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Substrate specificity of D -galactose oxidase
Car&hydrate
Research
193
Elsevier Poblkbing Company, Amsterdam Printed in Belgium
SUBSTRATE ROBERT A.
Department
SPECIFICITY
SCHLECEL**,
OF D-GALACTOSE
CLAIRE M. GERBECK?,
of Biochemistry,
qnicersity
AND
REX
OXIDASE*
MONTGOMERY~~
of Iowa. Iowa City, Iotaa 52240 (U. S. A.)
(ReceivedDecember 11 th, 1967; in revisedform, February 25th, 1968)
ABSTRACT
The rate of oxidation of methyl ethers of D-galactose and 2-amino-2-deoxy-Dgalactose, and of oligosaccharides and polysaccharides containing D-galactosyl residues having a free hydroxyl group at C-6, has been followed by various procedures that depend either upon the hydrogen peroxide or the aldehyde groups produced, or upon the unoxidized D-galactose residues remaining in the reaction mixture. Derivatives of D-galactose having substituents on the hydroxyl group at C-4 are not oxidized. 2-Amino-2-deoxy-D-galactose residues having glycosyl substituents at C-3 are not oxidized by the enzyme, and therefore, neither are chondroitin 4-sulfate nor dermatan sulfate. No completely satisfactory procedure was found for following the oxidation reaction to termination, which, in none of the cases studied, was 100% complete. INTRODUCTION
AND
DISCUSSION
D-Galactose oxidase (D-galactose:oxygen oxidoreductase EC l-1.3.9) has been stated to oxidize those residues of D-galactopyranose in which the primary hydroxyl group at C-6 is unsubstituted”“. The reaction has been applied widely to modify D-galactopyranosyl residues in carbohydrates3*4, to introduce a tritium label into such compounds 5-7, and to analyze for these residues2*‘-“. Several peculiarities of the reaction have gone unanswered, however. For example, the reaction does not go to completion2*5, unusual kinetics are sometimes observed’-‘, and analytical data are not consistent2. As a result, the value of application of this enzyme to structural studies is dubious. The present paper considers the influence of substituents on the rate of oxidation of the D-galactopyranosyl residues, in an attempt to clarify some of the above questions. The effect of chloride, sulfate, phosphate, cyanide, formate, hydrazine, and *This investigation was supported by U.S. Public Health Service Research Grants from the National Heart Institute (HE-06717) and the National Institute of General Medical Sciences (GM-14013). **Present address: Department of Biochemistry, Harvard University, Boston, Massachusetts. TPresent address: Department of Biochemistry, Presbyterian-St. Lukes Hospital, Chicago, Illinois. TfTo whom correspondence should be addressed. Curbohyd_ Res., 7 (1968) 193-199
194
R. A. SCHLEGEL,
C. M. GERBECK,
R. MONTGOMERY
hydroxylamine was first studied. Of these, the last four completely inhibit the enzyme, although the influence of hydroxylamine16 on the coupled peroxidase system complicates its evaluation as an inhibitor. Phosphate of concentrations up to 0.1~ at pH 7.0 was not inhibiting, but, at pH 5.6, a diminution in the rate of oxidation was noted. Sodium sulfate was not inhibiting at concentrations up to O.~M,whereas both sodium chloride and potassium chloride inhibited the reaction noticeably at 0.16hi and to the extent of 45% at 0.4~. The inhibition by chloride may well explain the variability noted by some workers in determining the D-galactose in carbohydrate polymers not oxidized by D-galactose oxidase “*‘*_ The oxidized polymer was hydrolyzed with hydrochloric acid, and the residual D-galactose was determined, after neutralization, by using D-galactose oxidase assay. Such difficuhies would appear to be avoided if sulfuric acid is used for the hydrolysis. Tne extent of oxidation of available D-gaiactose residues in a carbohydrate can be estimated by several methods, all of which have certain disadvantages. Perhaps the simplest is that in which the hydrogen peroxide produced by the oxidation reaction is coupled, through a peroxidase, to a chromogen”. Such a system serves well in estimation of the initial course of the oxidation, but suffers from several disadvantages when the reaction is followed to completion. The color produced is diminished in the presence of other electron acceptor?‘, and is affected by the precipitation of chromogen from solution13. Such side reactions decrease the absorbance value for the final oxidation, although, for o-nitrophenyl c+D-galactopyranoside, 1.5~anhydroD-galactitol, and raffinose, the results so obtained, calcuIated from a standard curve determined with hydrogen peroxide, are of the same order of magnitude as that calculated by a determination of D-galactose residues that were unoxidized (see Table I). As has been noted by Avigad et nL2 and several other workers, the initial velocities of the oxidation reaction differ for various substrates. In certain cases, this factor in itself presents the problem of determining the percentage of D-galactose residues having free hydroxyl groups at C-6 in an unknown carbohydrate. The problem is complicated, however, by the fact that some of the D-galactosyl residues in a carbohydrate having such a free hydroxyl group at C-6 are not oxidized. As may be noted in Table I, a D-galactose residue substituted at O-4 by a methyl group is not a substrate for D-galactose oxidase. This is true for 4-O-methyl-D-galactose and 2,4-di-O-methyl-D-galactose, but not for D-galactose substituted by methyl groups at O-2 or O-3. It may also be noted that 2-amino-2-deoxy-D-galactose residues having the hydroxyl group at C-3 substituted by a methyl group are oxidized a little more slowly than 2-amino-2-deoxy-D-gallactose, which itself is oxidized more slowly than D-galactose. If, however, the hydroxyl group at C-3 of 2-amino-2-deoxy-D-galactose is substituted by a D-glucopyranosyl or a D-glucopyranosyluronic acid residue, the oxidation is completely checked. This inhibition may be the result of steric effects, as can be seen for the slow oxidation of 2’-O-(a-L-fucopyranosyl)lactose and the resistance of uridine 5’-(ac-D-galactopyranosyl pyrophosphate) to oxidation2. From these results, it is therefore not surprising that chondroitin Qsulfate and dermatan Cnr6+‘cL Res.,
7
(1968) 193-199
195
SUBSTRATE SPECIFICITY OF D-GALACTO8E OXIDASE TABLE
I
SUB!XRATE
SPECIFICITY
CIF D-GALACTOSE
OXIDASE
Substratea
Relative rate of oxidation
% oxidized, as determined by Formation
of IY~O~
Park-
.rohllson
Residual D-gdaClOSC
procedure (24 II)
D-Galactose o-Nitrophenyl p-o-galactopyranoside Methyl a-o-galactopyranoside I-O-(a-IJ-GaiactopyranosyB-m_ro-inositol’ 2-O-Methyl-o-galactoseb 3-0-Methyl-o-galactose= 4-0-Methyl-o-galactoseb 2,4-Di-O-methyl-o-galactose’~c 2,3-Di-0-methyl-o-galactoseb
100
57 118 37 50 172 0 0 76
1,2:3,4-Di-0-isopropylidenea-~-galactosc I.5Anhydro-o-galactitol 0-wo-Galactopyranosyl-( I43)-D-galactoseC Raffinose 2’-O-(a-D-Ft~copyranosyl)lactose~ 2-Amino-2-deoxy-o-galactose Methyl 2-acetamido-2-deoxy-3-O-methyk-ogalactopyranosidee Chondrosinee O-fl-D-Glucopyranosyk(1+3)-2-acctamido2-deoxy-o-galactosee O-,%D-Galactopyranosyl-(1+3)-2-acetamido2-deoxy-D-gaiactoser
0 134 48 145 4 71 54
Guaran Ovomucoid Chondroitin 4-sulfatef Dermatan sulfatef
1.50 87 0 0
0 0
86 84 84 60 68
78
86
96
20
95
96
46
98
3
95
0 0
75
0 0
39 76 86
“Reaction solution contained 100 mg of available D-galactose/l. bProvided by Dr. 9. A. Lewis of the University of Minnesota. CProvided by Dr. G. F. Springer of Northwestern University. aProvided by Dr. V. Ginsberg of the National Institutes of Health. eprovided by Dr. R. W. Jeanloz of Harvard University Medical School. fProvided by Dr. J. A. Cifonelli, LaRabida Institute, Chicago.
sulfate, both of which have a free hydroxyl group at C-6 of their 2-amino-2-deoxy-Dgalactose residues, but have substituents at O-3 and O-4, are not oxidized by o-galactose oxidase. Several carbohydrate-containing biopolymers are oxidizable to some extent by D-galactose oxidase, but serious questions must now be raised concemingany structural deductions that can be made from suchinformation. If oxidation can be noted by any of the methods mentioned above, it would seem clear that some of the D-galactopyranosyl residues have free hydroxyl groups at C-6. However, a negative result in no way excludes the possibility of occurrence, within the molecule, Carbohyd. Res., 7 (1968) 193-199
196
R. A. SCHLEGEL,
C. M. GERBECK,
R. MONTGOMERY
of D-galactopyranosyl residues that might have been expected to be oxidized. Further work is continuing on this subject. For a while, it was thought that the incomplete oxidation of the substrates for D-galactose oxidase was due to inhibition by the product. It would seem, however, that this is not the case. A reaction that has come to a halt cannot be significantly reactivated by the addition of fresh n-galactose oxidase, and the extent of oxidation is not significantly different if the initial concentrations of substrate are varied. In the latter instance, it would have been expected that the concentration of product in the reactioa systems wouId have varied; and, with it, the extent of the product inhibition. This behavior was, however, not noted for the oxidation of o-nitrophenyl B-Dgalactopyranoside in concentrations that were varied several-fold. This also is an area for further study. EXPERIMENTAL
Materials. - D-Galactose oxidase was obtained from Worthington Biochemical Company and the Miles Laboratories, Inc. Catalase (beef Iiver, 185,000 units per ml) and peroxidase (horseradish, 400 units per mg) from the Worthington Biochemical Company were used without further purification. #I-D-Galactosidase was obtained from CaIbiochem. The various derivatives of D-galactose were either purchased from Pfanstiehl Chemical Company and used without purification, or were donated as indicated by the footnotes in Table I. DEAE-Cellulose was successively washed with 0.5M hydrochloric acid and 0.5~ sodium hydroxide folowed, each time, by a thorough washing with water. After regeneration with alkali, the DEAE-cellulose was equilibrated with 0.02~ phosphate buffer, pH 7.0. Parr$cation of D-galactose oxidase. Crude D-galactose oxidase (41 mg) from Miles Laboratories was dissoIved in approximately 25 ml of 0.02M phosphate buffer, pH 7.0, and the dark solution was passed through a column (7.0 x 0.9 cm) of DEAE-c&uIose equilibrated with the same buffer. The coIumn was then washed with buffer solution (twice the initial volume). A dark pigment remained at the top of the column. The eluate was collected immediately after addition of the sample, and was divided into two fractions to separate the more-concentrated peak from the tailing fractions. In a typical experiment, D-gaiactose oxidase (17,000 units) was added to a column; the first two-thirds of the eluate contained 13,000 units (250 units per mI) and the remaining one-third of the eluate contained 1,500 units (60 units per ml). Assay of D-galactose oxiabse activity. To one ml of enzyme solution was added 0.4 ml of o-tolidine (recrystahized from acetone-water, 100 mg/l of water), 0.4 ml of peroxidase (100 mg/l of water), and 0.2 ml of 0.25~ D-galactose solution. Ail of the solutions were preheated to 30” and kept at this temperature during the assay, which was continuously recorded at 420 nm with a GiIford spectrophotometer, Model 2000, with a l-cm &&t-path. One unit of euzymic activity corresponds to Cmhhyd.
Res., 7 (1968)
193-199
SUBSTRATE SPECIFICITY OF D-GALACTOSE OXIDASE
197
a net absorbancy of 1.0 at 420 nm after reaction for 10 min at 30”. This value, when converted into moles of hydrogen peroxide produced (and, therefore, D-galactose oxidized) can be redefined as 30 nmoles of D-galactose oxidized per min at 30”. Inhibitor studies. - The inhibitor studies were conducted with a Galactostat kit (Worthington Biochemical Company), from which the e&yme, dissolved in 25 ml of distilled water, and the chromogen solution were kept separated. According to the above assay, the enzyme solution was equivalent to 11.3 units per ml. Studies on the effect of potassium chloride, sodium chloride, sodium sulfate, and sodium formate were made with D-galactose (1.39 x 10w4~) as the substrate. Corresponding studies with sodium cyanide, sodium bisulfite, hydrazine sulfate, and hydroxylamine hydrochloride were made with methyl a-D-galactopyranoside (1.39 x 10V4~) as the substrate. In each instance, 0.4 ml of chromogen solution, 0.4 ml of enzyme solution, and appropriate volumes of the inhibitor solution and the substrate solution (to make a final volume of 1.6 ml) were mixed and allowed to react at 30” or 37”. As in the enzyme assay, the absorbance at 420 nm was measured periodically during 1 h. Relative rates of oxiaktion. The rates of oxidation of various substrates (see Table I) were determined in relation to that of D-galactose. In each case, 0.5 ml of D-galactose oxidase (70 units, putied on DEAE-cellulose), 0.5 ml of peroxidase (100 mg/l), 0.5 ml of o-tolidine (200 mg/l), and 0.5 ml of the substrate solution (the concentration of the reaction solution was 1.39 x 10p4~) were mixed at 30”. The rates of oxidation were followed spectrophotometrically for 10 min at 30”, at 420 nm in a l-cm cell. Extent of oxidation. - Attempts were made to measure the extent of the final oxidation of various substrates by (a) using the color produced by the chromogen, as read against a standard curve determined with hydrogen peroxide, (b) measuring the aldehyde formed, with use of the Park-Johnson reagentslg, or (c) measuring the D-galactose remaining after oxidation had apparently ceased. For o-nitrophenyl j?-D-galactopyranoside, the residual substrate was determined by hydrolysis with j?-D-galactosidase according to the procedure of Avigad’. These various methods will be illustrated below (A-0) by considering the particular substrate, o-nitrophenyl j?-D-galactopyranoside. A. The reaction solution was prepared exactly as already described for the determination of relative rates of oxidation. The color produced by the o-tolidine chromogen was determined continuously in the Gilford spectrophotometer at 420 nm until such time as the color produced was at the maximum. The equivalence of this color to the hydrogen peroxide produced by the reaction was derived from a standard curve. The latter was determined by mixing 0.5 ml of o-tolidine solution (200 mg/l), 0.5 ml of peroxidase solution (100 mg/l), and 1.0 ml of solutions containing various amounts (O-230 nmoles) of hydrogen peroxide in water; the color so produced was read immediately against an appropriate blank. The maximum color, equivalent to 84% of complete oxidation, was obtained in approximately 1 h with o-nitrophenyl /?-D-galactopyranoside. B. By the procedure of Avigad’, a mixture containing 0.5 ml of D-galactose CarbOh~CL Res., 7 (1968) 193-199
198
R. A. SCHLEGEL,
C. M. GBRBECK,
R. MONTGOMERY
oxidase (125 units per ml, purified by DEAE-cellulose chromatography), 0.5 ml of catalase (0.1 ml of commercial suspension diluted to 25 ml with water), 0.5 ml of o-nitrophenyl B-D-galactopyranoside (5.56 x 10m4~), and 0.5 ml of water was allowed to react for 24 h at 30”. A blank determination was made by omitting the glycoside. To 1 ml of the final reaction solution was added an equal volume of B-D-galactosidase (240 mg/l) in 0.01~ phosphate buffer, pH 7.0, which was mu in magnesium chloride. The amount of o-nitrophenol released was determined continuously at 405 nm, and read after 30-45 mi’n when the absorbance was at the maximum. The correspondence of this color to the original concentration of unoxidized o-nitrophenyl B-Dgalactopyranoside was d’etermined from a standard curve. Eight separate determinations gave results that varied from 71-77% of complete oxidation; the average value was 75%. C. The extent of oxidation, followed by the formation of aldehyde groups, was measured by an oxidation system similar to that already described for Avigad’s procedure5, except that samples were taken periodically from the reaction mixture and analyzed by the Park-Johnson procedure lg . The resulting color, read at 690 nm in a Coleman Jr. calorimeter, was converted into equivalents of D-galactose from a preconstructed, standard curve determined with D-galactose. The extent of oxidation, as determined by this procedure, was 75-85% ; average value 78%. D. The alternative procedure of determining the amount of residual D-galactose, after oxidation and hydrolysis with sulfuric acid, used an oxidation system similar to that already described for Avigad’s procedure5. When maximal oxidation had been achieved, as judged by the results by that procedure, 1 ml of the solution was mixed with 1 ml of ISM sulfuric acid, and the mixture was heated for 6 h at 100”. The solution was then de-ionized by passing it through a bed of mixed resin containing Amberlite G-45 (OH-) and Rexyn-101 (H+). The eluate was evaporated to dryness ir; LXZCUU, ihe residue was dissolved in 0.6 ml of distilled water. and the content of D-galactose was determined by means of D-galactose oxidase. The results obtained with other substrates are summarized in Table I. ACKNOWLEDGMENT
The authors are indebted to the colleagues noted in Table I for various derivatives of D-galactose. REFERENCES 1 G.
A-GAD,
4 (1961)
C. ASENSIO, D. ALIARAL,
AND
B. L. HORECKER.
Biochem.
Biophys.
Res.
Comnum.,
474.
2 G. AVIGAD,
D. AMARAL,
C. ASENSIO, AND
B. L. HORECKER,
J. BioI. Chem.,
237 (1962)
2736.
3 J. C. ROBINSON AND J. E. PIERCE, Arch. Biochem. Biophys., 106 (1964) 3484 G. F. SPRINGER, Y. NAGAI, AND H. TEGThlEYER, Biochemisfry, 5 (1966) 3254. 5 G. AVIGAD, Carbohyd. Res., 3 (1967) 430. 6 J. E. G. BARNEXT, Carbohyd. Res., 4 (1967) 7 A.G.
MORELL,C.J.A.VANDENHA~~ER,
241 (1966) Carbohyd_
3745.
Res.,
7 (1968)
193-199
267.
I.H. SCHEINBERG,
AND
G. ASHWELL,
J. BioI.
Chem.,
SUBSTRATE
SPECIFICKY
OF D-GALACTOSE
199
OSIDASE
8 S. A. BARKER, G. PARDOE, AF~DM. STACEY, 1Varrrre.197 (1963) 231. 9 R. M. BRADLEY AND J. N. KANFER, Biochim. Biophys. Acta, 84 (1964) 210. 10 C. H. DEVERDIERAND M. HJELhr, Clin. Chinr. Acta, 7 (1962) 742. 11 W. FISCHERAND J. ZAPF, 2. Ph_wiol. Chem., 337 (1964) 186. 12 W. FISCHERAND J. ZAPF, Z. Physiol. Chem., 339 (1964) 54. 13 M. HELM, Cfin. C/rim. Acta, 15 (1967) 87. 14 E. ROREM AND C. LEWIS, Anal. Biochem., 3 (1962) 230. 15 J. SELIPERE, C. GANCEDO, AND C. A.SENSIO,And. Biochenr., 12 (1965) 590. 16 D. &ARAL, L. BERNSTEIN,D. MORSE, AND B. L. HORECKER,J. Biol. Chent., 238 (1963) 2281. 17 W. BUTLER AND L. CuNNtxGHAh~, J. Bid. Chem.. 241 (1966) 3882. 18 U. LINDAHL, Biochim. Bioph_vs. Acts, 130 (1966) 361. 19 J. T. PARK AND M. J. JOHNSON, J. Biol. Chem., 181 (1949) 149.
Curbohyd.
Res., 7 (1968) 193-199
Research
193
Elsevier Poblkbing Company, Amsterdam Printed in Belgium
SUBSTRATE ROBERT A.
Department
SPECIFICITY
SCHLECEL**,
OF D-GALACTOSE
CLAIRE M. GERBECK?,
of Biochemistry,
qnicersity
AND
REX
OXIDASE*
MONTGOMERY~~
of Iowa. Iowa City, Iotaa 52240 (U. S. A.)
(ReceivedDecember 11 th, 1967; in revisedform, February 25th, 1968)
ABSTRACT
The rate of oxidation of methyl ethers of D-galactose and 2-amino-2-deoxy-Dgalactose, and of oligosaccharides and polysaccharides containing D-galactosyl residues having a free hydroxyl group at C-6, has been followed by various procedures that depend either upon the hydrogen peroxide or the aldehyde groups produced, or upon the unoxidized D-galactose residues remaining in the reaction mixture. Derivatives of D-galactose having substituents on the hydroxyl group at C-4 are not oxidized. 2-Amino-2-deoxy-D-galactose residues having glycosyl substituents at C-3 are not oxidized by the enzyme, and therefore, neither are chondroitin 4-sulfate nor dermatan sulfate. No completely satisfactory procedure was found for following the oxidation reaction to termination, which, in none of the cases studied, was 100% complete. INTRODUCTION
AND
DISCUSSION
D-Galactose oxidase (D-galactose:oxygen oxidoreductase EC l-1.3.9) has been stated to oxidize those residues of D-galactopyranose in which the primary hydroxyl group at C-6 is unsubstituted”“. The reaction has been applied widely to modify D-galactopyranosyl residues in carbohydrates3*4, to introduce a tritium label into such compounds 5-7, and to analyze for these residues2*‘-“. Several peculiarities of the reaction have gone unanswered, however. For example, the reaction does not go to completion2*5, unusual kinetics are sometimes observed’-‘, and analytical data are not consistent2. As a result, the value of application of this enzyme to structural studies is dubious. The present paper considers the influence of substituents on the rate of oxidation of the D-galactopyranosyl residues, in an attempt to clarify some of the above questions. The effect of chloride, sulfate, phosphate, cyanide, formate, hydrazine, and *This investigation was supported by U.S. Public Health Service Research Grants from the National Heart Institute (HE-06717) and the National Institute of General Medical Sciences (GM-14013). **Present address: Department of Biochemistry, Harvard University, Boston, Massachusetts. TPresent address: Department of Biochemistry, Presbyterian-St. Lukes Hospital, Chicago, Illinois. TfTo whom correspondence should be addressed. Curbohyd_ Res., 7 (1968) 193-199
194
R. A. SCHLEGEL,
C. M. GERBECK,
R. MONTGOMERY
hydroxylamine was first studied. Of these, the last four completely inhibit the enzyme, although the influence of hydroxylamine16 on the coupled peroxidase system complicates its evaluation as an inhibitor. Phosphate of concentrations up to 0.1~ at pH 7.0 was not inhibiting, but, at pH 5.6, a diminution in the rate of oxidation was noted. Sodium sulfate was not inhibiting at concentrations up to O.~M,whereas both sodium chloride and potassium chloride inhibited the reaction noticeably at 0.16hi and to the extent of 45% at 0.4~. The inhibition by chloride may well explain the variability noted by some workers in determining the D-galactose in carbohydrate polymers not oxidized by D-galactose oxidase “*‘*_ The oxidized polymer was hydrolyzed with hydrochloric acid, and the residual D-galactose was determined, after neutralization, by using D-galactose oxidase assay. Such difficuhies would appear to be avoided if sulfuric acid is used for the hydrolysis. Tne extent of oxidation of available D-gaiactose residues in a carbohydrate can be estimated by several methods, all of which have certain disadvantages. Perhaps the simplest is that in which the hydrogen peroxide produced by the oxidation reaction is coupled, through a peroxidase, to a chromogen”. Such a system serves well in estimation of the initial course of the oxidation, but suffers from several disadvantages when the reaction is followed to completion. The color produced is diminished in the presence of other electron acceptor?‘, and is affected by the precipitation of chromogen from solution13. Such side reactions decrease the absorbance value for the final oxidation, although, for o-nitrophenyl c+D-galactopyranoside, 1.5~anhydroD-galactitol, and raffinose, the results so obtained, calcuIated from a standard curve determined with hydrogen peroxide, are of the same order of magnitude as that calculated by a determination of D-galactose residues that were unoxidized (see Table I). As has been noted by Avigad et nL2 and several other workers, the initial velocities of the oxidation reaction differ for various substrates. In certain cases, this factor in itself presents the problem of determining the percentage of D-galactose residues having free hydroxyl groups at C-6 in an unknown carbohydrate. The problem is complicated, however, by the fact that some of the D-galactosyl residues in a carbohydrate having such a free hydroxyl group at C-6 are not oxidized. As may be noted in Table I, a D-galactose residue substituted at O-4 by a methyl group is not a substrate for D-galactose oxidase. This is true for 4-O-methyl-D-galactose and 2,4-di-O-methyl-D-galactose, but not for D-galactose substituted by methyl groups at O-2 or O-3. It may also be noted that 2-amino-2-deoxy-D-galactose residues having the hydroxyl group at C-3 substituted by a methyl group are oxidized a little more slowly than 2-amino-2-deoxy-D-gallactose, which itself is oxidized more slowly than D-galactose. If, however, the hydroxyl group at C-3 of 2-amino-2-deoxy-D-galactose is substituted by a D-glucopyranosyl or a D-glucopyranosyluronic acid residue, the oxidation is completely checked. This inhibition may be the result of steric effects, as can be seen for the slow oxidation of 2’-O-(a-L-fucopyranosyl)lactose and the resistance of uridine 5’-(ac-D-galactopyranosyl pyrophosphate) to oxidation2. From these results, it is therefore not surprising that chondroitin Qsulfate and dermatan Cnr6+‘cL Res.,
7
(1968) 193-199
195
SUBSTRATE SPECIFICITY OF D-GALACTO8E OXIDASE TABLE
I
SUB!XRATE
SPECIFICITY
CIF D-GALACTOSE
OXIDASE
Substratea
Relative rate of oxidation
% oxidized, as determined by Formation
of IY~O~
Park-
.rohllson
Residual D-gdaClOSC
procedure (24 II)
D-Galactose o-Nitrophenyl p-o-galactopyranoside Methyl a-o-galactopyranoside I-O-(a-IJ-GaiactopyranosyB-m_ro-inositol’ 2-O-Methyl-o-galactoseb 3-0-Methyl-o-galactose= 4-0-Methyl-o-galactoseb 2,4-Di-O-methyl-o-galactose’~c 2,3-Di-0-methyl-o-galactoseb
100
57 118 37 50 172 0 0 76
1,2:3,4-Di-0-isopropylidenea-~-galactosc I.5Anhydro-o-galactitol 0-wo-Galactopyranosyl-( I43)-D-galactoseC Raffinose 2’-O-(a-D-Ft~copyranosyl)lactose~ 2-Amino-2-deoxy-o-galactose Methyl 2-acetamido-2-deoxy-3-O-methyk-ogalactopyranosidee Chondrosinee O-fl-D-Glucopyranosyk(1+3)-2-acctamido2-deoxy-o-galactosee O-,%D-Galactopyranosyl-(1+3)-2-acetamido2-deoxy-D-gaiactoser
0 134 48 145 4 71 54
Guaran Ovomucoid Chondroitin 4-sulfatef Dermatan sulfatef
1.50 87 0 0
0 0
86 84 84 60 68
78
86
96
20
95
96
46
98
3
95
0 0
75
0 0
39 76 86
“Reaction solution contained 100 mg of available D-galactose/l. bProvided by Dr. 9. A. Lewis of the University of Minnesota. CProvided by Dr. G. F. Springer of Northwestern University. aProvided by Dr. V. Ginsberg of the National Institutes of Health. eprovided by Dr. R. W. Jeanloz of Harvard University Medical School. fProvided by Dr. J. A. Cifonelli, LaRabida Institute, Chicago.
sulfate, both of which have a free hydroxyl group at C-6 of their 2-amino-2-deoxy-Dgalactose residues, but have substituents at O-3 and O-4, are not oxidized by o-galactose oxidase. Several carbohydrate-containing biopolymers are oxidizable to some extent by D-galactose oxidase, but serious questions must now be raised concemingany structural deductions that can be made from suchinformation. If oxidation can be noted by any of the methods mentioned above, it would seem clear that some of the D-galactopyranosyl residues have free hydroxyl groups at C-6. However, a negative result in no way excludes the possibility of occurrence, within the molecule, Carbohyd. Res., 7 (1968) 193-199
196
R. A. SCHLEGEL,
C. M. GERBECK,
R. MONTGOMERY
of D-galactopyranosyl residues that might have been expected to be oxidized. Further work is continuing on this subject. For a while, it was thought that the incomplete oxidation of the substrates for D-galactose oxidase was due to inhibition by the product. It would seem, however, that this is not the case. A reaction that has come to a halt cannot be significantly reactivated by the addition of fresh n-galactose oxidase, and the extent of oxidation is not significantly different if the initial concentrations of substrate are varied. In the latter instance, it would have been expected that the concentration of product in the reactioa systems wouId have varied; and, with it, the extent of the product inhibition. This behavior was, however, not noted for the oxidation of o-nitrophenyl B-Dgalactopyranoside in concentrations that were varied several-fold. This also is an area for further study. EXPERIMENTAL
Materials. - D-Galactose oxidase was obtained from Worthington Biochemical Company and the Miles Laboratories, Inc. Catalase (beef Iiver, 185,000 units per ml) and peroxidase (horseradish, 400 units per mg) from the Worthington Biochemical Company were used without further purification. #I-D-Galactosidase was obtained from CaIbiochem. The various derivatives of D-galactose were either purchased from Pfanstiehl Chemical Company and used without purification, or were donated as indicated by the footnotes in Table I. DEAE-Cellulose was successively washed with 0.5M hydrochloric acid and 0.5~ sodium hydroxide folowed, each time, by a thorough washing with water. After regeneration with alkali, the DEAE-cellulose was equilibrated with 0.02~ phosphate buffer, pH 7.0. Parr$cation of D-galactose oxidase. Crude D-galactose oxidase (41 mg) from Miles Laboratories was dissoIved in approximately 25 ml of 0.02M phosphate buffer, pH 7.0, and the dark solution was passed through a column (7.0 x 0.9 cm) of DEAE-c&uIose equilibrated with the same buffer. The coIumn was then washed with buffer solution (twice the initial volume). A dark pigment remained at the top of the column. The eluate was collected immediately after addition of the sample, and was divided into two fractions to separate the more-concentrated peak from the tailing fractions. In a typical experiment, D-gaiactose oxidase (17,000 units) was added to a column; the first two-thirds of the eluate contained 13,000 units (250 units per mI) and the remaining one-third of the eluate contained 1,500 units (60 units per ml). Assay of D-galactose oxiabse activity. To one ml of enzyme solution was added 0.4 ml of o-tolidine (recrystahized from acetone-water, 100 mg/l of water), 0.4 ml of peroxidase (100 mg/l of water), and 0.2 ml of 0.25~ D-galactose solution. Ail of the solutions were preheated to 30” and kept at this temperature during the assay, which was continuously recorded at 420 nm with a GiIford spectrophotometer, Model 2000, with a l-cm &&t-path. One unit of euzymic activity corresponds to Cmhhyd.
Res., 7 (1968)
193-199
SUBSTRATE SPECIFICITY OF D-GALACTOSE OXIDASE
197
a net absorbancy of 1.0 at 420 nm after reaction for 10 min at 30”. This value, when converted into moles of hydrogen peroxide produced (and, therefore, D-galactose oxidized) can be redefined as 30 nmoles of D-galactose oxidized per min at 30”. Inhibitor studies. - The inhibitor studies were conducted with a Galactostat kit (Worthington Biochemical Company), from which the e&yme, dissolved in 25 ml of distilled water, and the chromogen solution were kept separated. According to the above assay, the enzyme solution was equivalent to 11.3 units per ml. Studies on the effect of potassium chloride, sodium chloride, sodium sulfate, and sodium formate were made with D-galactose (1.39 x 10w4~) as the substrate. Corresponding studies with sodium cyanide, sodium bisulfite, hydrazine sulfate, and hydroxylamine hydrochloride were made with methyl a-D-galactopyranoside (1.39 x 10V4~) as the substrate. In each instance, 0.4 ml of chromogen solution, 0.4 ml of enzyme solution, and appropriate volumes of the inhibitor solution and the substrate solution (to make a final volume of 1.6 ml) were mixed and allowed to react at 30” or 37”. As in the enzyme assay, the absorbance at 420 nm was measured periodically during 1 h. Relative rates of oxiaktion. The rates of oxidation of various substrates (see Table I) were determined in relation to that of D-galactose. In each case, 0.5 ml of D-galactose oxidase (70 units, putied on DEAE-cellulose), 0.5 ml of peroxidase (100 mg/l), 0.5 ml of o-tolidine (200 mg/l), and 0.5 ml of the substrate solution (the concentration of the reaction solution was 1.39 x 10p4~) were mixed at 30”. The rates of oxidation were followed spectrophotometrically for 10 min at 30”, at 420 nm in a l-cm cell. Extent of oxidation. - Attempts were made to measure the extent of the final oxidation of various substrates by (a) using the color produced by the chromogen, as read against a standard curve determined with hydrogen peroxide, (b) measuring the aldehyde formed, with use of the Park-Johnson reagentslg, or (c) measuring the D-galactose remaining after oxidation had apparently ceased. For o-nitrophenyl j?-D-galactopyranoside, the residual substrate was determined by hydrolysis with j?-D-galactosidase according to the procedure of Avigad’. These various methods will be illustrated below (A-0) by considering the particular substrate, o-nitrophenyl j?-D-galactopyranoside. A. The reaction solution was prepared exactly as already described for the determination of relative rates of oxidation. The color produced by the o-tolidine chromogen was determined continuously in the Gilford spectrophotometer at 420 nm until such time as the color produced was at the maximum. The equivalence of this color to the hydrogen peroxide produced by the reaction was derived from a standard curve. The latter was determined by mixing 0.5 ml of o-tolidine solution (200 mg/l), 0.5 ml of peroxidase solution (100 mg/l), and 1.0 ml of solutions containing various amounts (O-230 nmoles) of hydrogen peroxide in water; the color so produced was read immediately against an appropriate blank. The maximum color, equivalent to 84% of complete oxidation, was obtained in approximately 1 h with o-nitrophenyl /?-D-galactopyranoside. B. By the procedure of Avigad’, a mixture containing 0.5 ml of D-galactose CarbOh~CL Res., 7 (1968) 193-199
198
R. A. SCHLEGEL,
C. M. GBRBECK,
R. MONTGOMERY
oxidase (125 units per ml, purified by DEAE-cellulose chromatography), 0.5 ml of catalase (0.1 ml of commercial suspension diluted to 25 ml with water), 0.5 ml of o-nitrophenyl B-D-galactopyranoside (5.56 x 10m4~), and 0.5 ml of water was allowed to react for 24 h at 30”. A blank determination was made by omitting the glycoside. To 1 ml of the final reaction solution was added an equal volume of B-D-galactosidase (240 mg/l) in 0.01~ phosphate buffer, pH 7.0, which was mu in magnesium chloride. The amount of o-nitrophenol released was determined continuously at 405 nm, and read after 30-45 mi’n when the absorbance was at the maximum. The correspondence of this color to the original concentration of unoxidized o-nitrophenyl B-Dgalactopyranoside was d’etermined from a standard curve. Eight separate determinations gave results that varied from 71-77% of complete oxidation; the average value was 75%. C. The extent of oxidation, followed by the formation of aldehyde groups, was measured by an oxidation system similar to that already described for Avigad’s procedure5, except that samples were taken periodically from the reaction mixture and analyzed by the Park-Johnson procedure lg . The resulting color, read at 690 nm in a Coleman Jr. calorimeter, was converted into equivalents of D-galactose from a preconstructed, standard curve determined with D-galactose. The extent of oxidation, as determined by this procedure, was 75-85% ; average value 78%. D. The alternative procedure of determining the amount of residual D-galactose, after oxidation and hydrolysis with sulfuric acid, used an oxidation system similar to that already described for Avigad’s procedure5. When maximal oxidation had been achieved, as judged by the results by that procedure, 1 ml of the solution was mixed with 1 ml of ISM sulfuric acid, and the mixture was heated for 6 h at 100”. The solution was then de-ionized by passing it through a bed of mixed resin containing Amberlite G-45 (OH-) and Rexyn-101 (H+). The eluate was evaporated to dryness ir; LXZCUU, ihe residue was dissolved in 0.6 ml of distilled water. and the content of D-galactose was determined by means of D-galactose oxidase. The results obtained with other substrates are summarized in Table I. ACKNOWLEDGMENT
The authors are indebted to the colleagues noted in Table I for various derivatives of D-galactose. REFERENCES 1 G.
A-GAD,
4 (1961)
C. ASENSIO, D. ALIARAL,
AND
B. L. HORECKER.
Biochem.
Biophys.
Res.
Comnum.,
474.
2 G. AVIGAD,
D. AMARAL,
C. ASENSIO, AND
B. L. HORECKER,
J. BioI. Chem.,
237 (1962)
2736.
3 J. C. ROBINSON AND J. E. PIERCE, Arch. Biochem. Biophys., 106 (1964) 3484 G. F. SPRINGER, Y. NAGAI, AND H. TEGThlEYER, Biochemisfry, 5 (1966) 3254. 5 G. AVIGAD, Carbohyd. Res., 3 (1967) 430. 6 J. E. G. BARNEXT, Carbohyd. Res., 4 (1967) 7 A.G.
MORELL,C.J.A.VANDENHA~~ER,
241 (1966) Carbohyd_
3745.
Res.,
7 (1968)
193-199
267.
I.H. SCHEINBERG,
AND
G. ASHWELL,
J. BioI.
Chem.,
SUBSTRATE
SPECIFICKY
OF D-GALACTOSE
199
OSIDASE
8 S. A. BARKER, G. PARDOE, AF~DM. STACEY, 1Varrrre.197 (1963) 231. 9 R. M. BRADLEY AND J. N. KANFER, Biochim. Biophys. Acta, 84 (1964) 210. 10 C. H. DEVERDIERAND M. HJELhr, Clin. Chinr. Acta, 7 (1962) 742. 11 W. FISCHERAND J. ZAPF, 2. Ph_wiol. Chem., 337 (1964) 186. 12 W. FISCHERAND J. ZAPF, Z. Physiol. Chem., 339 (1964) 54. 13 M. HELM, Cfin. C/rim. Acta, 15 (1967) 87. 14 E. ROREM AND C. LEWIS, Anal. Biochem., 3 (1962) 230. 15 J. SELIPERE, C. GANCEDO, AND C. A.SENSIO,And. Biochenr., 12 (1965) 590. 16 D. &ARAL, L. BERNSTEIN,D. MORSE, AND B. L. HORECKER,J. Biol. Chent., 238 (1963) 2281. 17 W. BUTLER AND L. CuNNtxGHAh~, J. Bid. Chem.. 241 (1966) 3882. 18 U. LINDAHL, Biochim. Bioph_vs. Acts, 130 (1966) 361. 19 J. T. PARK AND M. J. JOHNSON, J. Biol. Chem., 181 (1949) 149.
Curbohyd.
Res., 7 (1968) 193-199