- Email: [email protected]
A modified procedure for the colorimetric, ultramicro determination of reducing sugars with the alkaline ferricyanide reagent
94
NOTES
REFERENCES 1 M. L. WOLFIXOM,A. THOMPSON, AND D. R. LINEBACK, J. Org. Chem., 27 (1962) 2563 ; M. L- WOLFROM, G. FR~ENKEL, D. R. LINEBACK, AND F. KOMITSKY, JR.,ibid., 29 (1964) 457; H. EL L(HADEh% M. L. WOLFROM, Z. M. EL SHAFEEI,AND S. H. EL ASHRY, Carbuhyd. Res., 4 (1967) 225. 2 T. PO~TERNAK, Biochem. Prep& 2 (1952) 57. 3 H. E. CARTER, C. BELINSKEY,R. K. CLARK. JR.. E. H. FLYNN, B. LYTLE, G. E. McCMuNo, AND M. ROBBINS, J. Biol. Chem., 174 (1948) 415. 4 S. J. ANGYALAND N.K. MATHJZTON, J. Chem. Sot., (1950)3349. 5 T. POSIFRNAK, Cyciitofs, Holden-Day, Inc., San Francisco, Calif., 1965, p. 159. 6 A. J. FATIADI AND H. S. ISBELL,Curbohyd. Res., 5 (1967) 302. 7 A. J. FATIADI,J. Res. NatI. Bur. Std., 71A (1967) 277. 8 B. COXON, H. J. JENNINGS,AND K. A. MCLAUCHLAN, Tetrahedron,23 (1967) 2395. 9 F. W. LICHTENTHALER,H. LEINERT,AND R.SNAUI, Chem.Ber., 100 (1967) 2383. 10 J. BUCKMGHAMAND R.D.GIJTHRIE, Chem. Common., (1967) 1241. 11 J. BUCKWGHAM AND R. D. GLWHRIE, Chem. Cummun., (1966) 781; J. Chem. SW. (C), (1967) 1700; A. HA~SNER AND R. CATSOIJLACOS,Chem. Commun., (1967) 121; Tetrahedron Lett.. (1967) 489. 12 L. CAGLIOTI, G. ROSINI, AND F. Rossr,J. Am. Chem. Sot., 86 (1967) 3865. Received January 5Lh, 1968) < Car6ohyd. Res., 7 (1968) 89-94
A modified procedure for the calorimetric. ultramicro determination of reducing sugars with the alkaline ferricyanide reagent
Oxidation in an alkaline ferricyanide solution’ is the basis for a convenient method commonIy employed for the quantitative assay of reducing sugars and of end groups in polysaccharides. A variety of procedures is available for measuring the extent of reduction of ferricyanide (see refs. 2-12 for selected procedures and applications). The sensitive, calorimetric method for the determination of ferrocyanide by formation of the Prussian Blue complex is often used in biochemical research8-12. This method, however, suffers from some disadvantages inherent in the colloidal nature of the colored complex; its stability in solution is highly dependent on ionic strength, pH, and the presence of other hydrocolloids8-‘0~12. An alternative method for the calorimetric determination of ferrocyanide formed as a result of the oxidation of a sugar is based on an indirect assay involving formation of ferrous ions and their subsequent interaction with a reagent for iron’3n14. Following this approach, conditions have now been defined for a sensitive assay of reducing sugars that uses 2,4,6-tripyridyl-s-triazine as the reagent for iron’s*‘6.
EXPERIMENTAL
Reagents. - 1. Potassium ferricyanide. This reagent (0.7 m&r) is made with 10 mu sodium hydroxide that contains 2% of sodium carbonate; kept in a brown bottle, it is stable for at least two weeks at room temperature. Curbohyd. Res., 7 (1968) 94-97
95
NOES
2. Ferric cizloride. This reagent (2 nm) is made with a solution containing citric acid and 2M sodium acetate in 5&racetic acid. It is advisable to dissolve the ferric salt in a small volume of the acid citrate-acetate solution, so as to provide amounts of reagents suflicient for only a day or two of use. Blank values tend to increase somewhat with older solutions. 3. 2,4,6-TrQyridyZ-s-triazine (TPTZ). This compound was obtained from G. F. Smith Chemical Co., P-0. Box 5906 Station D, Columbus, Ohio 43222. The reagent employed is 2.5 rnhf (0.78 mg per ml) in 3M acetic acid; it is stable for at least a week at room temperature. Procedure. - Reactions should be shielded from direct light. Aliquots (1.0 ml) of solutions containing 2 to 100 nmoIes of reducing sugar are placed in test tubes (1.5 x 12.5 cm). Reagent 1 (1.0 ml) is added, and the tubes are kept for 10 min in a boiling-water bath. Reagent 2 (I -0 ml) and reagent 3 (2.0 ml) are then added immediately, without cooling. The solutions are stirred vigorously with a Vibromixer and, after 5 min in the dark, the absorbance at 595 nm (A& of the Fe(TPTZ)z+ is read against a reagent blank: 0.2M
COMMENTS
The color produced is stable for at least 6 h when the mixture is kept in the dark, and also when it is diluted with several volumes of water. Exposure to light may cause a slow increase in the blank values, due to photoreduction of ferricyanide. A standard curve for the determination of D-glucose, and comparative vaIues for other sugars, are presented in Fig. I_ The molar extinction coefficient for D-ghCOSe obtained in the present procedure was 75,000 f3%, corresponding to 3.3 moIe equivalents of the violet Fe(TPTZ)$’ ( see Refs. 15 and 16). The reagents alone had A 595 0.15 to 0.25 for different batches of reagents and solutions; this value is probably attributable to presence of ferrous ions and other reducing contaminants in the reagents. If desired, most of these contaminations can be removed by prior reaction with TPTZ and extraction of the complex with nitrobenzene’5*‘6. Samples taken from biological solutions after deproteinization with trichloroacetic acid or perchloric acid can be assayed without difficulty. Mercaptans, Cuzt, Agt, CN-, NO-, or high concentrations of (ethylenediamine)tetraacetic acid may interfere with formation of color, but other ions commonly found in biological solutions do not affect the reaction15*i6. The violet Fe(TPTZ)g* complex can be extracted into organic solvents, a property which can be advantageous in the determination of reducing sugars and of end groups of polysaccharides in turbid solutions, in suspensions, or in solutions in nonpolar solvents. In general, the alkaline ferricyanide method for the determination of reducing sugars lacks specil?city, and is susceptibIe to interference if other reducing components are present in the solution. The present procedure for the assay of the resulting ferrocyanide affords a stable, soluble color; this property has obvious advantages over measurements based on the unstable, Prussian Blue colloid. The method also proCarhhyd.
Res., 7 (1968) 94-97
96
NOTES
vides one of the most sensitive assays available for the determination of reducing sugars. By further lessening the volumes of reagents used, amounts even smaller Id
1
Fig. 1. Standard curve for o-glucose. In comparison with D-gIucose (lOO%j, the relative molar yields of color with representative sugars were: D-fructose, 104; 2-deoxy-D-arabino-hexose, 80; D-xylose, 95; D-glucuronic acid, 100; 2-amino-2-deoxy-D-glucose, 135; maltose, 140; melibiose, 110; D-glucitol -z 0.1; methyl cr-D-glucoside, c 0.2; and sucrose, -z 0.2%. Development of the violet Fe(TPTiZ)f+ resulting from the oxidation of D-ghtcose, was conducted as described in the text, in a final volume of 5.0 ml. Measurements were made with a Gilford model 300 and a Cat-y 14 spectrophotometers. with cuvettes of 1.O-cm light-path.
2 nmoles of sugar can be assayed. However, at these low levels, control of the reproducibility and accuracy of color formation is much more difficult, owing to the relatively larger contribution of trace contaminants in the reagents. The use of reagent solutions prepurified “Jo by treatment with TPTZ is, under these circumstances, obligatory. than
ACKNOWLEDG-
I thank Dr. S. Englard for heIpfu1 discussions. This work was supported by Grant GM-04428 from the National Institutes of Health, United States Public Health Service. Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461 (U. S. A.)
GAD AVIGAD*
REFERENCES 1 J. G. GENTELE, Dinglers Polytech. L, 152 (1860) 68; Chem. Zentr., 2 H. C. HAGEDORN AND B. N. JENSEN, Biochem Z. 135 (1923) 46. *On leave from the Hebrew University, Jerusalem, Israel. Carbohyd. Res., 7 (1968j 94-97
30 (1859) 504.
97
NOTES 3 4 5 6 7 8 9 10 11 12 13 14 15 16
W. Z. HASSID, 2nd. Eng. Chem. Anal. Ed., 9 (1937) 228. R. L. WHISRER AND J. N. BEMILLER, Methods Carbohyd_ Chem., 1 (1962) 395. T. J. SCHOCH, Methods Carbohyd- Chem., 4 (1964) 64. T.E. FRIEDMAN,~. W. WEBER,AND N. F. WI-IT, AnaLBiochem.,4 (1962) 358. R. J. HENRY, Clinical Chemistry, Principles and Technics, Harper and Row, New York, 1964, p. 625. J. T. PARK AND M. J. JOHNSON, J. Bioi. Chem., 181 (1949) 149. G. T. A~HWELL, Methods Enzymol., 3 (1957) 86. R. I. MATELES, Natwe, 187 (1960) 241. R. G. SPIRO,Methods Enrymol., 8 (1966) 3. J. M. GHUYSEN, D. J-TIPPER.AND J. L.STROMINGER, Methods EnzymoI.,S (1966) 685. D. ~.KROGMANAND A.T.JAGENDORF, PIantPhysioi.,32 (1957) 373. M. AVRON AND N. SHAVIT,Anal. Biochem., 6 (1963) 549. D. F. COLLINS, H. DIEHL, AND E. F. Shrnw, Anal. Chem., 31 (1959) 1862. H. DIEHL, E. F. Shtrrn, L. MCBRIDE, AND R. CRYBERG, The Iron Reagents, G. F. Smith Chemical Co., Columbus, Ohio, 1965, p. 41.
(Received February 26th, 1968) Carbohyd. Res., 7 (1968) 94-97
Laminaran
of fshige
okamurai’
Previously, Handa and Nisizawa’ reported that the soluble laminaran isolated from ,?%&a bicydis Setchell was composed of J-(1 +3)- and B-(1+6)-linked D-glucose residues in an approximate ratio of 2: 1. This finding was mainly based on the isolation of a series of gentio- and laminari-oligosaccharides from a partial acid hydrolyzate of laminaran. Moreover, the /3-D-(1+6)-linked residues were assumed to be located near the center of the molecule on the basis of alkali degradation. The structure of Eisenia-laminaran was thus considerably different from the structures of laminarans of the genus Laminaria’, in spite of their belonging to the same family. In addition, no “insoluble” laminaran was found in Eisenia. From the determination of the amount of 2,3,4-tri-O-methyl-D-glucose obtained after hydrolysis of fully methylated laminaran and of the formaldehyde produced by the periodate oxidation following the Smith degradation procedure, it was found3 that the B-D-linked units form a straightchain structure which constitute about one-quart of the whole molecule. The absence of 2,4-di-O-methyl-D-glucose in the methylation products was also evidence for a nonbranched structure of this laminaran. In the present work, the laminaran of Ishige okamurai Yendo, which belongs to the Chondariales, was investigated in order to compare its structure with that of other laminarans; difFerences may be expected, since this alga belongs to a family far remote from the Laminariales. Ishige grows at an intertidal zone and is distributed from the middle to the southern part of Japan. Finely crushed dry fronds of Ishige (100 g) were extracted with about a IO-fold volume cf 0.09M hydrochloric acid for 2 h at room temperature by the countercurrent *Contribution No. Tokyo, Japan.
170 from the Shimoda
Marine Biological
Station, Tokyo
Kyoiku
University,
Carbohyd. Res., 7 (1968) 97-99
NOTES
REFERENCES 1 M. L. WOLFIXOM,A. THOMPSON, AND D. R. LINEBACK, J. Org. Chem., 27 (1962) 2563 ; M. L- WOLFROM, G. FR~ENKEL, D. R. LINEBACK, AND F. KOMITSKY, JR.,ibid., 29 (1964) 457; H. EL L(HADEh% M. L. WOLFROM, Z. M. EL SHAFEEI,AND S. H. EL ASHRY, Carbuhyd. Res., 4 (1967) 225. 2 T. PO~TERNAK, Biochem. Prep& 2 (1952) 57. 3 H. E. CARTER, C. BELINSKEY,R. K. CLARK. JR.. E. H. FLYNN, B. LYTLE, G. E. McCMuNo, AND M. ROBBINS, J. Biol. Chem., 174 (1948) 415. 4 S. J. ANGYALAND N.K. MATHJZTON, J. Chem. Sot., (1950)3349. 5 T. POSIFRNAK, Cyciitofs, Holden-Day, Inc., San Francisco, Calif., 1965, p. 159. 6 A. J. FATIADI AND H. S. ISBELL,Curbohyd. Res., 5 (1967) 302. 7 A. J. FATIADI,J. Res. NatI. Bur. Std., 71A (1967) 277. 8 B. COXON, H. J. JENNINGS,AND K. A. MCLAUCHLAN, Tetrahedron,23 (1967) 2395. 9 F. W. LICHTENTHALER,H. LEINERT,AND R.SNAUI, Chem.Ber., 100 (1967) 2383. 10 J. BUCKMGHAMAND R.D.GIJTHRIE, Chem. Common., (1967) 1241. 11 J. BUCKWGHAM AND R. D. GLWHRIE, Chem. Cummun., (1966) 781; J. Chem. SW. (C), (1967) 1700; A. HA~SNER AND R. CATSOIJLACOS,Chem. Commun., (1967) 121; Tetrahedron Lett.. (1967) 489. 12 L. CAGLIOTI, G. ROSINI, AND F. Rossr,J. Am. Chem. Sot., 86 (1967) 3865. Received January 5Lh, 1968) < Car6ohyd. Res., 7 (1968) 89-94
A modified procedure for the calorimetric. ultramicro determination of reducing sugars with the alkaline ferricyanide reagent
Oxidation in an alkaline ferricyanide solution’ is the basis for a convenient method commonIy employed for the quantitative assay of reducing sugars and of end groups in polysaccharides. A variety of procedures is available for measuring the extent of reduction of ferricyanide (see refs. 2-12 for selected procedures and applications). The sensitive, calorimetric method for the determination of ferrocyanide by formation of the Prussian Blue complex is often used in biochemical research8-12. This method, however, suffers from some disadvantages inherent in the colloidal nature of the colored complex; its stability in solution is highly dependent on ionic strength, pH, and the presence of other hydrocolloids8-‘0~12. An alternative method for the calorimetric determination of ferrocyanide formed as a result of the oxidation of a sugar is based on an indirect assay involving formation of ferrous ions and their subsequent interaction with a reagent for iron’3n14. Following this approach, conditions have now been defined for a sensitive assay of reducing sugars that uses 2,4,6-tripyridyl-s-triazine as the reagent for iron’s*‘6.
EXPERIMENTAL
Reagents. - 1. Potassium ferricyanide. This reagent (0.7 m&r) is made with 10 mu sodium hydroxide that contains 2% of sodium carbonate; kept in a brown bottle, it is stable for at least two weeks at room temperature. Curbohyd. Res., 7 (1968) 94-97
95
NOES
2. Ferric cizloride. This reagent (2 nm) is made with a solution containing citric acid and 2M sodium acetate in 5&racetic acid. It is advisable to dissolve the ferric salt in a small volume of the acid citrate-acetate solution, so as to provide amounts of reagents suflicient for only a day or two of use. Blank values tend to increase somewhat with older solutions. 3. 2,4,6-TrQyridyZ-s-triazine (TPTZ). This compound was obtained from G. F. Smith Chemical Co., P-0. Box 5906 Station D, Columbus, Ohio 43222. The reagent employed is 2.5 rnhf (0.78 mg per ml) in 3M acetic acid; it is stable for at least a week at room temperature. Procedure. - Reactions should be shielded from direct light. Aliquots (1.0 ml) of solutions containing 2 to 100 nmoIes of reducing sugar are placed in test tubes (1.5 x 12.5 cm). Reagent 1 (1.0 ml) is added, and the tubes are kept for 10 min in a boiling-water bath. Reagent 2 (I -0 ml) and reagent 3 (2.0 ml) are then added immediately, without cooling. The solutions are stirred vigorously with a Vibromixer and, after 5 min in the dark, the absorbance at 595 nm (A& of the Fe(TPTZ)z+ is read against a reagent blank: 0.2M
COMMENTS
The color produced is stable for at least 6 h when the mixture is kept in the dark, and also when it is diluted with several volumes of water. Exposure to light may cause a slow increase in the blank values, due to photoreduction of ferricyanide. A standard curve for the determination of D-glucose, and comparative vaIues for other sugars, are presented in Fig. I_ The molar extinction coefficient for D-ghCOSe obtained in the present procedure was 75,000 f3%, corresponding to 3.3 moIe equivalents of the violet Fe(TPTZ)$’ ( see Refs. 15 and 16). The reagents alone had A 595 0.15 to 0.25 for different batches of reagents and solutions; this value is probably attributable to presence of ferrous ions and other reducing contaminants in the reagents. If desired, most of these contaminations can be removed by prior reaction with TPTZ and extraction of the complex with nitrobenzene’5*‘6. Samples taken from biological solutions after deproteinization with trichloroacetic acid or perchloric acid can be assayed without difficulty. Mercaptans, Cuzt, Agt, CN-, NO-, or high concentrations of (ethylenediamine)tetraacetic acid may interfere with formation of color, but other ions commonly found in biological solutions do not affect the reaction15*i6. The violet Fe(TPTZ)g* complex can be extracted into organic solvents, a property which can be advantageous in the determination of reducing sugars and of end groups of polysaccharides in turbid solutions, in suspensions, or in solutions in nonpolar solvents. In general, the alkaline ferricyanide method for the determination of reducing sugars lacks specil?city, and is susceptibIe to interference if other reducing components are present in the solution. The present procedure for the assay of the resulting ferrocyanide affords a stable, soluble color; this property has obvious advantages over measurements based on the unstable, Prussian Blue colloid. The method also proCarhhyd.
Res., 7 (1968) 94-97
96
NOTES
vides one of the most sensitive assays available for the determination of reducing sugars. By further lessening the volumes of reagents used, amounts even smaller Id
1
Fig. 1. Standard curve for o-glucose. In comparison with D-gIucose (lOO%j, the relative molar yields of color with representative sugars were: D-fructose, 104; 2-deoxy-D-arabino-hexose, 80; D-xylose, 95; D-glucuronic acid, 100; 2-amino-2-deoxy-D-glucose, 135; maltose, 140; melibiose, 110; D-glucitol -z 0.1; methyl cr-D-glucoside, c 0.2; and sucrose, -z 0.2%. Development of the violet Fe(TPTiZ)f+ resulting from the oxidation of D-ghtcose, was conducted as described in the text, in a final volume of 5.0 ml. Measurements were made with a Gilford model 300 and a Cat-y 14 spectrophotometers. with cuvettes of 1.O-cm light-path.
2 nmoles of sugar can be assayed. However, at these low levels, control of the reproducibility and accuracy of color formation is much more difficult, owing to the relatively larger contribution of trace contaminants in the reagents. The use of reagent solutions prepurified “Jo by treatment with TPTZ is, under these circumstances, obligatory. than
ACKNOWLEDG-
I thank Dr. S. Englard for heIpfu1 discussions. This work was supported by Grant GM-04428 from the National Institutes of Health, United States Public Health Service. Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461 (U. S. A.)
GAD AVIGAD*
REFERENCES 1 J. G. GENTELE, Dinglers Polytech. L, 152 (1860) 68; Chem. Zentr., 2 H. C. HAGEDORN AND B. N. JENSEN, Biochem Z. 135 (1923) 46. *On leave from the Hebrew University, Jerusalem, Israel. Carbohyd. Res., 7 (1968j 94-97
30 (1859) 504.
97
NOTES 3 4 5 6 7 8 9 10 11 12 13 14 15 16
W. Z. HASSID, 2nd. Eng. Chem. Anal. Ed., 9 (1937) 228. R. L. WHISRER AND J. N. BEMILLER, Methods Carbohyd_ Chem., 1 (1962) 395. T. J. SCHOCH, Methods Carbohyd- Chem., 4 (1964) 64. T.E. FRIEDMAN,~. W. WEBER,AND N. F. WI-IT, AnaLBiochem.,4 (1962) 358. R. J. HENRY, Clinical Chemistry, Principles and Technics, Harper and Row, New York, 1964, p. 625. J. T. PARK AND M. J. JOHNSON, J. Bioi. Chem., 181 (1949) 149. G. T. A~HWELL, Methods Enzymol., 3 (1957) 86. R. I. MATELES, Natwe, 187 (1960) 241. R. G. SPIRO,Methods Enrymol., 8 (1966) 3. J. M. GHUYSEN, D. J-TIPPER.AND J. L.STROMINGER, Methods EnzymoI.,S (1966) 685. D. ~.KROGMANAND A.T.JAGENDORF, PIantPhysioi.,32 (1957) 373. M. AVRON AND N. SHAVIT,Anal. Biochem., 6 (1963) 549. D. F. COLLINS, H. DIEHL, AND E. F. Shrnw, Anal. Chem., 31 (1959) 1862. H. DIEHL, E. F. Shtrrn, L. MCBRIDE, AND R. CRYBERG, The Iron Reagents, G. F. Smith Chemical Co., Columbus, Ohio, 1965, p. 41.
(Received February 26th, 1968) Carbohyd. Res., 7 (1968) 94-97
Laminaran
of fshige
okamurai’
Previously, Handa and Nisizawa’ reported that the soluble laminaran isolated from ,?%&a bicydis Setchell was composed of J-(1 +3)- and B-(1+6)-linked D-glucose residues in an approximate ratio of 2: 1. This finding was mainly based on the isolation of a series of gentio- and laminari-oligosaccharides from a partial acid hydrolyzate of laminaran. Moreover, the /3-D-(1+6)-linked residues were assumed to be located near the center of the molecule on the basis of alkali degradation. The structure of Eisenia-laminaran was thus considerably different from the structures of laminarans of the genus Laminaria’, in spite of their belonging to the same family. In addition, no “insoluble” laminaran was found in Eisenia. From the determination of the amount of 2,3,4-tri-O-methyl-D-glucose obtained after hydrolysis of fully methylated laminaran and of the formaldehyde produced by the periodate oxidation following the Smith degradation procedure, it was found3 that the B-D-linked units form a straightchain structure which constitute about one-quart of the whole molecule. The absence of 2,4-di-O-methyl-D-glucose in the methylation products was also evidence for a nonbranched structure of this laminaran. In the present work, the laminaran of Ishige okamurai Yendo, which belongs to the Chondariales, was investigated in order to compare its structure with that of other laminarans; difFerences may be expected, since this alga belongs to a family far remote from the Laminariales. Ishige grows at an intertidal zone and is distributed from the middle to the southern part of Japan. Finely crushed dry fronds of Ishige (100 g) were extracted with about a IO-fold volume cf 0.09M hydrochloric acid for 2 h at room temperature by the countercurrent *Contribution No. Tokyo, Japan.
170 from the Shimoda
Marine Biological
Station, Tokyo
Kyoiku
University,
Carbohyd. Res., 7 (1968) 97-99