COMMUNICATION
/. Biochem. 82, 1785-1788 (1977)
from Normal Human Urine1 Masai KOSEKI and Koichi TSURUMI Department of Biochemistry, Fukushima Medical College, Fukushima, Fukushima 960 Received for publication, October 5, 1977
A simple method for the preparation of 3'-sialyllactose from normal human urine is described. The method for the collection of sialooligosaccharides is based on adsorption on charcoal followed by elution with a mixture of ethanol, pyridine, and water. The sialooligosaccharide mixture is then fractionated by means of gel filtration on Sephadex G-25 and the low molecular weight fraction is further fractionated on Dowex 1 (eluted with pyridinium acetate) to give five fractions. The last fraction contains almost pure 3'-sialyllactose.
As today there is widespread interest in studies of sialidases in animal tissues and in microorganisms, especially studies of the enzymology of sialidases and of sialidase-producing viruses, good substrates for these enzymes are being sought continuously. 3'-Sialyllactose which has been isolated only from bovine colostrum (1-3), has been used as one of these substrates. However, it is not easy to obtain a colostrum sample which can be used as the source of 3'-sialyllactose. In our laboratory, it has been observed that normal human urine was one of the good sources of 3'-sialyllactose (4). The present communication describes a convenient and economical method for the isolation of 3'-sialyllactose from normal human urine. About 5 liters of normal human urine was added to a column (5 cm x 60 cm) of activated granular charcoal (Wako Pure Chemicals Indus1
This work was supported in part by a research grant from the Ministry of Education, Science and Culture of Japan. Vol. 82, No. 6, 1977
1785
tries, Ltd., Osajca, Japan). After washing the column with 3 liters of distilled water, sialic acidcontaining materials in the human urine were eluted with 5 liters of a mixture of ethanol, pyridine, and distilled water (2 : 1>: 2, by vol.). The eluate was concentrated to dryness under reduced pressure, and the residue was extracted with three 50 ml portions of distilled water. The combined extract from 16 liters of human urine was applied to a column (2.5 cm x 30 cm) of Dowex 50 (hydrogen form, 200-400 mesh) connected to a column (2.5 cm x40 cm) of Dowex 1 x 2 (acetate form, 2CKM00 mesh). After washing the connected columns with 500 ml of distilled water, the Dowex 50 column was removed, and the Dowex 1 column was washed further with 300 ml of distilled water. The acidic fraction adsorbed on the Dowex 1 column was eluted with 700 ml of 1.0 M acetic acid (adjusted to pH 5.3 with pyridine). The eluate was concentrated to dryness, and the residue was extracted three times with 20 ml of distilled water. The extracts were combined, and a 5 ml aliquot of this solution was subjected to gel filtra-
Downloaded from https://academic.oup.com/jb/article-abstract/82/6/1785/784170 by University of Winnipeg user on 18 January 2019
A Convenient Method for the Isolation of 3'-Sialyllactose
1786
COMMUNICATION
4.0
was applied to a column (1.5 cm x 78 cm) of Dowex 1x2 (acetate form, 200-400 mesh), and the column was washed with 300 ml of distilled water. Then sialic acid-containing oligosaccharides were eluted carefully with 0.082 M pyridine adjusted to pH 4.9 with glacial acetic acid (the light absorbancy of this solution at 225 nm was 352) at a flow-rate of 10 ml per hour. 10 ml fractions were collected, and the sialic acid concentration of the fractions was determined by the orcinol method of Bohm (6). As shown in Fig. 2 sialic acid-containing oligosaccharides were fractionated into five fractions (D,, D,, D3, D4, and D6). Each fraction was separately combined, and sialic acid-containing oligosaccharides were recovered in the same manner as mentioned above. The oligosaccharide composition of these fractions was examined by thinlayer chromatography. Figure 3-A shows the chromatograms of fractions D,, D4, and D,. The results show that a reducing oligosaccharide which ran together with the authentic 3'-sialyllactose was the sole component of fraction D5 (see Fig. 3-A). Figure 3-B shows the thin-layer chromatograms of the neutral oligosaccharides which were obtained by mild acid hydrolysis of fractions D5, D4, and D 3 (hydrolyzed at 80°C for
32
4.0
o.e
10
s.
20 30 Tube Number (lOml / Tube)
4O
Fig. 1. Sephadex G-25 gel filtration of sialic acidcontaining materials obtained from normal human urine. Details of the procedures are given in the text. » Warren's method is recommended for the determination of urinary sialic acid, rather than the orcinol method of Bohm (6), since urinary pigments and uronides strongly interfere with the latter method.
10
20 30 40 50 60 Tube Number(lOral/Tuba)
70
Fig. 2. Dowex 1 x2 column chromatography of the sialooligosaccharide fraction, SG-2, in Fig. 1 obtained from normal human urine. Details of the procedures are given in the text. / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/82/6/1785/784170 by University of Winnipeg user on 18 January 2019
tion on a Sephadex G-25 (fine) column (2.5 cm X 90 cm) using 0.1 M acetic acid (adjusted to pH 5.3 with pyridine) as an eluent. The sialic acid concentrations of the fractions (10 ml per tube) were determined by the thiobarbituric acid method (5)* of Warren. As shown in Fig. 1, sialic acidcontaining materials in human urine were fractionated roughly into two fractions (SG-1 and SG-2). Chemical analysis of these fractions revealed that the former fraction was a mixture of many kinds of sialoglycopeptides, and the latter a mixture of several kinds of sialooligosaccharides. The latter fractions, SG-2, were combined and concentrated to dryness. The dried residue was dissolved in a small volume of aqueous methanol, and sialic acid-containing oligosaccharides were precipitated by the careful addition of ethanol and ether (about 920 mg of dry amorphous powder was obtained from 16 liters of human urine). The aqueous solution containing 450 mg of SG-2
ISOLATION OF 3-SIALYLLACTOSE FROM HUMAN URINE
1787
Sialic acid (mmol/100 g) (as NeuAc) Total hexose (mmol/100 g) (as lactose) Periodate consumption (mol/mol)
1 Di D, D, S,
Si D, tX, Do S»
.... i S
/. Biochem. 82, 1785-1788 (1977)
from Normal Human Urine1 Masai KOSEKI and Koichi TSURUMI Department of Biochemistry, Fukushima Medical College, Fukushima, Fukushima 960 Received for publication, October 5, 1977
A simple method for the preparation of 3'-sialyllactose from normal human urine is described. The method for the collection of sialooligosaccharides is based on adsorption on charcoal followed by elution with a mixture of ethanol, pyridine, and water. The sialooligosaccharide mixture is then fractionated by means of gel filtration on Sephadex G-25 and the low molecular weight fraction is further fractionated on Dowex 1 (eluted with pyridinium acetate) to give five fractions. The last fraction contains almost pure 3'-sialyllactose.
As today there is widespread interest in studies of sialidases in animal tissues and in microorganisms, especially studies of the enzymology of sialidases and of sialidase-producing viruses, good substrates for these enzymes are being sought continuously. 3'-Sialyllactose which has been isolated only from bovine colostrum (1-3), has been used as one of these substrates. However, it is not easy to obtain a colostrum sample which can be used as the source of 3'-sialyllactose. In our laboratory, it has been observed that normal human urine was one of the good sources of 3'-sialyllactose (4). The present communication describes a convenient and economical method for the isolation of 3'-sialyllactose from normal human urine. About 5 liters of normal human urine was added to a column (5 cm x 60 cm) of activated granular charcoal (Wako Pure Chemicals Indus1
This work was supported in part by a research grant from the Ministry of Education, Science and Culture of Japan. Vol. 82, No. 6, 1977
1785
tries, Ltd., Osajca, Japan). After washing the column with 3 liters of distilled water, sialic acidcontaining materials in the human urine were eluted with 5 liters of a mixture of ethanol, pyridine, and distilled water (2 : 1>: 2, by vol.). The eluate was concentrated to dryness under reduced pressure, and the residue was extracted with three 50 ml portions of distilled water. The combined extract from 16 liters of human urine was applied to a column (2.5 cm x 30 cm) of Dowex 50 (hydrogen form, 200-400 mesh) connected to a column (2.5 cm x40 cm) of Dowex 1 x 2 (acetate form, 2CKM00 mesh). After washing the connected columns with 500 ml of distilled water, the Dowex 50 column was removed, and the Dowex 1 column was washed further with 300 ml of distilled water. The acidic fraction adsorbed on the Dowex 1 column was eluted with 700 ml of 1.0 M acetic acid (adjusted to pH 5.3 with pyridine). The eluate was concentrated to dryness, and the residue was extracted three times with 20 ml of distilled water. The extracts were combined, and a 5 ml aliquot of this solution was subjected to gel filtra-
Downloaded from https://academic.oup.com/jb/article-abstract/82/6/1785/784170 by University of Winnipeg user on 18 January 2019
A Convenient Method for the Isolation of 3'-Sialyllactose
1786
COMMUNICATION
4.0
was applied to a column (1.5 cm x 78 cm) of Dowex 1x2 (acetate form, 200-400 mesh), and the column was washed with 300 ml of distilled water. Then sialic acid-containing oligosaccharides were eluted carefully with 0.082 M pyridine adjusted to pH 4.9 with glacial acetic acid (the light absorbancy of this solution at 225 nm was 352) at a flow-rate of 10 ml per hour. 10 ml fractions were collected, and the sialic acid concentration of the fractions was determined by the orcinol method of Bohm (6). As shown in Fig. 2 sialic acid-containing oligosaccharides were fractionated into five fractions (D,, D,, D3, D4, and D6). Each fraction was separately combined, and sialic acid-containing oligosaccharides were recovered in the same manner as mentioned above. The oligosaccharide composition of these fractions was examined by thinlayer chromatography. Figure 3-A shows the chromatograms of fractions D,, D4, and D,. The results show that a reducing oligosaccharide which ran together with the authentic 3'-sialyllactose was the sole component of fraction D5 (see Fig. 3-A). Figure 3-B shows the thin-layer chromatograms of the neutral oligosaccharides which were obtained by mild acid hydrolysis of fractions D5, D4, and D 3 (hydrolyzed at 80°C for
32
4.0
o.e
10
s.
20 30 Tube Number (lOml / Tube)
4O
Fig. 1. Sephadex G-25 gel filtration of sialic acidcontaining materials obtained from normal human urine. Details of the procedures are given in the text. » Warren's method is recommended for the determination of urinary sialic acid, rather than the orcinol method of Bohm (6), since urinary pigments and uronides strongly interfere with the latter method.
10
20 30 40 50 60 Tube Number(lOral/Tuba)
70
Fig. 2. Dowex 1 x2 column chromatography of the sialooligosaccharide fraction, SG-2, in Fig. 1 obtained from normal human urine. Details of the procedures are given in the text. / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/82/6/1785/784170 by University of Winnipeg user on 18 January 2019
tion on a Sephadex G-25 (fine) column (2.5 cm X 90 cm) using 0.1 M acetic acid (adjusted to pH 5.3 with pyridine) as an eluent. The sialic acid concentrations of the fractions (10 ml per tube) were determined by the thiobarbituric acid method (5)* of Warren. As shown in Fig. 1, sialic acidcontaining materials in human urine were fractionated roughly into two fractions (SG-1 and SG-2). Chemical analysis of these fractions revealed that the former fraction was a mixture of many kinds of sialoglycopeptides, and the latter a mixture of several kinds of sialooligosaccharides. The latter fractions, SG-2, were combined and concentrated to dryness. The dried residue was dissolved in a small volume of aqueous methanol, and sialic acid-containing oligosaccharides were precipitated by the careful addition of ethanol and ether (about 920 mg of dry amorphous powder was obtained from 16 liters of human urine). The aqueous solution containing 450 mg of SG-2
ISOLATION OF 3-SIALYLLACTOSE FROM HUMAN URINE
1787
Sialic acid (mmol/100 g) (as NeuAc) Total hexose (mmol/100 g) (as lactose) Periodate consumption (mol/mol)
1 Di D, D, S,
Si D, tX, Do S»
.... i S
