Isolation and Characterization of Oligosaccharides from Canine Submaxillary Mucin
|
|
- Derrick Flowers
- 5 years ago
- Views:
Transcription
1 Eur. J. Biochem. 49, (1974) Isolation and Characterization of Oligosaccharides from Canine Submaxillary Mucin Christian G. LOMBART and Richard J. WINZLERP Department of Biochemistry, Faculty of Health Sciences, State University of New York at Buffalo Dedicated to the memory of R. J. WINZLER with affection and respect (Received April 9/July 2, 1974) Pure mucin isolated from canine submaxillary glands was treated with alkaline borohydride and products of degradation were fractionated by a procedure that included ion-exchange chromatography, gel filtration, high-voltage electrophoresis and paper chromatography. Seven acidic, reduced oligosaccharides were isolated being divided into two distinct types : one, containing sialic acid and no glucosamine or sulfate (type A) and the other containing glucosamine and sulfate but no sialic acid (type B). The most complete oligosaccharide of type A designated component (I) was a tetrasaccharide a-l-fucopyranosyl-( 1 + 2)-~-galactopyranosyl- [N-acetylneuraminyl-(2-i 6)]-2-acetamido-2-deoxy-~galactitol. The most complex oligosaccharide of type B designated component (V) has a hexasaccharide with the following structure : a-l-fucopyranosyl-(i -i2)-p-~-galactopyranosyl-(l+3, 4, or 6)-~-2-acetamido-2-deoxy-~-glucopyranosyl 3 or 4 sulfate-(1 +6)-[a-~-fucopyranosyl-(l~2)]-~-~galactopyranosyl-(1 -i3) 2-acetamido-2-deoxy-~-galactitol. All the other acidic isolated oligosaccharides were derived from these two main components. No oligosaccharide containing both sialic acid and glucosamine sulfate could be demonstrated. Based on these structural features, a possible biosynthetic pathway leading to the two different oligosaccharide chains is discussed. Variations in ratios of sugars observed in canine submaxillary mucin and especially the reciprocal relationship between sialic acid and fucose noted earlier (1962) by Dische, Pallavicini, Kavasaki, Smirnow, Cizek and Chien may reflect variations in the proportions of these two types of oligosaccharides. Mucins from the submaxillary glands of a variety of species have been shown to consist of a polypeptide chain with many relatively small oligosaccharide side chains linked to the peptide through glycosidic bonds between the anomeric carbon of N-acetylgalactosamine and the hydroxyl group of serine or threonine [I]. The oligosaccharides from ovine and bovine submaxillary mucins have been shown to be mainly t Deceased (September 28, 1972). Abbreviations. Fuc, L-fucose; Gal, D-galaCtOSe; AcNeu, N-acetylneuraminic acid; GlcNAc, N-acetylglucosamine (2- deoxy-2-acetamide-~-glucose); GalNAcol, N-acetylgalactosaminitol(2-deoxy-2-acetamido-galactitol). Enzymes (CBN Recommendations 1972). a-i,2-~-fucosidase (EC ); P-N-acetyl-glucosaminidase (EC ); 8-galactosidase (EC ); neuraminidase (EC ). sialyl-n-acetylgalactosamine [I 1. The oligosaccharides of porcine submaxillary mucin are more complex, consisting of structures up to the size of pentasaccharides, some having blood group A activity [2-51. The structures of oligosaccharides from canine submaxillary mucin have not been investigated. However, this is a problem of considerable interest, since Dische [6,7] has found that the ratios of sialic acid to fucose in the submaxillary secretions from the dog may vary by a factor of 1 depending upon the nature of the stimulus eliciting the secretion. Although the ratio of the sugars varies widely, their sum remains constant. It was originally suggested that the two sugars, which are both terminal at the non-reducing ends of oligosaccharide chains, had a reciprocal relationship in which, under one set of secretory conditions, oligosaccharides were mostly terminated with
2 78 Oligosaccharides from Canine Submaxillary Mucin sialic acid and in other situations, mainly with fucose. Subsequently, some evidence for the production of different mucins was advanced [8]. In order to elucidate the metabolic relationship between sialic acid and fucose in canine submaxillary mucin, it will be necessary to establish the structures of the oligosaccharides of this mucin. The present paper presents data on the acidic oligosaccharides from mucins isolated from pooled canine submaxillary glands. MATERIALS AND METHODS Mucin was isolated from the pooled submaxillary glands of dogs as previously described [9]. N-Acetylgalactosaminitol and galactitol were prepared by reduction of acetyl-galactosamine and galactose with sodium borohydride. Threosaminitol was prepared as previously described [ 11. P-D-Galactosidase and a(1 + 2)-~-fucosidase from Aspergillus niger were provided by Dr. P. Bahl (State University of New York at Buffalo). P-N-Acetylglucosaminidase was a gift from Dr B. Weismann (University of Illinois College of Medicine, Chicago). Neuraminidase from Vibrio cholera was purchased from Behringwerke (Marburg, Germany). Hexoses were determined by the phenol - sulfuric acid method of Dubois et al. [I I], and by gas-liquid chromatography of the alditol acetates [12]. Bound sialic acid was determined by the Ehrlich method of Werner and Odin [13] and free sialic acid by the method of Aminoff [14]. Protein was determined by the method of Lowry et al. [15]. Amino sugars and amino sugar alcohols, determined with the Beckman amino acid analyser [16]. Uronic acids were determined by the carbazole method [17]. Glycerol was determined by gas-liquid chromatography. Sulfate was determined by the method of Antonopoulos [18]. Descending analytical paper chromatography was performed on Whatman no. 1 and preparative paper chromatography on Whatman no. 3M with the following solvents. Solvent 1, ethyl acetate - pyridine - water (1: 4: 3) ; solvent 2, n-butanol - pyridine - water (6: 4: 3) ; solvent 3, n-butanol- acetic acid- water (4: 1 : 5) ; solvent 4, n-butanol - n-propanol -.1 N HCl (2: 1 :2); solvent 5, n-butanol-pyridine-.1 N HCl(5 : 3 : 2) (all v/v/v). After development of preparative chromatograms, oligosaccharides were located by spraying marginal strips containing standards and reference substances with a periodate- benzidine reagent [19]. The remaining paper was cut appropriately, the strips sprayed with water, and the material eluted by centrifugation into conical tubes. Any paper lint was remov- ed by passing the supernatant through a short Biogel P-2 column, and the samples were then lyophilized. Thin-layer chromatography of monosaccharides and oligosaccharides was performed on silicagel H using the following solvents : solvent 6, n-propanolwater (7: 3); solvent 7, n-propanol- water (6: 3); solvent 8, n-propanol- ethyl acetate - water (7 : 5 : 2) (all v/v). Plates were usually developed twice in the same solvent, dried at 11 "C for 3 min and sugars located by the periodate- benzidine stain. High-voltage electrophoresis was carried out in.1 M pyridine acetate buffer at ph 4.7 on Whatman 3M paper for 9 min at 5 V/cm. Carbohydrates were detected by means of the periodate - benzidine reagent. Per ioda te Oxidat ion Approximately.5 ymol oligosaccharide (based on galactose) dissolved in.3 ml of water was oxidized at 4 "C with 5 ymol sodium metaperiodate in 5 pl water. The consumption of periodate was followed spectrophotometrically [2] by diluting 2-p1 aliquots to 2 ml with distilled water and determining absorbance at nm. Smith Degradation A sample containing 1 to 1.5 ymol oligosaccharide was incubated with 2 ymol periodate in.2 ml for 24 to 36 h at 4 "C in the dark. Excess periodate was destroyed by adding.1 ml.3 M sodium arsenite, and the product reduced with.2 ml 1.3 M NaBH,. After 24 h at 4 "C the reaction mixture was neutralized with 2 p1 12 N HC1 and the salts removed by gel filtration on a Biogel P-2 column (1 x 36 cm). Fractions containing salt-free material were pooled, lyophilized and hydrolyzed for 2 h at 1 "C with 1OOyl of Dowex 5x2 (H') resin (4 suspension w/v) in.1 N HCl. A rabitol (.14 pm) was added as internal standard and the mixture was passed through a microcolumn (.25 x.5 cm) of Dowex 1x2 (HCO;). The column was washed 8 times with.2 ml of water and the combined eluates, after lyophilization, were analyzed for neutral sugars by gas chromatography. The amino sugars and the amino sugar alcohols were eluted from the resin with 1.5 ml of.5 N HCl and analyzed with the amino acid analyzer as described by Weber and Winzler [16]. Enzymatic Hydrolysis Hydrolysis with P-galactosidase and P-N-acetylglucosaminidase were carried out in.5 M citrate buffer at ph 4.6. Digestion with a(1+2)-~-fucosidase
3 C. G. Lombart and R. J. Winzler 79 was performed in.5 M citrate buffer at ph 3.9. Incubation with neuraminidase was in.8 % saline-.1 M sodium acetate buffer at ph 5.5. Approximately.5 pm oligosaccharide was used in each experiment. In sequential enzymatic hydrolysis, the first enzyme and 2 pl toluol was added and aliquots of 5 to 1 pl taken at different times and analyzed for free monosaccharides. Before adding a new enzyme to the mixture, the preceding one was inactivated by immersing the preparation for 3 min in a boiling water bath. The ph was adjusted if necessary, and the new enzyme and 5 pl toluol added. For extended incubation periods more enzyme was added periodically. In cases where a glycosidase did not act on the substrate, the activity of the enzyme at the end of incubation was checked by adding the appropriate p-nitrophenylglycosides as substrates. Qualitative demonstration of the released sugars was performed by thinlayer chromatography in solvents 6, 7 and 8 by paper chromatography by using solvents 2, 3 and 5. Quantitative analysis of the release of neutral sugars during enzymatic hydrolysis was carried out with gas-liquid chromatography of the alditol acetates, or by the Nelson-Somogyi colorimetric method as modified by Bahl [21] for micro determination. Release of acetylglucosamine during enzymatic hydrolysis was followed by the method of Reissig et al. [22] as modified by Bahl [21]. RESULTS Isolation oj Reduced Oligosaccharides Reduced oligosaccharides were removed from canine submaxillary mucin by treatment with alkaline borohydride essentially by the procedure of Carlson [3]. In order to determine the optimum time for release of reduced oligosaccharides from canine submaxillary mucin by alkaline borohydride, 12 mg was dissolved in 1 ml of.373 M NaBH, in.1 M KOH and incubated at 45 "C in a screw-cap tube under nitrogen. 1-p1 aliquots were taken at different time intervals and neutralized with 2 pl of 2.5 N acetic acid. After the addition of 1 ml of water, the solution was lyophilized. The dried material was hydrolyzed in 25 pl of 4 N HCI at 1 "C for 7 h. The whole hydrolysate was applied directly on a long column of the amino acid analyzer equilibrated with a citrate - borate buffer at ph 5.29 for amino sugar analysis [ 161. The kinetics of oligosaccharide release during the alkaline borohydride treatment of canine submaxillary mucin was followed by measuring destruction of galactosamine and the appearance of galactosaminitol. Fig. 1 shows that at 24 h, 88 % of the galac w Time (h) Fig. 1. Rate of decrease of bound galactosamine and rate of' increase of galactosaminitol during alkaline borohydride treutment of canine submaxillary mucin. Alkaline conditions were.1 M KOH and.375m NaBH4 at 45 C. ( O d ) Galactosalinitol; (O--O) galactosamine, ( A4) galactosaminitol + galactosamine tosamine originally present in the mixture is destroyed and recovered almost quantitatively (85 %) as galactosaminitol. For subsequent studies on a larger scale a 16-h incubation period was chosen, since under these conditions no degradation of hexoses or sialic acid was observed. Lyophilized canine submaxillary mucin (1.6 g) was dissolved in 1 ml of.375 M NaBH, and.1 N KOH. The reaction mixture was incubated 16 h at 45 "C in sealed tubes under nitrogen. The reaction was stopped by adding 4 N acetic acid to ph 5. The mixture was then desalted in two batches on a column of Biogel P-2 (5.65 x 43 cm). The fractions containing the released sugars from both columns were pooled and lyophilized, yielding 1.32 g of salt-free material. The fractions containing salts were discarded. The lyophilized material was then dissolved in 2 ml of distilled water and fractionated on a Sephddex G-25 column (5.65 x 4 cm). The elution profile is shown in Fig. 2. Fractions 4-61, containing larger peptides and glycopeptides, were not studied further. The retarded fractions (62-79) containing low-molecularweight reduced oligosaccharides representing 6 yo of the original carbohydrate were pooled, lyophilized, dissolved in 1 ml of water and passed through a column (2 x 16 cm) of Dowex 5x8 (2-4 mesh, H') to remove peptides. The fractions eluted with water were pooled and lyophilized. The reduced oligosaccharides were then separated into neutral and acidic fractions by dissolving in 1 ml of water and passing over a column of Dowex 1x2
4 8 Oligosaccharides from Canine Submaxillary Much E 1.O t P Ln a * c m n m L : n Q.4 - m v) 2.4 L 73 )r c f s w E L.2 9 u.- u._ m In Fraction number Fig. 2. Gel filtration of the alkaline borohydride degradation products of canine submaxillary mucin. A sample containing 1.32 g hydrolysate was applied to a column (5.65 x 4 cm) of Sephadex G-25 and eluted with distilled water. Fractions (9.5 nil) were collected and aliquots (.1 ml) were analysed by the phenol-sulfuric reaction (-) and.2 ml were taken for the Ehrlich test for sialic acid (-); results are given as absorbance at 49 nm and 57 nm respectively (C1-) (1 x 15 cm) and washing with 2 ml water. The neutral fraction was obtained in the water eluate and the acidic fraction was eluted from the column with.2 M NaCl. This fraction was desalted by chromatography on Biogel P-2. The acidic fraction was designated by the letter A and the neutral by the letter N. Each fraction after lyophilization was dissolved in water and fractionated on a long Biogel P-2 column (2.6 x 16 cm). Two runs were employed for the separation of the neutral and 1 runs for the acidic fractions. Characteristic profiles for each group of oligosaccharides are shown in Fig. 3 and 4. Fractions were pooled as indicated and lyophilized. Most of the reduced oligosaccharides released by alkaline borohydride are acidic. Thus, from 1.6 g of canine submaxillary mucin, 537 mg (6%) of neutral sugars was released as reduced oligosaccharides. Of this, 22 mg was finally recovered as acidic oligosaccharides and 37 mg as neutral oligosaccharides after gel filtration on Biogel P-2 columns described in Fig. 3 and 4. The acidic fractions (except minor fraction 4) were each submitted to preparative electrophoresis in.1 M pyridine-acetate buffer at ph 4.7, with 4 to 5 mg of material being applied in each run, and Fraction number Fig. 3. Elution pattern of the acidic oligosaccharide fractions from a column of Biogel P-2, 2-4 mesh (2.6~ 16 cm). A 25-mg sample was applied to the column and eluted with water; 1.7-ml fractions were collected; a.1-ml aliquot of each fraction was analysed with the phenol - sulfuric acid reaction for carbohydrates (o--+) and.2 ml with the Ehrlich reaction for sialic acid (M). A1 to A5 represent pooled fractions. The column was calibrated with known standards (stachyose, raffinose, maltose and galactose) with their elution volume as indicated on the chromatogram Fraction number 2 27 Fig. 4. Elution pattern of the neutral oligosacclzaride fractions from a column of Biogel P-2, 2-4 mesh (2.6 x 16 cm). A 4-mg sample was applied to the column and eluted with water. The fractions (1.75 ml) were analysed by the phenolsulfuric acid reaction. N1 to N6 represent pooled fractions. The standards are the same as in Fig. 3
5 C. G. Lombart and R. J. Winzler 81 tion, Smith degradation and sequential enzymatic hydrolysis. m II 6) AI-AZ-A~-A~-A~-ACN~U - Fig. 5. High-voltuge electrophoresis on paper Whatman no. 1 oj the pooled fraction A1 - AS from the Biogel P-2 column. The electrophoresis was carried out in.1 M pyridine acetate buffer ph 4.7 during 9 min at 5 V/cm. The position of the sugar-containing material was revealed with the benzidineperiodate reagent. Numbers I to VIII designate purified oligosaccharides according to our classification (see text). Letters GP represent glycopeptides N-acetylneuraminic acid present as a reference for mobility measurements. Each fraction yield rather well-separated zones with characteristic mobilities relative to N-acetylneuraminic acid (Fig. 5). Such zones from a number of runs were eluted, pooled, and submitted to paper Chromatography in solvents 1 and 2 to check the purity. Further purification of some of the acidic acid components was necessary, and was achieved by repeated chromatography in solvent 2. Seven reduced acidic oligosaccharides and free sialic acid were isolated in pure form from the pooled fractions of Fig. 3. The composition and yields of these components are tabulated in Table 1, and their localization in the electrophoretic fractions shown in Fig. 5. CHARACTERIZATION OF ACIDIC OLIGOSACCHARIDES Studies were carried out on the structure of each of the acidic oligosaccharides using periodate oxida- Component I The structure assigned to component I is CI-L- Fuc( 1 + 2)-P-Gal-( 1 + 3)- [AcNeu-(2 + 6)]-GalNAcol (Fig. 7). This structure is based on the following observations. a) The molecular weight of I by gel filtration on Biogel P-2 corresponds approximately to a tetrasaccharide (Fig. 3 and 5). b) 1 has equimolar amounts of sialic acid, fucose, galactose and N-acetylgalactosaminitol (Table 1). c) Treatment of I with a(l + 2)-fucosidase releases fucose quantitatively, and produces a compound which has the same electrophoretic mobility and R, (solvent 4) as component I1 (Table 2). d) After treatment of I with cc(1.+ 2)-fucosidase, P-galactosidase releases all of the galactose and an acidic component having one mole each of sialic acid and acetyl-galactosaminitol (Table 2) ; therefore, fucose is linked to galactose. Since the enzyme is specific for a(1 + 2)-fucosyl linkages [21], the fucose must be linked to the 2 position of galactose and the sialic acid must be linked to acetyl-galactosaminitol. e) Treatment of I with neuraminidase or with.5 M HCI at 8 "C for 1 h releases sialic acid quantitatively, resulting in the formation of a neutral oligosaccharide (which did not migrate in paper electrophoresis) (Table 2). f) Smith degradation of I followed by resin hydrolysis destroys sialic acid, fucose, and galactose, and converts the N-acetylgalactosaminitol to threosaminito1 (Table 3). This shows that the N-acetylgalactosaminitol is unsubstituted at carbon 4 and 5, and indicates that the sialic acid and galactose are linked to carbon 3 and 6. g) Smith degradation and resin hydrolysis of desialyzed I also produces threosaminitol (Table 3), showing that galactose must be linked to carbon 3 and sialic acid to carbon 6 of acetylgalactosaminitol. h) 6 mol periodate are consumed when 1 mol I (based on galactose) is subjected to quantitative periodate oxidation (Fig. 6). Component II Component I1 is assigned the structure /?-Gal- (1.+3)-[AcNeu-2(+6)]-GalNAcol (Fig. 7). This structure is based on the following observations. a) I1 has equimolar amounts of galactose, sialic acid and N-acetyl galactosaminitol (Table 1). b) Treatment of I with a(1 + 2)-fucosidase produces an acidic oligosaccharide which co-chromatog-
6 82 Oligosaccharides from Canine Submaxillary Mucin Table 1. Composition of acidic oligosaccharides The yield was calculated as the percentage by weight of the acidic oligosaccharide mixture. The molar ratio is expressed with AcNeu or GlcNAc taken as unity Compo- Electrophoretic Yield Molar ratio nent mobility relative to AcNeu Gal Fuc GalNAcol GlcNAc AcNeu so4 I I I o IV o V o 1.5 VI o.96 VII o.91 ~ VIII o 1.1 Table 2. Treatment of reduced oligosaccharides with glycosidases Substrate Enzyme Sugars Product released (amount) (mol/mol) I I-FUC I I1 I1 V V-FW V-Fuc-Gal VI a-fucosidase p-galactosidase neuraminidase neuraminidase b-galactosidase a-fucosidase /I-galactosidase p-acetylglucosaminidase a-fucosidase Fuc (1) Gal (1) AcNeu (1) AcNeu (1) Gal (1) Fuc (2) Gal (1) acidic GlcNAc (1) Fuc (1) I-FUC" sialyl-galnacol neutral oligosaccharide n.s.b sialyl-galnacol (V)-FUC (V)-Fuc-Gal (V)-Fuc-Gal-GlcNAc (neutral oligosaccharide) (VI)-Fuc VI-FUC p-galactosidase Gal (1) (V1)-Fuc-Gal VI-Fuc-Gal /I-acetylglucosaminidase acidic (V1)-Fuc-Gal-GlcNAc GlcNAc (1) VI-Fuc-Gal-GlcNAc VII P-galactosidase 11-acetylglucosaminidase Gdl (1) acidic n.s. (VI1)-GlcNAc VII a-fucosidase GlcNAc (1) Fuc (1) (VI1)-Fuc(identical with VIII by paper chromatog- VII-Fuc-GlcNAc j-galactosidase Gal (1) raphy) n.s. VIII /I-acetylglucosaminidase acidic neutral oligosaccharide containing only Gal and GlcNAc (1) GalNAcol a R, in solvent 4 and high-voltage electrophoretic mobility identical with 11. Not studied. Table 3. Eff.ct of Smith degradation followed by resin hydrolysis on reduced oligosaccharides Component Sugars destroyed Sugars not destroyed Amino sugar produced I Fuc, Gal, GalNAcol, AcNeu - threosaminitol Desialized I Fuc, Gal, GalNAcol - threosaminitol I1 Gal, GalNAcol, AcNeu - threosaminitol Desialyzed I1 Gal, GalNAcol - threoaaminitol I11 GalNAcol, AcNeu - serinol V Fuc, Gal, GalNAcol GlcNAc threosaminitol VI Fuc, Gal, GalNAcol GlcNAc threosaminitol VII Fuc, Gal, GalNAcol GlcNAc threosaminitol VIII Gal, GalNAcol GlcNAc threosaminitol
7 C. G. Lombart and R. J. Winzler 83 7 li o f. '. ' '...I:.. '. o a244a Time (h) Fig. 6. Periodate consumption curves for acidic oligosaccharides. Component I (A---A); component V (--); component VI (-); component VII (M). Oligosaccharides (.3 to.5 pm) were incubated with 5 pm sodium metaperiodate in a final volume of 35 pl in the dark at 4 "C. At the indicated time intervals, 2 p1 was removed, diluted to 2 ml with distilled water and measured spectrophotometrically at nm raphs (solvent 4) and also coelectrophoreses with I1 (Table 2). c) Digestion of I1 with P-galactosidase or neuraminidase releases galactose or sialic regardless of the order of digestion (Table 2). Therefore, both are terminal non-reducing sugars. d) Smith degradation of I1 or of desialyzed I1 followed by resin hydrolysis destroys galactose-acetylgalactosaminitol and sialic acid, and produces threosaminitol. Therefore, the acetylgalactosaminitol must be unsubstituted on carbon 4 and 5, galactose is on carbon 3, and sialic acid is on carbon 6. Componen t III al I. Fuc -Gal -GalNAcol II. m. Gal pl-3,4,or6 ta2-6 Ac Neu p1-3 Gal -GalNAcol lap-6 Ac Neu Gal NAcol la2-6 Ac Neu p1-6 p1-3 GlcNAc -Gal -GalNAcol la,-2 3r4 So, lal-2 Fuc Fuc =. P1--3,4,or6 p1-6 GlCNAc -Gal lal-2 3w4so4 p1-3 -GalNAcol Fuc m. GlcNAc B1--6Gal81-3GalNAcol ma. 3or4S4 lal-2 Fuc pl-6 GlcNAc -Gal -" -'GalNAcd 3r 4%4 \ I TYP~ A - Type Fig. I. Structure of reduced oligosaccharides from canine submaxillavy mucin Component IV Component IV was identified as free N-acetylneuraminic acid by its electrophoresis and chromatographic (solvent 4) mobilities. It reacts with thiobarbituric acid [I41 to give the same color yield as standard N-acetylneuraminic acid. Based on the following observations component I11 has been assigned the structure AcNeu-(2 + 6)- GalNAcol (Fig. 7). a) I11 has an elution volume between disaccharides and trisaccharides during gel filtration on Biogel P-2 (Fig. 3 and 5). b) I11 contains equimolar amounts of N-acetylneuraminic acid and N-acetylgalactosaminitol (Table 1). c) Smith degradation of 111 followed by resin hydrolysis destroys sialic acid and N-acetylgalactosaminitol with production of serinol but not threosaminitol. Thus cabon 3 and 4 of GalNAcol are unsubstituted in component 111. d) Component I11 and free galactose are produced from component I1 by digestion with P-galactosidase. Component V Component V was assigned the structure a-fuc- (1 + 2)-P-Gal-(l + 3,4 or 6)-P-GlcNAc-3 or 4 sulfate- (1 + 6)-[a-Fuc-(l + 2)]-P-Gal-(l + 3)-GalNAcol (Fig. 7), based on the following observations. a) V contains fucose, galactose, glucosamine, glucosaminitol and sulfate in the proportions 2: 2: 1 : 1 : 1 (Table 1). b) In Biogel P-2 gel filtration columns, V moves with an elution volume suggesting that it is at least a tetrasaccharide (Fig. 3 and 5). c) 2 mol fucose, 1 mol galactose and 1 mol N- acetylglucosamine are enzymatically released from V only if a(1 + 2)-fucosidase, P-galactosidase and P-N-
8 84 Oligosaccharides from Canine Submaxillary Mucin acetylglucosaminidase are used sequentially in the order given (Table 2). This indicates that one galactose has only an a(1 + 2)-fucose substitution whereas the other galactose has an additional substitution at carbon 3, 4 or 6. Also, the data indicates that both fucosyls are nonreducing termini, that one galactose is penultimate, and that this galactose is attached to glucosamine. d) The acetylglucosamine released in the above treatment was strongly bound to Dowex 1 resins and therefore was acidic. It is presumed that this is due to the presence of a sulfate ester on carbon 3, 4 or 6. Location of the sulfate on carbon 3 or 4 is indicated in discussion of component VII. e) Under the standard conditions for the Smith degradation (4 "C), one galactose and both fucoses were destroyed while one galactose and the N-acetylglucosamine were left intact. Determination of periodate consumption at 4 "C showed 7 mol periodate taken up per rnol glucosamine (Fig. 6). The consumption of 7 mol periodate could not be reconciled with periodate uptake on component VII (see below); therefore the oxidation was carried out at 2 "C. This lead to the consumption of 8 mol periodate per mol V and the destruction of all galactose but not of glucosamine. Thus, one pair of vicinal hydroxyl groups appears to be more resistant than the other seven to periodate oxidation. These results indicate that both galactoses in V are unsubstituted on carbons 3 and 4, and that the acetylglucosamine is protected by substitution at carbons 3 or 4. The acetylglucosamine is linked to the 6 position of galactose. f) In the Smith degradation of IV followed by acid hydrolysis, galactosaminitol was converted to threosaminitol, showing tha GalNAcol was substituted on carbon 3 and not on carbon 4. Component VI Component VI was assigned the structure a-fuc- (I 4 2)-j-Gal(l + 3, 4 or 6)-J-GlcNAc-3 or 4 sulfate (1 + 6)-j-Gal(l + 3)-GalNAcol (Fig. 7). This structure was based on the following observations. a) VI contains 2 rnol galactose, and 1 mol each of fucose, glucosamine, acetyl-glucosaminitol, and sulfate (Table 1). b) Sequential treatment of VI with a(1 -+ 2)-fucosidase, P-galactosidase, j-acetylglucosaminidase and J-galactosidase resulted in the stepwise liberation of 1 mol of each monosaccharide per mol (based on glucosamine) in sequence. Other sequences of enzyme treatment were ineffective in removing monosaccharides. This establishes the terminal sequence of VI as a-fuc( 1 + 2)-j-Gal-J-GlcNAc-Gal. c) The acetylglucosamine released in this sequence was negatively charged and was bound to Dowex 1 resin columns. d) Smith degradation of VI followed by hydrolysis in 1 N HCl destroys all fucose, galactose but does not affect glucosamine. N-Acetylgalactosaminitol is converted to threosaminitol (Table 3). e) 7 mol periodate are consumed per mol (based on glucosamine), with the last periodate being taken up very slowly (Fig. 6). Component VI, therefore, appears to be the same as component V except for the absence of one fucose. Component VII Component VII was assigned the structure j-glc- NAc-3 or 4 sulfate (1 -+ 6)-[a-Fuc(l + 2)I-J-Gal- (1 + 3)-GalNAcol (Fig. 7). This structure is based on the following observations. a) VII contains equimolar amounts of galactose, fucose, acetylglucosamine and acetylgalactosaminitol (Table 2). b) N-Acetylglucosamine in cleaved from VII by j-n-acetylglucosaminidase. It is therefore at a nonreducing terminus. The released N-acetylglucosamine is strongly anionic by high-voltage electrophoresis and is adsorbed to columns of Dowex 1 resin. c) Fucose is removed from VII by incubation with a(1 + 2)-fucosidase. After treatment with both fucosidase and P-N-acetylglucosaminidase, galactose becomes susceptible to cleavage with p-galactosidase (Table 3). d) 5 mol periodate were consumed when VII was oxidized with periodate (Fig. 6). N-Acetylglucosamine was resistant to periodate oxidation. This indicates that the sulfate is on the 3 or 4 positions of acetylglucosamine. e) Smith degradation followed by acid hydrolysis destroys fucose and galactose. N-Acetylglucosamine is resistant to periodate oxidation, indicating that the sulfate group is at position 3 or 4. Acetylgalactosaminitol is converted to threosaminitol. Component VIII Component VIII has been assigned the structure : P-GlcNAc-3 or 4 sulfate-(1 -+ 6)-J-Gal(l -+ 3)-Gal- NAcol (Fig. 7). This assignment is based on the following information. a) VIII contains equimolar amounts of galactose, glucosamine, glucosaminitol and sulfate (Table 1). b) VII is converted into a component having the composition, chromatographic behavior (solvent 2) and electrophoretic behavior of VIII by digestion with a(1 + 2)-fucosidase (Table 2).
9 C. G. Lombart and R. J. Winzler 85 Neutral Oligosaccharides Most (85 o/,) of the oligosaccharides cleaved from canine submaxillary mucin by alkaline borohydride were acidic, and seven such oligosaccharides could be identified. The neutral fraction was also shown to have at least nine oligosaccharides as demonstrated by paper chromatography in solvents 1, 2 and 3. All of these contained galactosaminitol, galactose and fucose. Most appeared by gel filtration to be larger than the acidic oligosaccharides. However, no detailed studies of their quantitative composition or structure have been carried out. DISCUSSION It is clear from the data presented that most of the oligosaccharide chains released from canine submaxillary mucin by alkaline borohydride are acidic. It is striking that the acidic oligosaccharides are of two types, one (type A) containing sialic acid and no glucosamine or sulfate (components I to IV) and the other (type B) containing glucosamine and sulfate and no sialic acid (components V to VIII) (Fig. 7). No examples of acidic components containing both sialic acid and glucosamine were encountered. Components I and V are the largest oligosaccharides of the two types A and B, respectively. These appear to be the complete or parent oligosaccharides for all of the other acidic oligosaccharides isolated in these studies. Whether the smaller oligosaccharides were produced during the process of isolation or whether they were preexistent in the isolated mucin is not determinable from the experiments described. The latter seems the more likely possibility, in view of the many reports on microheterogeneity and incompleted oligosaccharides chains demonstrated in other mucins and glycoproteins [23]. The existence of two distinct types of oligosaccharides in canine submaxillary mucin indicates that there is not a single parent or complete acidic oligosaccharide in canine submaxillary mucin to which all other incomplete acid oligosaccharides are related. All, however, contain j-galactosyl-( l -+ 3)-N-acetylgalactosamine linked to serine or threonine in the peptide chain of the mucin. It would appear possible that elaboration of the oligosaccharide side chains of canine submaxillary mucin may be regulated at the level of the galactosyl-acetylgalactosamine peptide. One pathway, leading to type A chains, would follow the transfer of a sialyl group to carbon 6 of acetylgalactosamine. The other pathway, leading to type B chain, would follow the transfer of glucosamine to carbon 6 of galactose. The two pathways appear to be mutually exclusive. The reciprocal relationship between sialic acid and fucose noted by Dische [6-81 clearly is not due to reciprocal substitution of fucose or sialic acid on a single competitive site. However, the reciprocal relationship may be the result of variation in the relative proportion of the two types of oligosaccharides, since type A oligosaccharide containing sialic acid have less fucose than the type B oligosaccharide containing glucosamine and sulfate. If our hypothesis is correct, the nature of the stimulus which is known to change the fucose to sialic acid ratio in canine submaxillary mucin [6] would affect the relative activities of the two transferases; i.e. the sialyl and acetylglucosaminyl transferases toward their common substrate, the galactosyl acetylgalactosamine peptide. However, metabolic studies are needed to confirm such an hypothesis. This work was supported by National Institutes of Health Grant 2 RO 1 CA , American Cancer Society Grant BC-2 SE, and Armed Forces Epidemiological Board Grant MD 283. C.L. is a career investigator of Institut National de la Santt et de la Recherche mtdicale (France). REFERENCES 1. Pigman, W. & Gottschalk, A. (1966) in Glycoproteins (A. Gottschalk, ed.) p. 454, Elsevier, Amsterdam. 2. Carlson, D. M. (1966) J. Biol. Chem. 241, Carlson, D. M. (1968) J. Biol. Chem. 243, Katzman, R. L. & Eylar, E. H. (1968) Arch. Biochem. Biophys. 127, Baig, M. M. & Aminoff, D. (1972) J. Biol. Chem. 247, Dische, Z., Pallavicini, C., Kavasaki, H., Smirnow, N., Cizek, L. J. & Chien, S. (1962) Arch. Biochem. Biophys. 97, Dische, Z. (1963) Ann. N. Y. Acad. Sci. 16, Dische, Z., Burgher, C., Danilchenko, A. & Rothschild, C. (1969) Arch. Biochem. Biophys. 135, Lombart, C. & Winzler, R. J. (1972) Biochem. J. 128, Thomas, D. B. & Winzler, R. J. (1969) J. Biol. Chem. 244, Dubois, M., Giles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. (1956) Anal. Chem. 28, Lehnhardt, W. F. & Winzler, R. J. (1968) J. Chvornatogr. 34, Werner, I & Odin, L. (1952) Acta Soc. Med. Ups. 57, Aminoff, D. (1961) Biochem. J. 81, Lowry,. H., Rosenbrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, Weber, P. & Winzler, R. J. (1969) Arch. Biochem. Biophys. 129, Bitter, T. & Muir, H. M. (1962) Anal. Biochem. 4,
10 86 C. G. Lombart and R. J. Winzler : Oligosaccharides from Canine Submaxillary Mucin 18. Antonopoulos, C. A. (1962) ActaChem. Scand. 16, Bahl,. P. (1969) J. Biol. Chem. 244, Reissig, J. L., Strominger, J. L. & Leloir, L. F. (1955) 19. Gordon, H. T., Thornburg, W. & Werum, L. N. (1956) J. Biol. Chem. 217, Anal. Chem. 28, Cunningham, L. W. (1971) in Glycoproteins of Blood 2. Dixon, J. S. & Lipkin, D. (1954) Anal. Chem. 26, 192- Cells and Plasma (G. A. Jamieson & T. J. Greenwalt, 193. eds) p. 16, J. B. Lippincott, Co., Philadelphia. C. G. Lombart s present address : Laboratoire de Biochimie, U.E.R. Biomkdicale des Saint-Peres, 45 Rue des Saints-Peres, F-7527 Paris-Cedex 6, France
J. Biosci., Vol. 3, Number 4, December 1981, pp Printed in India.
J. Biosci., Vol. 3, Number 4, December 1981, pp. 343-360. Printed in India. Studies on carbohydrate moieties of the glycoprotein, glucoamylase II of Aspergillus niger: Nature of carbohydratepeptide linkage
More informationPeriodate Oxidation of Glycopeptides from Ovalbumin*
The Journal of Biochemistry, Vol. 60, No. 3, 1966 Periodate Oxidation of Glycopeptides from Ovalbumin* By MAYUMI MAKING and IKUO YAMASHINA (From the Department of Biological Chemistry, Faculty of Pharmaceutical
More information189,311, , ,561, ,639, ,679, Ch13; , Carbohydrates
Lecture 31 (12/8/17) Reading: Ch7; 258-267 Ch10; 371-373 Problems: Ch7 (text); 26,27,28 Ch7 (study-guide: applying); 2,5 Ch7 (study-guide: facts); 6 NEXT (LAST!) Reading: Chs4,6,8,10,14,16,17,18; 128-129,
More informationMucus Glycoprotein Secretion by Tracheal Explants: Effects of Pollutants
Environmental Health Perspectives Vol. 35, pp. 131-138, 198 Mucus Glycoprotein Secretion by Tracheal Explants: Effects of Pollutants by Jerold A. Last* and Tokio Kaizu* Tracheal slices incubated with radioactive
More informationGlycoprotein Deglycosylation Kit Cat. No
Visit our interactive pathways at /pathways User Protocol 362280 Rev. 23 February 2006 RFH Page 1 of 5 Glycoprotein Deglycosylation Kit Cat. No. 362280 Note that this user protocol is not lot-specific
More information120. Paper Electrophoresis o f Hexosamines, N-Acetylhexosamines and N Acetylneuraminic Acid*l
534 [Vol. 39, 120. Paper Electrophoresis o f Hexosamines, N-Acetylhexosamines and N Acetylneuraminic Acid*l By Seiichi OHKUMA and Toshiaki SHINOHARA Biochemical Laboratory, Scientific Police Research Institute,
More information189,311, , ,561, ,639, ,679, Ch13; , Carbohydrates. Oligosaccharides: Determination of Sequence
Lecture (2//7) Reading: Chs4,6,8,0,4,6,7,8; 28-29, 89,,77-80,555-557,56,62-622,69,662-66,679, 69-694 Ch; 497-50, 507-54 Problems: Ch (text); 5,6,9,0,22,24 Ch7 (study-guide: applying); 4 Ch7 (study-guide:
More informationSialic Acid Fluorescence Labeling Kit
Cat. # 4400 For Research Use Sialic Acid Fluorescence Labeling Kit Product Manual Table of content I. Description... 3 II. Components... 3 III. Storage... 3 VI. Procedure... 4 V. Experiment Example...
More informationKinetics analysis of β-fructofuranosidase enzyme. 1-Effect of Time Incubation On The Rate Of An Enzymatic Reaction
Kinetics analysis of β-fructofuranosidase enzyme 1-Effect of Time Incubation On The Rate Of An Enzymatic Reaction Enzyme kinetics It is the study of the chemical reactions that are catalyzed by enzymes.
More information774 [Vol. 39, *) The abbreviations used are: GIcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine;
774 [Vol. 39, 170. Separation and Identification of N-Acetylhexosamines and N Acetylneuraminic Acid by Two-dimensional Electrophoresis and Chromatography on Paper By Seiichi OHKUMA and Toshiaki SHINOHARA
More informationNew immunomodulators with antitumoral properties; Isolation of active naturally-occurring anti-mitotic components of MR>1KD from pollen extract T60
I M M U N O M O D U L A T O R S U P P O R T : GRAMINEX Flower Pollen Extract New immunomodulators with antitumoral properties; Isolation of active naturally-occurring anti-mitotic components of MR>1KD
More informationTECHNICAL BULLETIN. Sialic Acid Quantitation Kit. Catalog Number SIALICQ Storage Temperature 2 8 C
Sialic Acid Quantitation Kit Catalog Number SIALICQ Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description The Sialic Acid Quantitation Kit provides a rapid and accurate determination of total
More informationTable of content. I. Description...2. II. Kit Components...2. III.Storage...2. VI.Procedure...3. V. Experiment Example...3
Table of content I. Description...2 II. Kit Components...2 III.Storage...2 VI.Procedure...3 V. Experiment Example...3 VI. Application Example...11 VII. Related Products...14 VIII. References...14 1 I.
More informationExtraction and Purification of Sialic Acid from Submandibular Mucin of Goat
Article International Journal of Modern Biochemistry, 2015, 4(1): 1-9 International Journal of Modern Biochemistry Journal homepage: www.modernscientificpress.com/journals/ijbiochem.aspx ISSN: 2169-0928
More informationExperiment 1. Isolation of Glycogen from rat Liver
Experiment 1 Isolation of Glycogen from rat Liver Figure 35: FIG-2, Liver, PAS, 100x. Note the presence of a few scattered glycogen granules (GG). Objective To illustrate the method for isolating glycogen.
More informationTHE RELATIONSHIP BETWEEN TWO METHODS FOR EVALUATING FIVE-CARBON SUGARS IN EUCALYPTUS EXTRACTION LIQUOR
THE RELATIONSHIP BETWEEN TWO METHODS FOR EVALUATING FIVE-CARBON SUGARS IN EUCALYPTUS EXTRACTION LIQUOR Congcong Chi, a,b* Zeng Zhang, a Weiwei Ge, a and Hasan Jameel b Alkaline pre-extraction and hydrothermal
More informationConsequently, lipoprotein fractions have been analyzed
THE PHOSPHOLIPID COMPOSITION OF HUMAN SERUM LIPOPROTEIN FRACTIONS SEPARATED BY ULTRACENTRIFUGATION * BY GERALD B. PHILLIPS (From the Departments of Biochemistry and Medicine, College of Physicians and
More informationB. 1% (w/v) Salicin Substrate Solution (Salicin) (Prepare 50 ml in Reagent A using Salicin, Sigma Prod. No. S-0625.)
SIGMA QUALITY CONTROL TEST PROCEDURE (Q]\PDWLFÃ$VVD\ÃRIÃ */8&26,'$6( PRINCIPLE: 'Glucoside + H 2 O Glucosidase > D-Glucose + an Alcohol CONDITIONS: T = 37 C, ph = 5.0, A 540nm, Light path = 1 cm METHOD:
More informationInositol Phosphate Phosphatases of Microbiological Origin: the Inositol Pentaphosphate Products of Aspergillus ficuum
JOURNAL OF BACTERIOLOGY, OCt. 1972, p. 434-438 Copyright 1972 American Society for Microbiology Vol. 112, No. 1 Printed in U.S.A. Inositol Phosphate Phosphatases of Microbiological Origin: the Inositol
More informationGlycosaminoglycans, Proteoglycans, and Glycoproteins
Glycosaminoglycans, Proteoglycans, and Glycoproteins Presented by Dr. Mohammad Saadeh The requirements for the Pharmaceutical Biochemistry I Philadelphia University Faculty of pharmacy I. OVERVIEW OF GLYCOSAMINOGLYCANS
More informationKE-SIALIQ Sialic Acid Quantitation Kit. SialiQuant Sialic Acid Quantitation Kit
SialiQuant Sialic Acid Quantitation Kit Part Number KE-SIALIQ Certification of Analysis Lot Number 706.1A Kit Storage Kits should be stored at 4 C. Kit Contents Kit contains all the reagents to quickly
More informationΒ-FRUCTOFURANOSIDASE ENZYME
KINETICS ANALYSIS OF Β-FRUCTOFURANOSIDASE ENZYME 2-The effects of enzyme concentration on the rate of an enzyme catalyzed reaction. Systematic names and numbers β-fructofuranosidase (EC 3.2.1.26) Reactions
More informationAnalytical Method for 2, 4, 5-T (Targeted to Agricultural, Animal and Fishery Products)
Analytical Method for 2, 4, 5-T (Targeted to Agricultural, Animal and Fishery Products) The target compound to be determined is 2, 4, 5-T. 1. Instrument Liquid Chromatograph-tandem mass spectrometer (LC-MS/MS)
More informationAmino Acid Composition of Hypertensin II.-- EXPERIMENTAL
THE AMINO ACID COMPOSITION OF HYPERTENSIN II AND ITS BIOCHEMICAL RELATIONSHIP TO HYPERTENSIN I BY KENNETH E. LENTZ, PH.D., LEONARD T. SKEGGS, JR., PH.D., KENNETH R. WOODS, PH.D., JOSEPH R. KAHN, M.D.,
More informationTECHNICAL BULLETIN. Enzymatic Protein Deglycosylation Kit. Catalog Number EDEGLY Storage Temperature 2 8 C
Enzymatic Protein Deglycosylation Kit Catalog Number EDEGLY Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description The EDEGLY kit contains all the enzymes and reagents needed to completely remove
More informationEnzymatic Assay of ß-GLUCOSIDASE (EC )
PRINCIPLE: ß-D-Glucoside + H 2 O ß-Glucosidase > D-Glucose + an Alcohol CONDITIONS: T = 37 C, ph = 5.0, A 540nm, Light path = 1 cm METHOD: Colorimetric 1 REAGENTS: A. 100 mm Sodium Acetate Buffer, ph 5.0
More informationON THE NATURE OF THE TRANSALDOLASE-DIHYDROXYACETONE
VOL. 47, 1961 BIOCHEMISTRY: HORECKER ET AL. 1949 3 Bonsignore, A., S. Pontremoli, E. Grazi, and M. Mangiarotti, Biochem. Biophys. Research Communs., 1, 79 (1959). 4 Venkataraman, R., and E. Racker, J.
More informationN-Glycosidase F Deglycosylation Kit
For life science research only. Not for use in diagnostic procedures. FOR IN VITRO USE ONLY. N-Glycosidase F Deglycosylation Kit Kit for the deglycosylation of asparagine-linked glycan chains on glycoproteins.
More informationEnzymatic Assay of POLYGALACTURONASE (EC )
PRINCIPLE: Polygalacturonic Acid + H 2 O PG > Reducing Sugars Abbreviations: PG = Polygalacturonase CONDITIONS: T = 30 C, ph 5.0, A 540nm, Light path = 1 cm METHOD: Colorimetric REAGENTS: A. 50 mm Sodium
More information130327SCH4U_biochem April 09, 2013
Option B: B1.1 ENERGY Human Biochemistry If more energy is taken in from food than is used up, weight gain will follow. Similarly if more energy is used than we supply our body with, weight loss will occur.
More informationNo Water-soluble Carbohydrates of Zizyphi Fructus. II.1 Isolation of Two Polysaccharides and Structure of an Arabinan
No.4 707 Chem. Pharm. Bull. 21(4) 707-711 (1973) UDC 547. 458. 02. 05 : 581. 192 : 615. 322. 011. 5 Water-soluble Carbohydrates of Zizyphi Fructus. II.1 Isolation of Two Polysaccharides and Structure of
More informationQuestions- Carbohydrates. A. The following structure is D-sorbose. (Questions 1 7) CH 2 OH C = O H C OH HO C H H C OH
Questions- Carbohydrates A. The following structure is D-sorbose. (Questions 1 7) CH 2 C = O H C HO C H H C CH 2 1. 2. 3. 4. 5. Which characteristic is different when comparing the open-chain forms of
More informationA STUDY OF THE METABOLISM OF THEOBROMINE, THEOPHYLLINE, AND CAFFEINE IN MAN* Previous studies (1, 2) have shown that after the ingestion of caffeine
A STUDY OF THE METABOLISM OF THEOBROMINE, THEOPHYLLINE, AND CAFFEINE IN MAN* BY HERBERT H. CORNISH AND A. A. CHRISTMAN (From the Department of Biological Chemistry, Medical School, University of Michigan,
More informationRAFFINOSE/ SUCROSE/ GLUCOSE
www.megazyme.com RAFFINOSE/ SUCROSE/ GLUCOSE ASSAY PROCEDURE K-RAFGL 06/5 (20 Assays per Kit) Megazyme International Ireland 205 INTRODUCTION: Grain legumes are an important component of both human and
More informationEnzyme Derived Pig Livers. By Shoei ISEKI and Haruko YAMAMOTO Department of Legal Medicine, School of Medicine, Gunma University, Maebashi
No. 4] Proc. Japan Acad., 44 (1968) 269 62. A-Decomposing Human and Enzyme Derived Pig Livers from By Shoei ISEKI and Haruko YAMAMOTO Department of Legal Medicine, School of Medicine, Gunma University,
More informationThe Occurrence of Repetitive Glycopeptide Sequences in Bovine Submaxillary Glycoprotein
Eur. J. Biochem. 32,4854 (973) The Occurrence of Repetitive Glycopeptide Sequences in Bovine Submaxillary Glycoprotein Ward PIGMAN, John MOSCHERA, Michael WEISS, and Guido TETTAMANTI Biochemistry Department,
More informationOligosaccharide Analysis by High-Performance Anion- Exchange Chromatography with Pulsed Amperometric Detection
Oligosaccharide Analysis by High-Performance Anion- Exchange Chromatography with Pulsed Amperometric Detection Jeff Rohrer, Ph.D. Director, Applications Development, Dionex Products 1 The world leader
More informationfertilization, we have assayed a series of normal
SIALIC ACID IN HUMAN SEMEN AND IN THE MALE GENITAL TRACT BY LEONARD WARREN (From the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service,
More informationBiosynthesis of N and O Glycans
TechNote #TNGL101 Biosynthesis of N and O Glycans These suggestions and data are based on information we believe to be reliable. They are offered in good faith, but without guarantee, as conditions and
More informationStudies on the Glucanase of Sclerotinia libertiana. EBATA and Yukio SATOMURA
Studies on the Glucanase of Sclerotinia libertiana By Junko EBATA and Yukio SATOMURA Faculty of Science, Osaka City University, Osaka Received December 13, 1962 The digestion of yeast cells with the glucanase
More informationBiochemistry: A Short Course
Tymoczko Berg Stryer Biochemistry: A Short Course Second Edition CHAPTER 10 Carbohydrates 2013 W. H. Freeman and Company Chapter 10 Outline Monosaccharides are aldehydes or ketones that contain two or
More informationLudgerPure TM APTS Labelled IgG Glycan Library
Certificate of Analysis LudgerPure TM APTS Labelled IgG Glycan Library Cat. #: CAPTS-IgG-0 Batch #. B-0 Size: approx. 0 pmol Description and: Source A mixture of APTS labelled fucosylated bi-antennary
More informationFor example, monosaccharides such as glucose are polar and soluble in water, whereas lipids are nonpolar and insoluble in water.
Biology 4A Laboratory Biologically Important Molecules Objectives Perform tests to detect the presence of carbohydrates, lipids, proteins, and nucleic acids Recognize the importance of a control in a biochemical
More informationHeparin Sodium ヘパリンナトリウム
Heparin Sodium ヘパリンナトリウム Add the following next to Description: Identification Dissolve 1 mg each of Heparin Sodium and Heparin Sodium Reference Standard for physicochemical test in 1 ml of water, and
More informationPurification and Some Properties of Milk-clotting Enzyme from Aspergillus niger
J. gen. Microbiol. (1969), 59, 131-135 Printed in Great Britain Purification and Some Properties of Milk-clotting Enzyme from Aspergillus niger By H. G. OSMAN, A. F. ABDEL-FATTAH AND SOUHAIR S. MABROUK
More informationEnzymatic Assay of PROTEASE (EC )
Enzymatic Assay of PROTEASE PRINCIPLE: Hemoglobin + H 2 O Protease > Amino Acids CONDITIONS: T = 37 C, ph = 2.8, A 660nm, Light path = 1 cm METHOD: Colorimetric REAGENTS: A. 50 mm Potassium Phthalate Buffer,
More informationStructures of the Oligosaccharide Chains in Swine Trachea Mucin Glycoproteins*
THE JOURNAL OF BOLOGCAL CHEMSTRY 0 1984 by The American Society of Biological Chemists, nc Vol. 259, No. ssue Structures of the Oligosaccharide Chains in Swine Trachea Mucin Glycoproteins* (Received for
More informationBlotGlyco O-GLYCAN SAMPLE PREPARATION KIT BS-45450Z
BlotGlyco O-GLYCAN SAMPLE PREPARATION KIT BS-45450Z CONTENTS Introduction... 3 Safety Information... 4 Notice... 4 Kit Components... 5 Required Equipment, Labware, and Reagents... 6 Reagents for BlotGlyco
More informationBiochemical Studies on Carbohydrates. Hajime Masamune, Zensaku Yosizawa
The Tohoku Journal of Experimental Medicine, Vol. 65, Nos. 2-3, 1957 Biochemical Studies on Carbohydrates CXCVI. Comparison among Blood Group A, B and 0 Mucopolysaccharides from Human Gastric Juices By
More informationExperiment 3: Activity Determination
Experiment 3: Activity Determination Introduction: Specific activity is a method for measuring enzymatic activity and the enzyme purity in a mixture. In order to determine the specific activity of an enzyme,
More informationThe addition of sugar moiety determines the blood group
The addition of sugar moiety determines the blood group Sugars attached to glycoproteins and glycolipids on the surfaces of red blood cells determine the blood group termed A, B, and O. The A and B antigens
More informationGlycosaminoglycans: Anionic polysaccharide chains made of repeating disaccharide units
Glycosaminoglycans: Anionic polysaccharide chains made of repeating disaccharide units Glycosaminoglycans present on the animal cell surface and in the extracellular matrix. Glycoseaminoglycans (mucopolysaccharides)
More informationAbdullah zurayqat. Bahaa Najjar. Mamoun Ahram
9 Abdullah zurayqat Bahaa Najjar Mamoun Ahram Polysaccharides Polysaccharides Definition and Structure [Greek poly = many; sacchar = sugar] are complex carbohydrates, composed of 10 to up to several thousand
More informationSupplementary Data: Monosaccharide Composition and Linkage Analysis of RPS
Supplementary Data: Monosaccharide Composition and Linkage Analysis of RPS Methods: Glycosyl composition analysis was done by gas chromatography-mass spectrometry (GC-MS) of the monosaccharide alditol
More informationSUPPLEMENTARY MATERIAL
SUPPLEMENTARY MATERIAL Purification and biochemical properties of SDS-stable low molecular weight alkaline serine protease from Citrullus Colocynthis Muhammad Bashir Khan, 1,3 Hidayatullah khan, 2 Muhammad
More informationMammalian-type Glycosylation l in LEXSY
Mammalian-type Glycosylation l in LEXSY Case Study: Recombinant hu Erythropoietin Jena Bioscience GmbH Loebstedter Str. 80 07749 Jena, Germany Tel.: +49-3641-628-5000 Fax: +49-3641-628-5100 628 e-mail:
More informationOligosaccharide Profiling of O-linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2-AA)
Oligosaccharide Profiling of O-linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2-AA) Elisabeth A. Kast and Elizabeth A. Higgins GlycoSolutions Corporation, Worcester, MA Data originally presented
More informationCaution: For Laboratory Use. A product for research purposes only. Eu-W1024 ITC Chelate & Europium Standard. Product Number: AD0013
TECHNICAL DATA SHEET Lance Caution: For Laboratory Use. A product for research purposes only. Eu-W1024 ITC Chelate & Europium Standard Product Number: AD0013 INTRODUCTION: Fluorescent isothiocyanato-activated
More informationCERTIFICATE OF ANALYSIS
CERTIFICATE OF ANALYSIS PRODUCT NAME: PRODUCT CODE: LOT NUMBER: PACK SIZE: PURITY: FORM: STORAGE: EXPIRATION: GLYKO ASIALO, GALACTOSYLATED, BIANTENNARY COMPLEX N-GLYCAN, CORE-SUBSTITUTED WITH FUCOSE (NA2F)
More informationIntroduction to Biochemistry Midterm exam )ومن أحياها(
Introduction to Biochemistry Midterm exam 2016-2017 )ومن أحياها( 1. Which of the following amino (in a peptide chain) would probably be found at a beta bend or turn? a. lysine * b. Gly c. arg d. asn 2.
More informationDELFIA Tb-N1 DTA Chelate & Terbium Standard
AD0029P-1 (en) 1 DELFIA Tb-N1 DTA Chelate & AD0012 Terbium Standard For Research Use Only INTRODUCTION DELFIA Tb-N1 DTA Chelate is optimized for the terbium labeling of proteins and peptides for use in
More informationBiochemical Techniques 06 Salt Fractionation of Proteins. Biochemistry
. 1 Description of Module Subject Name Paper Name 12 Module Name/Title 2 1. Objectives Understanding the concept of protein fractionation Understanding protein fractionation with salt 2. Concept Map 3.
More informationThe Oligosaccharide Moieties of the Epidermal Growth Factor Receptor in A-431 Cells
~ THE JOURNAL OF BIOLOGICAL CHEMlSTRV 1985 by The American Society of Biological Chemists, Inc. Vol. 26, No. 22, Issue of October 5, pp, 11944-11952 1985 Printed in ~s.a. The Oligosaccharide Moieties of
More information1 Preparation and Characterization of Lignin-Carbohydrate Complexes
1 Preparation and Characterization of Lignin-Carbohydrate Complexes To explain the difficulty in separating lignin from carbohydrates in wood, Erdman (1866) hypothesized that the two combined chemically
More informationDELFIA Eu-DTPA ITC Chelate & Europium Standard
AD0026P-3 (en) 1 DELFIA Eu-DTPA ITC Chelate & AD0021 Europium Standard For Research Use Only INTRODUCTION DELFIA Eu-DTPA ITC Chelate is optimized for the europium labelling of proteins and peptides for
More informationLANCE Eu-W1024 ITC Chelate & Europium Standard AD0013 Development grade
AD0017P-4 (en) 1 LANCE Eu-W1024 ITC Chelate & Europium Standard AD0013 Development grade INTRODUCTION Fluorescent isothiocyanato-activated (ITC-activated) Eu-W1024 chelate is optimized for labelling proteins
More informationThe Role of a Dolichol-Oligosaccharide as an Intermediate in Glycoprotein Biosynthesis
University of South Carolina Scholar Commons Faculty Publications Chemistry and Biochemistry, Department of 6-1974 The Role of a Dolichol-Oligosaccharide as an Intermediate in Glycoprotein Biosynthesis
More informationEXPERIMENT 4 DETERMINATION OF REDUCING SUGARS, TOTAL REDUCING SUGARS, SUCROSE AND STARCH
Practical Manual Food Chemistry and Physiology EXPERIMENT 4 DETERMINATION OF REDUCING SUGARS, TOTAL REDUCING SUGARS, SUCROSE AND STARCH Structure 4.1 Introduction Objectives 4.2 Experiment 4a: Reducing
More informationStudy of On-Resin Convergent Synthesis of N-Linked. Glycopeptides Containing a Large High Mannose N- Linked Oligosaccharide
Supporting Information Study of On-Resin Convergent Synthesis of N-Linked Glycopeptides Containing a Large High Mannose N- Linked Oligosaccharide Rui Chen and Thomas J. Tolbert* Department of Chemistry,
More informationRAPID SAMPLE PREPARATION METHODS FOR THE ANALYSIS OF N-LINKED GLYCANS
RAPID SAMPLE PREPARATION METHODS FOR THE ANALYSIS OF N-LINKED GLYCANS Zoltan Szabo, András Guttman, Tomas Rejtar and Barry L. Karger Barnett Institute, Boston, MA, USA PCT Workshop,Boston, 21 May, 2010.
More informationGLYCOGEN BEFORE THE LAB YOU HAVE TO READ ABOUT:
GLYCGEN BEFRE THE LAB YU HAVE T READ ABUT:. Glycogen structure. 2. Glycogen synthesis and degradation (reactions with structural formulas and enzymes). 3. The role of glycogen in liver and muscles. INTRDUCTIN
More informationChemical characterization of neutral glycolipids in the human myeloid leukemias1
Chemical characterization of neutral glycolipids in the human myeloid leukemias1 John C. Klock, Jay L. D Angona, and Bruce A. Macher Cancer Research Institute and the Department of Medicine,* and Department
More informationCaution: For Laboratory Use. A product for research purposes only. Eu-W1284 Iodoacetamido Chelate & Europium Standard. Product Number: AD0014
TECHNICAL DATA SHEET Lance Caution: For Laboratory Use. A product for research purposes only. Eu-W1284 Iodoacetamido Chelate & Europium Standard Product Number: AD0014 INTRODUCTION: Iodoacetamido-activated
More informationON THE DIFFERENCE IN ADSORPTION ON SEPHADEX GEL OF THE DEXTRANSUCRASE OF STREPTOCOCCUS BOVIS GROWN ON SUCROSE AND GLUCOSE MEDIA
J. Gen. App!. Microbiol., 34, 213-219 (1988) ON THE DIFFERENCE IN ADSORPTION ON SEPHADEX GEL OF THE DEXTRANSUCRASE OF STREPTOCOCCUS BOVIS GROWN ON SUCROSE AND GLUCOSE MEDIA TOSHIRO HAYASHI, RYO IOROI,*
More informationTRACP & ALP Assay Kit
Cat. # MK301 For Research Use TRACP & ALP Assay Kit Product Manual Table of Contents I. Description...3 II. III. IV. Introduction...3 Components...4 Materials Required but not Provided...4 V. Storage...4
More informationSubstrate Specificity and Salt Inhibition of Five Proteinases Isolated from the Pyloric Caeca and Stomach of Sardine
Agric. Biol. Chem., 46 (6), 1565~1569, 1982 1565 Substrate Specificity and Salt Inhibition of Five Proteinases Isolated from the Pyloric Caeca and Stomach of Sardine Minoru Noda, Thanh Vo Van, Isao Kusakabe
More informationMechanism of Salivary Amylase Action
Proceedings of the Iowa Academy of Science Volume 57 Annual Issue Article 22 1950 Mechanism of Salivary Amylase Action John H. Pazur Iowa State College Dexter French Iowa State College Doris W. Knapp Iowa
More informationA COLORIMETRIC METHOD FOR THE ESTIMATION OF SERUM GLYCATED PROTEINS BASED ON DIFFERENTIAL REDUCTION OF FREE AND BOUND GLUCOSE BY SODIUM BOROHYDRIDE
CHAPTER V A COLORIMETRIC METHOD FOR THE ESTIMATION OF SERUM GLYCATED PROTEINS BASED ON DIFFERENTIAL REDUCTION OF FREE AND BOUND GLUCOSE BY SODIUM BOROHYDRIDE 132 INTRODUCTION The measurement of glycated
More informationCertificate of Analysis
Certificate of Analysis Human IgG Glycoprotein Standard Cat. #: GCP-IGG-50U Batch: B13T-06 Nominal size: 50μg Expiry: Dec 2020 Description: A glycoprotein standard for use during glycan release and labeling.
More information-can be classified by the number of sugars that constitute the molecules: -how to differentiate between glucose and galactose?
Carbohydrates (Also called: saccharides) -can be classified by the number of sugars that constitute the molecules: 1- monosaccharides: -General formula: (CH2O)n -Contain one sugar molecule -Contain two
More informationStudent Handout. This experiment allows you to explore the properties of chiral molecules. You have
Student Handout This experiment allows you to explore the properties of chiral molecules. You have learned that some compounds exist as enantiomers non-identical mirror images, such as your left and right
More informationDetection and Quantification of Alpha-Galactosidase (α-gal) Activity in Tissue extracts, Cell lysate, Cell culture media and Other
Alpha-Galactosidase Microplate Assay Kit User Manual Catalog # MBS8243211 Detection and Quantification of Alpha-Galactosidase (α-gal) Activity in Tissue extracts, Cell lysate, Cell culture media and Other
More informationTLC SEPARATION OF AMINO ACIDS
TLC SEPARATION OF AMINO ACIDS LAB CHROM 7 Adapted from Laboratory Experiments for Organic and Biochemistry. Bettelheim & Landesberg (PA Standards for Sci & Tech 3.1.12.D; 3.4.10.A; 3.7.12.B) INTRODUCTION
More informationSTREPTOCOCCAL L FORMS
STREPTOCOCCAL L FORMS II. CHEMICAL COMPOSITION' CHARLES PANOS, S. S. BARKULIS, AND J. A. HAYASHI Department of Biological Chemistry, University of Illinois College of Medicine, Chicago, Illinois Received
More informationWork-flow: protein sample preparation Precipitation methods Removal of interfering substances Specific examples:
Dr. Sanjeeva Srivastava IIT Bombay Work-flow: protein sample preparation Precipitation methods Removal of interfering substances Specific examples: Sample preparation for serum proteome analysis Sample
More informationSialic Acid Assay Kit
Sialic Acid Assay Kit Catalog Number KA1655 100 assays Version: 04 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use... 3 Background... 3 Principle of the Assay...
More informationSYNOPSIS STUDIES ON THE PREPARATION AND CHARACTERISATION OF PROTEIN HYDROLYSATES FROM GROUNDNUT AND SOYBEAN ISOLATES
1 SYNOPSIS STUDIES ON THE PREPARATION AND CHARACTERISATION OF PROTEIN HYDROLYSATES FROM GROUNDNUT AND SOYBEAN ISOLATES Proteins are important in food processing and food product development, as they are
More informationSeparation of Saccharides Using TSKgel Amide-80, a Packing Material for High-performance Normal Phase Partition Chromatography (2) Table of Contents
No. 079 SEPARATION REPORT Separation of Saccharides Using TSKgel Amide-80, a Packing Material for High-performance Normal Phase Partition Chromatography (2) Table of Contents 1. Introduction 1 2. Comparison
More informationEuropium Labeling Kit
Europium Labeling Kit Catalog Number KA2096 100ug *1 Version: 03 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use... 3 Background... 3 Principle of the Assay...
More informationGlycoprotein monosaccharide analysis using HPAE-PAD with manually prepared eluent
TECHNICAL NOTE 75 Glycoprotein monosaccharide analysis using HPAE-PAD with manually prepared eluent Authors Sachin Patil and Jeffrey Rohrer, Thermo Fisher Scientific, Sunnyvale, CA, USA Keywords Glycoprotein,
More informationTECHNICAL BULLETIN. R 2 GlcNAcβ1 4GlcNAcβ1 Asn
GlycoProfile II Enzymatic In-Solution N-Deglycosylation Kit Product Code PP0201 Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description Glycosylation is one of the most common posttranslational
More informationARABINAN
www.megazyme.com ARABINAN ASSAY PROCEDURE K-ARAB 08/18 (100 Assays per Kit) Megazyme 2018 INTRODUCTION: In the processing of apples and pears, the yield of juice can be dramatically improved by using enzymes
More informationChapter PURIFICATION OF ALKALINE PROTEASES
Chapter PURIFICATION OF ALKALINE PROTEASES E /xtracellular alkaline proteases produced by Bacillus sp. K 25 and bacillus pumilus K 242, were purified and the homogeneity was examined by electrophoresis.
More informationFigure 2. Figure 1. Name: Bio AP Lab Organic Molecules
Name: Bio AP Lab Organic Molecules BACKGROUND: A cell is a living chemistry laboratory in which most functions take the form of interactions between organic molecules. Most organic molecules found in living
More informationPURIFICATION OF THE TOXIN IN A ZOAN PALYTHOA TUBERCULOSA.
Title PURIFICATION OF THE TOXIN IN A ZOAN PALYTHOA TUBERCULOSA Author(s) Kimura, Shoji; Hashimoto, Yoshiro Citation PUBLICATIONS OF THE SETO MARINE BIO LABORATORY (1973), 20: 713-718 Issue Date 1973-12-19
More informationGlycanPac AXR-1 Columns
CHRMATGRAPHY GlycanPac AXR- Columns For High Resolution Glycan Analysis Product Specifications The Thermo Scientific GlycanPac AXR- columns are highperformance, silica-based HPLC columns for simultaneous
More informationCLASS 11th. Biomolecules
CLASS 11th 01. Carbohydrates These are the compound of carbon, hydrogen and oxygen having hydrogen and oxygen in the same ratio as that of water, i.e. 2 : 1. They are among the most widely distributed
More informationQualitative test of protein-lab2
1- Qualitative chemical reactions of amino acid protein functional groups: Certain functional groups in proteins can react to produce characteristically colored products. The color intensity of the product
More informationSequential Extraction of Plant Metabolites
ISSN: 2319-7706 Volume 4 Number 2 (2015) pp. 33-38 http://www.ijcmas.com Original Research Article Sequential Extraction of Plant Metabolites Shankar L. Laware* PG. Department of Botany, Fergusson College
More information2. Which of the following amino acids is most likely to be found on the outer surface of a properly folded protein?
Name: WHITE Student Number: Answer the following questions on the computer scoring sheet. 1 mark each 1. Which of the following amino acids would have the highest relative mobility R f in normal thin layer
More information