Preparative Paper Chromatography Research Articles

Structural analysis of oligosaccharides isolated from the urine of a blood group A, secretor, woman during pregnancy and lactation.

Twenty different oligosaccharides have been isolated from urine collected from an A, Le(a- b+), secretor woman during her pregnancy and subsequent lactation. Nine of these have been found previously in the milk of Le (a- b+) individuals. Three new fucose-containing oligosaccharides denoted lacto-N-neotrifucoheptaose II (alpha-L-Fuc-(1 leads to 2)-beta-D-Gal-(1 leads to 4)-[alpha-L-Fuc-(1 leads to 3)]-beta-D-GlcNAc-(1 leads to 3)-[alpha-L-Fuc-(1 leads to 2)]-beta-D-Gal-(1 leads to 4)-D-Glc), lacto-N-neodfucohexaose I (alpha-L-Fuc-(1 leads to 2)-beta-D-Gal-(1 leads to 4)-[alpha-L-Fuc-(1 leads to 3)]beta-D-GlcNAc-(1 leads to 3)-beta-D-Gal-(1 leads to 4)-D-Glc), and lacto-N-difucohexaose IV (alpha-L-Fuc-(1 leads to 2)-beta-D-Gal-(1 leads to 3)-beta-D-GlcNAc-(1 leads to 3)-[alpha-L-Fuc-(1 leads to 2)]-beta-D-Gal-(1 leads to 4)-D-Glc) are described. Three new myo-inositol-containing oligosaccharides which are characteristic for pregnancy have also been isolated. Their structures are: alpha-D-GalNAc-(1 leads to 3)-[alpha-L-Fuc-(1 leads to 2)]-beta-D-Gal-(1 leads to 0)-[alpha-L-Fuc-(1 leads to 0)]-myo-inositol; alpha-L-Fuc-(1 leads to 2)-beta-D-Gal-(1 leads to 0)-[alpha-D-Fuc-(1 leads to 0)]myo-inositol; and alpha-L-Fuc-(1 leads to 2)-beta-D-Gal-(1 leads to 0)-myo-inositol, respectively. Three oligosaccharides containing only glucose were found and partially characterized. In addition, two oligosaccharides described previously (a glucose tetrasaccharide (Hallgren, P., Hansson, G., Henriksson, K.-G., Häger, A., Lundblad, A., and Svensson, S. (1974) Eur. j. clin. Invest. 4, 429-433) and a urine A pentasaccharide (Lundblad, A., and Svensson, S. (1973) Carbohyd. Res. 30, 187-189) were also isolated and characterized. The isolation procedure included ultrafiltration, gel chromatography on Sephadex G-25, and preparative paper chromatography, Structural determination involved sugar and methylation analyses. Tri- and tetrasaccharides were investigated by combined gas-liquid chromatography-mass spectrometry. Sequence analyses of larger oligosaccharides were performed by combined gas-liquid chromatography-mass spectrometry of di- and trisaccharides obtained after partial acid hydrolysis of the parent compound. Anomeric configurations were deduced from optical rotations and by comparison with authentic samples.

Structural analysis of nine oligosaccharides isolated from the urine of a blood group O, nonsecretor, woman during pregnancy and lactation.

Nine oligosaccharides have been isolated from urine collected from an O(H), Le(a+ b-), nonsecretor, woman during her pregnancy and subsequent lactation. One of them is a new hexasaccharide denoted lacto-N-neo-difucohexaose II: beta-D-Gal-(1 leads to 4)-[alpha-L-Fuc-(1 leads to 3)]-beta-D-GlcNAc-(1 leads to 3)-beta-D-Gal-(1 leads to 4)-[alpha-L-Fuc-(1 leads to 3)]-D-Glc. Seven of the remainder were identified as the milk oligosaccharides lacto-N-difucohexaose II, lacto-N-fucopentaose II and III, lacto-N-tetraose, 6'-galactosyllactose, 3-fucosyllactose, and lactose. The ninth component is a glucose-containing tetrasaccharide, alpha-D-Glc-(1 leads to 6)-alpha-D-Glc-(1 leads to 4)-alpha-D-Glc-(1 leads to 4)-D-Glc, previously found in normal human urine. The oligosaccharides were isolated by gel chromatography, preparative zone electrophoresis, and paper chromatography. The structures were determined by sugar and methylation analyses. Sequence analysis of tri- and tetra-saccharides was carried out by gas-liquid chromatography-mass spectrometry of reduced and permethylated oligosaccharide derivatives. The monosaccharide sequence in penta- and hexasaccharides was deduced from gas chromatographic-mass spectrometric studies of di- and trisaccharides obtained from partial acid hydrolysis of the parent compound. Anomeric configurations were deduced from the optical rotation.

Identification of .DELTA.-valerobetaine and other betaines in the ovary of a shellfish Callista brevishiphonata.

Quaternary amines in the ovary of the shellfish Callista brevishiphonata were isolated by treatment on columns of ion-exchange resins and then by preparative paper chromatography. The presence of at least ten quaternary amines was recognized. Eight of these were identified by comparison with synthetic compounds by mass spectral analysis and by paper chromatography: Choline, glycine betaine, γ-butyrobetaine, δ-valerobetaine, stachydrine, valine betaine, homarine, and homoserine betaine.

Determination of hydroxylysine in urine.

A new method for colorimetric determination of urinary hydroxylysine is described. Approximately one-hundredth of human urine collected for 24 hr was diluted to 25 ml and titrated to pH 2.0 with 2 hcl, and then subjected to column chromatography on Dowex 50 X 4 (H+ form). Amino acids were eluted from the column with 1.5 N NH4OH. Hydroxylysine in the elute with 1.5 N NH4OH was separated from the other amino acids, especially from serine and threonine, by preparative paper chromatography. The paper corresponding to hydroxylysine was cut and eluted with water. An aliquot of the eluate with water was oxidized by sodium metaperiodate, and formaldehyde liberated from hydroxylysine was assayed by chromotropic acid reagents, after removing periodate and iodate with Dowex 1 X 8 (formate form). Excretion rate of hydroxylysine in urine of adults was shown to be approximately 110 mumoles per day.

Determination of free monosaccharides and detection of sugar alcohols in mature soybean seeds.

Although the oligosaccharide contents of soybeans are well documented, the exact values of monosaccharide contents have not been reported. Elaborate methods of preparative paper chromatography together with gas chromatography established the following data for one variety, Kyushu No. 12. The air-dried cotyledon part (admixed with hypocotyls) contained 0.030% glucose and 0.039% fructose. The hull part contained 0.018% galactose, 0.028% glucose, 0.023% fructose, 0.005% arabinose, and 0.002% xylose. Gas chromatograms of trimethylsilated monosaccharide fractions revealed the existence of minute amounts of sorbitol, arabitol, xylitol, and mannitol in decreasing order (about 0.03% to 0.001% of whole seeds).

Zinkporph yrin-Pigmente im Botryoidgewehe von Haemopis sanguisuga L. und ihre Lokalisation mit der Diaminobenzidin-H 22-O Reaktion

From isolated botrydiosomes of Haemopis sanguisuga L. a pigment was extracted, separated by preparative paper chromatography and analysed chemically. Chromatographically the pigment was found to be composed of two fractions, which has been identified as zincporphyrin compounds, which are able to oxidize of diaminobenzidine in the presence of H2O2. As proved by ultrahistochemical method, the product of hte diaminobenzidine-H2O2 reaction is associated in the botryoid cells with the tubules foth of the endoplasmic reticulum and of botrydiosomes. The proboble functions of the zincporphyrins in the oxidizing processes are discussed.

A simple and efficient method for the preparation of GDP-fucose

A crude extract from hog submaxillary gland was found to synthesize guanosine diphosphate (GDP)-fucose, when incubated with fucose, ATP and GTP, the two enzymatic steps (fucose ▪fucose-1-phosphate ▪GDP-fucose) proceeding without noticeable side reactions. Column chromatography on Dowex 1 (HCO 3 −), gel filtration on Sephadex G-15 and preparative paper chromatography gave pure GDP-fucose in an overall yield of 81%. The sugar nucleotide was shown to be active as a glycosyl donor in fucose-incorporating systems.

Structures of the oligosaccharides from the enzymic hydrolysis of hemicellulose by a hemicellulase of ceratocystis paradoxa

An endo-hemicellulase (HC-II) of Ceratocystis paradoxa degraded spear-grass hemicellulose B to a series of mixed oligosaccharides. Four neutral oligosaccharides (AraXyl 2, AraXyl 3, Xyl 2,and Xyl 3,),isolated by preparative paper chromatography, were shown by enzymic and methylation techniques to constitute a series of β(1 →4)-linked D-xylopyranosyl oligomers. The oligosaccharides AraXyl 2 and AraXyl 3 were identified as O-α- L-arabinofuranosyl-(1→3)- O-β- D-xylopyranosyl-(1→4)- O-β- D-xylopyranosyl-(1→4)- D-xylose and O-α- L-arabinofuranosyl-(1→3)-O-β- D-xylopyranosyl-(1→4)- D-xylose,respectively, the latter being a new compound.

Characterization of the peptide-bound flavin of a bacterial sarcosine dehydrogenase

As in the case of the succinate and sarcosine dehydrogenases of liver mitochondria, the flavin prosthetic group of the bacterial sarcosine dehydrogenase can be released from the enzyme by proteolytic digestion with trypsin and chymotrypsin. The flavin, isolated in the dinucleotide form and covalently bound to a peptide fragment, is converted to the mononucleotide and purified by sequential chromatography on Sephadex G-25, DEAE-Sephadex A-25, followed by preparative paper chromatography and high voltage electrophoresis. The absorption maxima of the purified flavin at pH 7 are found at 268, 350, and 447 nm, with 268:447 nm and 350:447 nm ratios of 3.08 and 0.79, respectively. The fluorescence excitation and emission maxima, 450 and 530 nm, respectively, are similar to those of flavin mononucleotide. The fluorescence of the flavin-peptide is maximal at pH 3.0–3.1. Amino acid analysis of the flavin-peptide (riboflavin form) gave the following molar ratios of amino acids to flavin: Lys(1), Asp(2), Thr(1), Ser(1), Glu(1), Gly(1), and Ala(1). Aspartic acid was the N-terminal amino acid. Upon more extensive hydrolysis, histidine was obtained in 71–84% yields. Employing aminopeptidase M, the partial sequence of amino acids in the flavin-peptide was determined to be as follows: Flavin ▪ -Asp-Lys-Ser-Glu-Gly-His-(Asp,Ala,Thr)-Evidence is presented that the isoalloxazine ring is linked covalently via its 8 α-methyl group to N-3 of histidine.

The nature of the human blood group P 1 determinant

A glycoprotein with blood group P1 specificity isolated from sheep hydatid cyst fluid was subjected to partial acid hydrolysis. The hydrolysis products were separated by preparative paper chromatography. One oligosaccharide with strong P1 serological activity was characterised as a trisaccharide composed of galactose and glucosamine in the molar ratio of 2:1. Reduction with sodium borohydride gave galactose and glucosaminitol. Methylation analysis and degradation with specific α- and β- galactosidases showed the structure of the P1 determinant to be D-galactosyl-α(l→4)-D-galactosyl-β(l→4)-N-acetyl-D-glucosamine.

Free amino acids and γ-glutamyl peptides in seeds of Fagus silvatica

The contents of amino acids and peptides have been investigated in seeds of Fagus silvatica L. (beechnuts). In addition to the common amino acids, the following compounds have been isolated and identified: 4-hydroxyproline (probably the cis- l-isomer), N 5-acetylornithine, 3-(2-furoyl)- l-alanine, methionine sulfoxide (probably an artefact), pipecolic acid (probably partially racemized d-isomer), l-willardiine (with a small amount of the d-isomer), N-(3-amino-3-carboxypropyl)azetidine-2-carboxylic acid, N-[ N-(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl]azetidine-2-carboxylic acid, 2( S),5( S),6( S)-5-hydroxy-6-methylpipecolic acid, 2( S),5( R),6( S)-5-hydroxy-6-methylpipecolic acid, γ-glutamylalanine, γ-glutamylglutamic acid, γ-glutamylisoleucine, γ-glutamylleucine, γ-glutamylmethionine sulfoxide (probably an artefact), γ-glutamylphenylalanine, γ-glutamyltyrosine, γ-glutamylvaline, glutathione, γ-glutamylwillardiine, and γ-glutamylphenylalanylwillardiine. γ-Glutamylphenylalanine and willardiine are the dominating components of the amino acid fraction. The isolations were performed by use of ion exchange chromatography, taking advantage of the different pK-values of the amino acids, mainly on acid resins in the 3-chloropyridinium form with aq. 3-chloropyridine as eluant and on basic resins in the acetate form with aqueous acetic acid as eluant. These methods in combination with preparative paper chromatography have permitted the isolation and identification of compounds present in amounts as low as 1/6000 of the dominant ninhydrin-reactive component. The implications of the occurrence of this large variety of compounds in the Fagaceae are briefly discussed.

Effect of ethyl alcohol on the biosynthesis of glucosamine and galactosamine in the human gastric mucosal tissue in vitro

The effect of ethanol on the biosynthesis of glucosamine and galactosamine by human gastric mucosa in vitro was studied. The slices of human gastric mucosa were incubated in medium containing glucose- 14C with or without ethanol. Hexosamines were isolated by using ion-exchange column chromatography and then they were purified and separated by means of preparative paper chromatography. It was shown that human gastric mucosa is able to synthesize both glucosamine and galactosamine from glucose in vitro. In the presence of ethanol the biosynthesis of galactosamine is distinctly decreased.

Structure of the antitumor protein neocarzinostatin: Purification, amino acid composition, disulfide reduction, and isolation and composition of tryptic peptides

The antitumor protein neocarzinostatin has been purified by repeated CM-cellulose chromatography and Sephadex G-50 gel filtration. The protein possesses 109 amino acid residues in a single chain, crosslinked by two disulfide bonds. Reduction and S-alkylation could not be accomplished in aqueous solution and required the use of dithiothreitol in liquid ammonia, followed by treatment with alkyl chloride. Tryptic digestion of (tetra- S-carboxymethyl)neocarzinostatin afforded five tryptic fragments which were fractionated by preparative paper chromatography and high-voltage paper electrophoresis, purified by Sephadex gel filtration and characterized by amino acid and amino end-group analysis. The total number of amino acid residues of these fragments account for the 109 residues of neocarzinostatin.

Cofactor Investigation of Bovine Plasma Amine Oxidase: NaBH4 REDUCTION OF ENZYME-SUBSTRATE MIXTURE

Abstract It has been reported that treatment of amine-diamine oxidase mixtures with sodium borohydride resulted in the formation of a stable substrate-pyridoxal phosphate complex. When this procedure was applied to the bovine plasma amine oxidase in the presence of 14C-labeled substrate, the enzyme was inactivated and became radioactive. A pure radioactive product was isolated from acid hydrolyzates by ion exchange chromatography and preparative paper chromatography of the hydrolyzate. The compound was shown to be e-N-benzyllysine by mass spectrometry. This adduct was apparently a reduced intermediate in the catalytic pathway of substrate oxidation by the enzyme. Formation of a substrate-pyridoxal phosphate complex could not be confirmed.

Enzymic degradation products of omega-heparin (whale heparin).

ω-Heparin isolated from whale organs was digested with an eliminase [heparin lyase, EC 4.2.2.7] prepared from crude heparinase, which was obtained from α-heparin-adapted flavobacteria. The digestion products were then fractionated into 16 fractions by ion-exchange column chromatography on Dowex-1. Subsequently, the resulting fractions were purified by rechromatography on Dowex-1 and or by preparative paper chromatography, yielding almost quantitatively purified compounds in fractions 2, 4, 5, 6, and 12. In addition, compounds 10a and 15a were isolated in a homogeneous state from fractions 10 and 15. Compounds 2, 4, 5, 6, 10a, 12, and 15a were characterized and identified as N-acetylglucosamine,Δ4, 5-hexosyluronic acid-N-acetylglucosamine, N-sulfated glucosamine, Δ4,5-hexosyluronic acid-N-sulfated glucosamine, N-, 6-O-disulfated glucosamine, and most probably sulfated Δ4, 5-hexosyluronic acid-N-sulfated glucosamine and sulfated Δ4,5-hexosyluronic acid-N-, 6-O-disulfated glucosamine, respectively, by chemical and enzymic studies together with infrared and ultraviolet spectral analyses. Furthermore, the major fractions (fractions 12, 13, 14, and 15) were each shown to contain two or three sulfate residues per unsaturated disaccharide unit. The total yield of unsaturated oligosaccharides was 66.6% of the starting material, while the major fractions accounted for 48.2%. In agreement with previous data, the major glucosaminidic linkages of ω-heparin appear to be 1-4. The present study suggests that ω-heparin has a more complex structure than α-heparin.

On the limited peptic digestion of horse heart cytochrome C. isolation of C-terminal peptide sequences.

Proteolysis of horse heart cytochrome C with pepsin for 3 min produces a large heme peptide, which was shown to be a mixture of the sequences 1–64 and 1–66. By ion exchange chromatography, gel filtration and preparative paper chromatography the heme‐free fraction was resolved into seven peptide fragments, corresponding to sequences 65–94, 65–96, 67–94, 67–96, 67–80, 95–104 and 97–104.

A Mannose-containing Trisaccharide Isolated from Urines of Three Patients with Mannosidosis

Abstract The urine from three patients with mannosidosis was found to contain abnormally high amounts of mannose-containing oligosaccharides. The highest yield (123 to 430 mg per liter of urine) was obtained of a trisaccharide, which was isolated and characterized. The isolation procedure included ultrafiltration, gel chromatography on Sephadex G-25, purification by preparative zone electrophoresis, and preparative paper chromatography. Sugar analysis showed that the trisaccharide consisted of 2 d-mannose moieties and 1 2-acetamido-2-deoxy-d-glucose residue. The linkages and sequential order of the sugar units were established by methylation analysis of the reduced trisaccharide. The partially methylated sugars obtained on hydrolysis of the methylated trisaccharide alditol were analyzed by gas-liquid chromatography and mass spectrometry. The anomeric configurations were determined by chromium trioxide oxidation followed by methylation analysis. Additional proof for the anomeric natures of the sugar units was obtained by enzymatic hydrolysis studies and by inhibition experiments using a precipitin system with concanavalin A. As a result of these studies, the structure of the trisaccharide could be established as α-d-mannopyranoside-(1→3)-β-d-mannopyranoside-(1→4)-2-acetamido-2-deoxy-d-glucose. The oligosaccharide is most probably a degradation product derived from the inner core of glycoprotein chains.

Alkali-labile oligosaccharides of brain glycoproteins

Treatment of glycopeptides prepared from rabbit brain glycoproteins with NaOH or NaOH-NaBH 4 released approximately 10% of the carbohydrate as low molecular weight oligosaccharides. After treatment with alkali alone, a disaccharide and a trisaccharide were isolated by gel filtration and preparative paper chromatography, and have been identified as N- acetylneuraminyl-(2→6)-O-2- acetamido-2- deoxy- D- galactose (Component B-I) and di-N- acetylneuraminyl- D- galactose (Component B-III). The N-acetylgalactosamine in Component B-I was obtained as preformed Morgan-Elson chromogen, and Components B-I and B-III were not obtained when the glycopeptides were treated with alkaline NaBH 4 under conditions which prevent peeling. These results indicate that the trisaccharide (Component B-III) was originally present as a substituent at C-3 of the N-acetylgalactosamine involved in the carbohydrate-peptide linkage, and was eliminated as a result of alkali treatment. A disaccharide (Component M) was isolated after alkaline NaBH 4 treatment of the desialylated glycopeptides, and yielded equimolar amounts of galactose and galactosaminitol after hydrolysis. Smith degradation of Component M gave galactose and threosaminitol, indicating that the disaccharide was galactopyranosyl-(1→3)-O-2- acetamido-2- deoxy- D- galactitol . From the isolation of Components B-I, B-III and M after NaOH and NaOH-NaBH 4 treatment of the glycopeptides, it is concluded that the major alkali-labile oligosaccharide of brain glycoproteins is the pentasaccharide di-N- acetylneuraminyl- D- galactopyranosyl-(1→3) [N- acetylneuraminyl-(2→6)]-O-2- acetamido-2- deoxy- D- galactose .

Comparative study of enzyme activities degrading sorbitol, ribitol, xylitol and gluconate in guinea pig tissues.

In guinea pig tissues the activities of the enzymes D-gluconokinase, sorbitol dehydrogenase, D-ribulo- and D-xylulokinase were measured. D-ribulose and D-xylulose were prepared by isomerisation of D-ribose and D-xylose in pyridine and separated by preparative paper chromatography. The activity patterns of the pentulokinases were identical in all tested organs. The highest activities of these two enzymes were found in adipose tissue, when referred to soluble cell protein, and was higher than the activity in liver and kidney. The high enzyme activities of the pentulokinases in adipose tissue may explain the antilipolytic effect of these pentitols and pentoses in diabetes. The activities of sorbitol dehydrogenase and gluconokinase showed a similar activity pattern in all tested organs of the guinea pig. The highest activities were found in liver and kidney and the lowest in the adipose tissue. The direct metabolism of gluconate in adipose tissue seems impossible. The activity of the pentulokinases are diminished in the tissues of the diabetic rat.

Thyonatan 4-Sulfate [(1 → 2)-α-l-Fucopyranan 4-Sulfate]: A SULFATED POLYSACCHARIDE FROM THYONE BRIAREUS CONNECTIVE TISSUE

Abstract Thyonatan 4-sulfate, a connective tissue polysaccharide, was isolated from the dermis of the sea cucumber Thyone briareus by proteolysis, cationic detergent precipitation, and precipitation from potassium acetate solution with ethanol. The fraction precipitating between 38 and 52% ethanol contained 47.5% l-fucose and 3.2% galactose residues, 27% sulfate groups, 1.2% nitrogen, and 6.5% of firmly bound, alkali-stable peptide. Thyonatan 4-sulfate showed [α]d20 - 155° (in water), an intrinsic viscosity of 0.6 dl g-1, and it absorbed strongly at 825 cm-1. It was homogeneous on cellulose acetate electrophoresis and stained metachromatically with toluidine blue. It was polydisperse on gel filtration on P-100 and P-200 resins and its molecular weight was estimated to be between 20,000 and 60,000. Strong acid hydrolysis of thyonatan 4-sulfate gave l-fucose, characterized as the 1-methyl-1-phenylhydrazone. Mild acid hydrolysis gave l-fucose 4-sulfate, isolated after partial acid hydrolysis (0.15 n H2SO4, 100°, 1 hour) by ion exchange chromatography, gel filtration, and preparative paper chromatography. l-erythro-Butan-1,2,3-triol was obtained in 56% yield from l-fucose 4-sulfate after periodate oxidation and sodium borohydride reduction, establishing the position of the sulfate group at carbon 4. Exhaustive methylation of the polysaccharide with dimethyl sulfate and NaOH yielded 3-O-methyl-l-fucose and 2-O-methyl-l-fucose in the ratio of 3:1. The above data suggest that the predominant structure for this polysaccharide is (1 → 2)-α-l-fucopyranan 4-sulfate.