinal Glycosidase Activities in one Adult and two Suckling Echidnas: Absence of a Neutral Lactase (b-D-Galactosidase).

Intestinal Glycosidase Activities in one Adult and two Suckling Echidnas: Absence of a Neutral Lactase (b-D-Galactosidase).

4,8-Anhydro-N-acetylneuraminic acid. Isolation from edible bird's nest and structure determination

Detection and quantification of peptide‐N⁴‐(N‐acetyl‐β‐glucosaminyl)asparagine amidases

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Stable thiobarbituric acid chromophore with dimethyl sulphoxide. Application to sialic acid assay in analytical de-O-acetylation

Cited by 268 publications

References 16 publications

Intestinal Glycosidase Activities in one Adult and two Suckling Echidnas: Absence of a Neutral Lactase (b-D-Galactosidase).

Intestinal Glycosidase Activities in one Adult and two Suckling Echidnas: Absence of a Neutral Lactase (b-D-Galactosidase).

4,8-Anhydro-N-acetylneuraminic acid. Isolation from edible bird's nest and structure determination

Detection and quantification of peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidases

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Detection and quantification of peptide‐N⁴‐(N‐acetyl‐β‐glucosaminyl)asparagine amidases