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Enzymic determination of d -galactose, d -arabinose, and their homologs
ANALYTICAL
Enzymic
BIOCHEMISTRY
25,221-227
Determination and
(1968)
of D-Galactose, Their Homologs
A. S. L. HU Department
of Biochemistry,
University Received
AIiD
S. GRANT
of Kedacky, November
D-Arabinose,
Lexington,
Kentucky
40506
24, 1967
In biological and chemical investigations of carbohydrates, it is often desirable to perform quantitative determinations of specific monosaccharides. The spectrophotometric determination of sugars by means of enzyme-catalyzed reactions is particularly useful in these investigations because it is generally rapid, sensitive, simple, and specific. At present D-ghCOSe is routinely determined by means of the reaction catalyzed by glucose oxidase (1)) and n-galactose determinations by means of galactose oxidase have become increasingly popular (2). The isolation and characterization of three sugar dehydrogenases from a pseudomonad has been reported (3-5). Two of these enzymes, n-galactose dehydrogenase (n-galactose:NADP oxidoreductase) and n-arabinose dehydrogenase (n-arabinose :NAD (P) oxidoreductnsc) , are quite specific and permit the quantimtive determination of n-galactose and its homologs and n-arabinose and its homologs, respectively. Similar dehydrogenases were first described in extracts of Pseudomonas saccharophila by Doudoroff and his co-workers (6,7). This communication deals with the use of t,he aforementioned enzymes in the quantitative determination of n-galactose and its homologs, n-arahinose and its homologs, and some oligosaccharides which are composed of these sugars. MATERIALS
ASD
METHODS
The sugars used in these studies were obt’ained from the following commercial sources: n-galactose, L-arabinose. n-arabinose, and lactose were obtained from Mann Biochemical Corp., while n-fucose, L-fucose, D-glycero-n-mannoheptosc and 3-O-,8-D-galactopyranosyl-D-arabinose were products of General Biochemicals, Inc. The sugars were chromatographed in a solvent system of butanol/pyridine/water (6/4/3) and were found to be free of det.cctahlc contamination after visualization with 221
HU AND GRANT
222
silver nitrate reagent (8). All sugar preparations were dried in a vacuum oven at 40°C for 24 to 48 hr before use. The preparation of n-galactose dehydrogenase and n-arabinose dehydrogenase from Pseudomonas extracts is described elsewhere (3). The preparation of crystalline ,&galactosidase from extracts of Escherichia coli K12 has also been described (9). The enzymes were dialyzed against 0.1 M Tris-acetate and stored at -20°C. The triphosphopyridine nucleotide (TPN) and Tris were obtained from the Sigma Chemical Co. All other chemicals employed were commercial reagent-grade preparations. The various analytical procedures described here are based on the generalized reaction: aldose
+ TPI\+
+ HCO -+ aldonic
acid
+ TPSH
+ H+
As the molar abaorbancy of the oxidized coenzyme at 340 mp is zero and that of the reduced coenzyme is 6.22 X 103, the amount of sugar oxidized may be calculated directly from the increased absorbancy at the wavelength. The equilibrium of the reaction greatly favors oxidation of the sugar, thus ensuring its quantitative oxidation. The assay system for the monosaccharides cont.ained 270 pmoles of Tris-acetate buffer, pH 8.5, 2 pmoles of TPN, 5 units of dehydrogenase (3), and sugar in a final volume of 3 ml. Reagent blanks contained the ident’ical ingredients with the exception of enzyme. Tubes containing the assay mixtures were incubated at room temperature for 30 min, after which the contents were transferred to 1 cm spectrophotometric cells; the increase of absorbancy at 340 111,~was measured in a Zeiss PMQII spectrophotomcter. As a check of whether the reaction had proceeded to completion, the absorbancy of each sample was generally measured again after a further incubation period of 15 min. Under the conditions described, the reaction was generally complete within 30 min. The P-galactosides were assayed in a system composed of 270 pmolcs of Tris-Cl buffer, pH 7.5, 2.7 ,kmoleeof MgCl,, 2 ,.moles of TPN, 5 units of dehydrogenase, 5000 units of P-galactosidase (91, and sugar. The ,&galactosidase was omitted from the blank tubes. The increase in absorbancy at 340 111~was checked after incubation at, room temperature for 30 min, and :,t 15 min intervals after that, unt’il no further increase was observed. Under these conditions, the reaction was usually complete in 45 min. IiESITLTS
AND
DISCI’SSIOS
As shown in Figure 1, galactose dehydrogenasc may be used to determine n-galactose, L-arabinose, and D-fUCOSequantitatively. The experimental values agree quite well with the calculated values. This enzyme
ENZTMIC
DETERMINATION
OF
223
SUGARS
in quite specific for D-galactose and its homologs and we found that D-glucose had no effect on the determination of D-galactose, even when D-glucose was present in great excess. Identical results were obtained when D-mannose was substituted for glucose. Galactose dehydrogenase will also catalyze the oxidation of 2-deoxy-Dgalactose and D-glycero-D-mannoheptose. In the case of the former, the large Michaelis constant (Table 1) requires that the quantity of enzyme
1.6 -
= 1.6E 0
: 1.4 lo ; 1.2 -
./
UJ " i.oc
‘
0
0.1
0.2
0.3
0.4
Micromoles
/
0.5
0.6
of
Suqar
0.7
0.9
0.9
Fro. 1. Increase in absorbance at 340 rnp as a function of increasing concentrations of n-galactose and its homologs. The linp is based on theoretical values while the points are experimental values. Symbols (0) IA-arabinose; (A) o-fucose; (0) n-galactose. Thr, assays are dpscribcd in the test.
used in the assay system be increased approximately IO-fold. The combination of large Michaelis constant and low maximal velocity makes the quantitative determination of D-glycero-D-mannoheptose quite difficult. In our hands the assay of that sugar has never been satisfactory. The enzyme D-arabinose dehydrogena$e catalyzed the quantitative oxidation of D-arabinose and L-fucose. The results were identical to t.hose shown in Figure 1. The measurements were not extended to L-galactose, although the kinetic constants in Table 1 indicate that D-arahinoee dehydrogcnase u~oulclcatalyze its oxidation at a sufficient rate to pe;mit its
224
HU
Apparent
AND
TABLE 1 Kinetic Constants of o-Arabinose and o-Calactose Dehgdrogenase D-Arabinose
SWW
5.4 1.1 3.0
Dehydrogenase
Dehydrogenase Relative
Km
o-arabinose L-Galactose L-Pucose n-Galaetose L-Arabinose n-Fucose 2-Deoxy-n-galactose n-Glycero-n-mannoheptose The reaction constant, K,,
GRANT
x lo-* x 10-z x 1OVj
F,
100 40 30 3.9 1.5 s 0 1.0 5 0
mixture and conditions and maximal velocity,
x x x x x
10-J 10-d 10-d 10-z 10-X
of assay are described in the text. The from Lineweaver-Burk
Ii,,,, were obtained
100 40 160 100 1.4 Michaelis plots*
determination also. Because of its potential importance for nucleic acid studies, the ability of the enzyme to catalyze the oxidation of 2-deoxyribose has been examined and found not to occur. As a test of the usefuhless of these dehydrogenases for determining sugars in biological fluids, D-gala&se and D-arabinose were dissolved in
quantitative
1.6. z 0 t I0
I .4 1.2.
0 1.0. 0 ” g 0.0. 0 & m 0.6. 2
0
0.4 -
0
/
/
0.2
0.4 Mlcromolrs
FIG. 2. Determination of n-galactose The sugars were dissolved in serum the assay described in the text.
of
0.6 Sugor
(0) and n-arabinose (0) and aliquots of the serum
0.
.
in human serum. were subjected to
EKZYMIC
DETERMINATION
OF
225
SUGARS
human serum and their concentration determined enzymically. The values obtained, shown in Figure 2, agreed well with the calculated values at the low concentration range. The deviation from the calculated values at higher concentrations was not further investigated, but was probably the result of a competing enzyme reaction, It is probably advisable to denature the proteins in a biological fluid before analysis. In this laboratory, n-galactose dehydrogenase has been utilized for I -30 -20; 0 (I) ul
=5
so-
-10;
E 40. : 0 + g 7; W
30.
-8 -
as 0
-6 _
z 0
20.
Hours
of
Growth
FIG. 3. Ctilization of n-galactosc I,- a psrudonomad. The ~~11s were cultured in a New Brunswick continuons-culture ;tpparatus at 30°C. More galactose was added at the times designated by the arrows. dliqnota were removed at time intervals for the determination of turbidity in a Bausch h Lomb Spectronic 20 calorimeter at 660 mp. Simultaneously, an aliquot was chilled in an ice bath, centrifuged, and the supernatant subjected to analysis as tlrscribt-d in the trot. Symbols: (0) galactose concrntration : (0) turbidity.
monitoring the utilization of n-gnlactose by bacteria. It was found, for instance, that) the n-galactose in a culture of Pseu~omo,~as disappeared rapidly even after the cessation of cell division (Fig. 3). Enzymic determinations are particularly useful where the sugar in question may be converted into other carbohydrate materials, which may interfere with the chemical determination of the sugar. When coupled with the proper hydrolytic enzymes, the dehydrogenaFes may help in elucidating t.he structure of some oligosaccharides. For example, when coupled with the enzyme /3-galactosidase, the dehydrogenases could be used to establish the /3-link of galactoeides and, in some cases,
226
HU
AND
GRANT
to check the nature of the hydrolytic products; the disaccharides lactose and 3-O-P-n-galactopyranosyl-n-arabinose were analyzed by this method, and the results found to agree well with the calculated values (Table 2’~. A similar method has been employed by Venkataraman and Reithel (lo), who coupled /3-galactosidase with hexokinase and glucose-6-phosphate dehydrogenase to measure lactose. Quite clearly, these dehydrogenases may be employed to study many reactions in which a suitable monosaccharide is liberated. Wallenfels has made use of the requirement for the ,&anomer by another n-galactose dehydrogenase to establish that ,8-n-galactose is liberated in the reaction catalyzed by P-galactosidase from Escherichia co& (11). Enzymic
TABLE 3 Assay of p-Galactosides -4mount added, pIllOk
u-Galactose found, j4mole
Lactose
0.20 0 50
0.19 0.48
3-O-p-D-Galactosyl-o-arabinose
0.20 0 60
0.19 0.49
Galactoside
The
assayed
assay
system
is described
D-Arabinose found. ~mole
0.21 0.51
in the text.
Since D-galactose dehydrogenase is specific for n-galactose and its homologs and n-arabinose dehydrogenase is specific for n-arabinose and its homologs, these two enzymes can be used to distinguish n-galactose from L-galactose, n-arabinose from L-arabinose, etc. It is clear, however, that the enzymes cannot be used to furnish quantitative information if homologs such as n-galactose and L-arabinose are both present in a mixture. In such cases a preliminary separat,ion of the homologs must first be performed. We have also found a n-glucose dehydrogenase in Pseudomonas (12), but have not as yet purified it extensively. Our preliminary examination of partially purified preparations indicates that this enzyme is also quite specific and would be a useful enzyme for studies of carbohydrates. SUMMARY
The enzymes n-galactose dehydrogenase and n-arabinose dehydrogenase were demonstrated to be applicable to the quantitative determination of n-galactose (and homologs) and n-arabinose (and homologs) , respectively. The enzymic reactions were quite specific. When coupled with ,8-galactosidase, n-galactose dehydrogenase could be used in the quantiiative determination of /3-galactosides.
ESZTMIC
DETERMINATIOS
OF
227
SUGARS
ACKNOWLEDGMENT This Health,
work United
1. HOU~II,
2. 3. 4. 5. 6, 7.
8. 9. 10. 11.
12.
was supported States Public
bp Grant GM-08816 Health Service.
from
the
Sational
Institute
of
I,., .\su Joxw, J. K. N., in “Methods in Carbohydrate Chemistry” (R. L. Whistler and M. I,. Wolfrom, rds.), Vol. I, p. 400. Academic Press, New York, 1962. ROTH, H.. SEG.~L. S., O-D BEHTOLI, D., Anal. Biochem. 10, 32 (1965). CLINE, A. L.. AND Hu, A. S. L., J. Riol. Chem. 240, 4458 (1965). CLINK, A. I,.. .AIVD Hu. =\. S. I,., J. Biol. Chrm. 240, 4493 (1965). CLINE, 8. I,., AND Hu, A. S. L., d. Biol. Chem. 240, 4498 (1965). DOUDOROFF, &I.. Cos~oI~ou~on, C. R., AND BURNS, S.. Proc. Intern. Symp. Enzyme Chem. Z’okyo Kyoto, 1957, 313 (1958). PALLEROKI. S. J., AND DOUDOROBF, M..J.Bact. 74, 180 (1957). TREVELx4N. If*. E., PROCTOR. D. P., AND HARRISON, J. S., Nature 166, 444 (1950). COLHT, C.. JR.. AND Hr. A. S. L., Riochim. Biophys. Acfa 157, 167 (1968). VEXKATARAMAN, R., .~ND REITHEL. F. J., ilrch. Biochem. Biophys. 75, 443 (1958). WALLENFELS, K., AND KURZ, G., Biochem. Z.335,559 (1962). Hu, A. S. L., ASD CLINE, A. L., Biochim. Biophys. Acta 93, 237 (1964).
Enzymic
BIOCHEMISTRY
25,221-227
Determination and
(1968)
of D-Galactose, Their Homologs
A. S. L. HU Department
of Biochemistry,
University Received
AIiD
S. GRANT
of Kedacky, November
D-Arabinose,
Lexington,
Kentucky
40506
24, 1967
In biological and chemical investigations of carbohydrates, it is often desirable to perform quantitative determinations of specific monosaccharides. The spectrophotometric determination of sugars by means of enzyme-catalyzed reactions is particularly useful in these investigations because it is generally rapid, sensitive, simple, and specific. At present D-ghCOSe is routinely determined by means of the reaction catalyzed by glucose oxidase (1)) and n-galactose determinations by means of galactose oxidase have become increasingly popular (2). The isolation and characterization of three sugar dehydrogenases from a pseudomonad has been reported (3-5). Two of these enzymes, n-galactose dehydrogenase (n-galactose:NADP oxidoreductase) and n-arabinose dehydrogenase (n-arabinose :NAD (P) oxidoreductnsc) , are quite specific and permit the quantimtive determination of n-galactose and its homologs and n-arabinose and its homologs, respectively. Similar dehydrogenases were first described in extracts of Pseudomonas saccharophila by Doudoroff and his co-workers (6,7). This communication deals with the use of t,he aforementioned enzymes in the quantitative determination of n-galactose and its homologs, n-arahinose and its homologs, and some oligosaccharides which are composed of these sugars. MATERIALS
ASD
METHODS
The sugars used in these studies were obt’ained from the following commercial sources: n-galactose, L-arabinose. n-arabinose, and lactose were obtained from Mann Biochemical Corp., while n-fucose, L-fucose, D-glycero-n-mannoheptosc and 3-O-,8-D-galactopyranosyl-D-arabinose were products of General Biochemicals, Inc. The sugars were chromatographed in a solvent system of butanol/pyridine/water (6/4/3) and were found to be free of det.cctahlc contamination after visualization with 221
HU AND GRANT
222
silver nitrate reagent (8). All sugar preparations were dried in a vacuum oven at 40°C for 24 to 48 hr before use. The preparation of n-galactose dehydrogenase and n-arabinose dehydrogenase from Pseudomonas extracts is described elsewhere (3). The preparation of crystalline ,&galactosidase from extracts of Escherichia coli K12 has also been described (9). The enzymes were dialyzed against 0.1 M Tris-acetate and stored at -20°C. The triphosphopyridine nucleotide (TPN) and Tris were obtained from the Sigma Chemical Co. All other chemicals employed were commercial reagent-grade preparations. The various analytical procedures described here are based on the generalized reaction: aldose
+ TPI\+
+ HCO -+ aldonic
acid
+ TPSH
+ H+
As the molar abaorbancy of the oxidized coenzyme at 340 mp is zero and that of the reduced coenzyme is 6.22 X 103, the amount of sugar oxidized may be calculated directly from the increased absorbancy at the wavelength. The equilibrium of the reaction greatly favors oxidation of the sugar, thus ensuring its quantitative oxidation. The assay system for the monosaccharides cont.ained 270 pmoles of Tris-acetate buffer, pH 8.5, 2 pmoles of TPN, 5 units of dehydrogenase (3), and sugar in a final volume of 3 ml. Reagent blanks contained the ident’ical ingredients with the exception of enzyme. Tubes containing the assay mixtures were incubated at room temperature for 30 min, after which the contents were transferred to 1 cm spectrophotometric cells; the increase of absorbancy at 340 111,~was measured in a Zeiss PMQII spectrophotomcter. As a check of whether the reaction had proceeded to completion, the absorbancy of each sample was generally measured again after a further incubation period of 15 min. Under the conditions described, the reaction was generally complete within 30 min. The P-galactosides were assayed in a system composed of 270 pmolcs of Tris-Cl buffer, pH 7.5, 2.7 ,kmoleeof MgCl,, 2 ,.moles of TPN, 5 units of dehydrogenase, 5000 units of P-galactosidase (91, and sugar. The ,&galactosidase was omitted from the blank tubes. The increase in absorbancy at 340 111~was checked after incubation at, room temperature for 30 min, and :,t 15 min intervals after that, unt’il no further increase was observed. Under these conditions, the reaction was usually complete in 45 min. IiESITLTS
AND
DISCI’SSIOS
As shown in Figure 1, galactose dehydrogenasc may be used to determine n-galactose, L-arabinose, and D-fUCOSequantitatively. The experimental values agree quite well with the calculated values. This enzyme
ENZTMIC
DETERMINATION
OF
223
SUGARS
in quite specific for D-galactose and its homologs and we found that D-glucose had no effect on the determination of D-galactose, even when D-glucose was present in great excess. Identical results were obtained when D-mannose was substituted for glucose. Galactose dehydrogenase will also catalyze the oxidation of 2-deoxy-Dgalactose and D-glycero-D-mannoheptose. In the case of the former, the large Michaelis constant (Table 1) requires that the quantity of enzyme
1.6 -
= 1.6E 0
: 1.4 lo ; 1.2 -
./
UJ " i.oc
‘
0
0.1
0.2
0.3
0.4
Micromoles
/
0.5
0.6
of
Suqar
0.7
0.9
0.9
Fro. 1. Increase in absorbance at 340 rnp as a function of increasing concentrations of n-galactose and its homologs. The linp is based on theoretical values while the points are experimental values. Symbols (0) IA-arabinose; (A) o-fucose; (0) n-galactose. Thr, assays are dpscribcd in the test.
used in the assay system be increased approximately IO-fold. The combination of large Michaelis constant and low maximal velocity makes the quantitative determination of D-glycero-D-mannoheptose quite difficult. In our hands the assay of that sugar has never been satisfactory. The enzyme D-arabinose dehydrogena$e catalyzed the quantitative oxidation of D-arabinose and L-fucose. The results were identical to t.hose shown in Figure 1. The measurements were not extended to L-galactose, although the kinetic constants in Table 1 indicate that D-arahinoee dehydrogcnase u~oulclcatalyze its oxidation at a sufficient rate to pe;mit its
224
HU
Apparent
AND
TABLE 1 Kinetic Constants of o-Arabinose and o-Calactose Dehgdrogenase D-Arabinose
SWW
5.4 1.1 3.0
Dehydrogenase
Dehydrogenase Relative
Km
o-arabinose L-Galactose L-Pucose n-Galaetose L-Arabinose n-Fucose 2-Deoxy-n-galactose n-Glycero-n-mannoheptose The reaction constant, K,,
GRANT
x lo-* x 10-z x 1OVj
F,
100 40 30 3.9 1.5 s 0 1.0 5 0
mixture and conditions and maximal velocity,
x x x x x
10-J 10-d 10-d 10-z 10-X
of assay are described in the text. The from Lineweaver-Burk
Ii,,,, were obtained
100 40 160 100 1.4 Michaelis plots*
determination also. Because of its potential importance for nucleic acid studies, the ability of the enzyme to catalyze the oxidation of 2-deoxyribose has been examined and found not to occur. As a test of the usefuhless of these dehydrogenases for determining sugars in biological fluids, D-gala&se and D-arabinose were dissolved in
quantitative
1.6. z 0 t I0
I .4 1.2.
0 1.0. 0 ” g 0.0. 0 & m 0.6. 2
0
0.4 -
0
/
/
0.2
0.4 Mlcromolrs
FIG. 2. Determination of n-galactose The sugars were dissolved in serum the assay described in the text.
of
0.6 Sugor
(0) and n-arabinose (0) and aliquots of the serum
0.
.
in human serum. were subjected to
EKZYMIC
DETERMINATION
OF
225
SUGARS
human serum and their concentration determined enzymically. The values obtained, shown in Figure 2, agreed well with the calculated values at the low concentration range. The deviation from the calculated values at higher concentrations was not further investigated, but was probably the result of a competing enzyme reaction, It is probably advisable to denature the proteins in a biological fluid before analysis. In this laboratory, n-galactose dehydrogenase has been utilized for I -30 -20; 0 (I) ul
=5
so-
-10;
E 40. : 0 + g 7; W
30.
-8 -
as 0
-6 _
z 0
20.
Hours
of
Growth
FIG. 3. Ctilization of n-galactosc I,- a psrudonomad. The ~~11s were cultured in a New Brunswick continuons-culture ;tpparatus at 30°C. More galactose was added at the times designated by the arrows. dliqnota were removed at time intervals for the determination of turbidity in a Bausch h Lomb Spectronic 20 calorimeter at 660 mp. Simultaneously, an aliquot was chilled in an ice bath, centrifuged, and the supernatant subjected to analysis as tlrscribt-d in the trot. Symbols: (0) galactose concrntration : (0) turbidity.
monitoring the utilization of n-gnlactose by bacteria. It was found, for instance, that) the n-galactose in a culture of Pseu~omo,~as disappeared rapidly even after the cessation of cell division (Fig. 3). Enzymic determinations are particularly useful where the sugar in question may be converted into other carbohydrate materials, which may interfere with the chemical determination of the sugar. When coupled with the proper hydrolytic enzymes, the dehydrogenaFes may help in elucidating t.he structure of some oligosaccharides. For example, when coupled with the enzyme /3-galactosidase, the dehydrogenases could be used to establish the /3-link of galactoeides and, in some cases,
226
HU
AND
GRANT
to check the nature of the hydrolytic products; the disaccharides lactose and 3-O-P-n-galactopyranosyl-n-arabinose were analyzed by this method, and the results found to agree well with the calculated values (Table 2’~. A similar method has been employed by Venkataraman and Reithel (lo), who coupled /3-galactosidase with hexokinase and glucose-6-phosphate dehydrogenase to measure lactose. Quite clearly, these dehydrogenases may be employed to study many reactions in which a suitable monosaccharide is liberated. Wallenfels has made use of the requirement for the ,&anomer by another n-galactose dehydrogenase to establish that ,8-n-galactose is liberated in the reaction catalyzed by P-galactosidase from Escherichia co& (11). Enzymic
TABLE 3 Assay of p-Galactosides -4mount added, pIllOk
u-Galactose found, j4mole
Lactose
0.20 0 50
0.19 0.48
3-O-p-D-Galactosyl-o-arabinose
0.20 0 60
0.19 0.49
Galactoside
The
assayed
assay
system
is described
D-Arabinose found. ~mole
0.21 0.51
in the text.
Since D-galactose dehydrogenase is specific for n-galactose and its homologs and n-arabinose dehydrogenase is specific for n-arabinose and its homologs, these two enzymes can be used to distinguish n-galactose from L-galactose, n-arabinose from L-arabinose, etc. It is clear, however, that the enzymes cannot be used to furnish quantitative information if homologs such as n-galactose and L-arabinose are both present in a mixture. In such cases a preliminary separat,ion of the homologs must first be performed. We have also found a n-glucose dehydrogenase in Pseudomonas (12), but have not as yet purified it extensively. Our preliminary examination of partially purified preparations indicates that this enzyme is also quite specific and would be a useful enzyme for studies of carbohydrates. SUMMARY
The enzymes n-galactose dehydrogenase and n-arabinose dehydrogenase were demonstrated to be applicable to the quantitative determination of n-galactose (and homologs) and n-arabinose (and homologs) , respectively. The enzymic reactions were quite specific. When coupled with ,8-galactosidase, n-galactose dehydrogenase could be used in the quantiiative determination of /3-galactosides.
ESZTMIC
DETERMINATIOS
OF
227
SUGARS
ACKNOWLEDGMENT This Health,
work United
1. HOU~II,
2. 3. 4. 5. 6, 7.
8. 9. 10. 11.
12.
was supported States Public
bp Grant GM-08816 Health Service.
from
the
Sational
Institute
of
I,., .\su Joxw, J. K. N., in “Methods in Carbohydrate Chemistry” (R. L. Whistler and M. I,. Wolfrom, rds.), Vol. I, p. 400. Academic Press, New York, 1962. ROTH, H.. SEG.~L. S., O-D BEHTOLI, D., Anal. Biochem. 10, 32 (1965). CLINE, A. L.. AND Hu, A. S. L., J. Riol. Chem. 240, 4458 (1965). CLINK, A. I,.. .AIVD Hu. =\. S. I,., J. Biol. Chrm. 240, 4493 (1965). CLINE, 8. I,., AND Hu, A. S. L., d. Biol. Chem. 240, 4498 (1965). DOUDOROFF, &I.. Cos~oI~ou~on, C. R., AND BURNS, S.. Proc. Intern. Symp. Enzyme Chem. Z’okyo Kyoto, 1957, 313 (1958). PALLEROKI. S. J., AND DOUDOROBF, M..J.Bact. 74, 180 (1957). TREVELx4N. If*. E., PROCTOR. D. P., AND HARRISON, J. S., Nature 166, 444 (1950). COLHT, C.. JR.. AND Hr. A. S. L., Riochim. Biophys. Acfa 157, 167 (1968). VEXKATARAMAN, R., .~ND REITHEL. F. J., ilrch. Biochem. Biophys. 75, 443 (1958). WALLENFELS, K., AND KURZ, G., Biochem. Z.335,559 (1962). Hu, A. S. L., ASD CLINE, A. L., Biochim. Biophys. Acta 93, 237 (1964).