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An NADH coupled assay system for galactose oxidase
ANALYTICAL
BIOCHEMISTRY
86,
An NADH Coupled
470-476 (1978)
Assay System for Galactose
Oxidase
GAD AVIGAD Department
of Biochemistry, College of Medicine and Dentistry of New Medical School, Piscataway. N. J. 08854
Jersey,
Rutgers
Received August 1, 1977; accepted December 16, 1977 A galactose oxidase (EC 1.1.3.9):NADH-peroxidase (EC 1.11.1.1) coupled assay system is used for the estimation of galactose oxidase activity. Spectrophotometric measurement of NADH consumption yields direct quantitative value of enzymic activity or can be used for the end-point determination of the amount of galactose oxidase substrate present in test solutions. Use of similar coupled systems is suggested for the assay of other H,O,-producing enzymes and their substrates.
Galactose oxidase (EC 1.1.3.9) from Dactylium dendroides is a useful tool for the detection and labeling of glycoconjugates and also is often employed for the specific quantitative analysis of galactose and galactosides (1,2). The common procedure for the quantitative assay of galactose oxidase activity is a spectrophotometric method which involves coupling of the enzyme with a horseradish peroxidase-chromogen system (3,4). Among the chromogens employed, o-dianisidine (3,4) is probably the most popular. Use of different chromogens such as o-cresol(5), benzidine (6), and o-tolidine (7,8) as well as others, has also been recommended. Alternative procedures introduced for the estimation of galactose oxidase activity were fluorimetric (9), manometric (10,l l), amperometric (12), coulometric (13), and polarographic using the Clark oxygen electrode (14- 16). These methods were useful for specific analytical purposes or for the study of enzyme mechanisms, but failed to be adopted for standard routine assays of enzyme activity or for the quantitative determination of substrates. This failure was due to several reasons, such as: a requirement for highly specialized equipment or for a relatively large volume of reaction mixture; the disadvantages of a discontinuous assay; in some cases, by a lower degree of sensitivity compared to the peroxidase-chromogen assay procedure: an inhibition of the enzyme by the H,O, produced in the reaction or by other reagents introduced into the system in order to carry out the titration. The peroxidase-coupled system was found to have several disadvantages. An important one is the difficulty in exactly estimating the specific molar extinction coefficient of the chromogen obtained in the peroxidase 0003.2697/78/0862-0470$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
470
GALACTOSE
OXIDASE
ASSAY
471
reaction (several values can be found in the literature). As a consequence, enzyme activities are usually recorded in arbitrary optical density units rather than in moles of substrate oxidized. In addition, the chromogen was often found to inhibit galactose oxidase, to be sensitive to light or to sulfhydryl reagents, to tend to precipitate, to require the introduction of organic solvent into the reaction mixture, and to be effected by pH. Many of these chromogens are known to be carcinogenic and their use is now severely restricted. It has also been noted that horseradish peroxidase itself can have an activating effect on galactose oxidase (16- 19). It is obvious that establishment of an alternative simple, sensitive, and inexpensive method for the quantitative determination of galactose oxidase activity would be of a significant benefit to the biochemist. In the present communication, the assay of galactose oxidase using a coupled system with NADH peroxidase (EC 1.11.1.1; Ref. 20) is recommended. The reaction is straightforward, involves the use of a relatively stable, commercially available NADH peroxidase, and employs standard uv-spectrophotometric procedures which are simple to quantify. The method is adaptable both for the estimation of rates of enzymic activity and for the quantitative determination of the amount of substrate oxidized. MATERIALS
AND METHODS
Streptococcus fuecalis NADH peroxidase was obtained from Boehringer-Mannheim Biochemicals as a suspension in ammonium sulfate solution. When assayed in 0.05 M acetate buffer, pH 5.5, it had a specific activity of 48 units/mg of protein. In 0.05 M phosphate buffer, pH 7.0, its specific activity was 9 units/mg, whereas in the same pH 7.0 buffer supplemented with 0.05 M sodium acetate its specific activity was 26 units/mg of protein (20). The enzyme was diluted in 1% bovine serum albumin (BSA) in 0.1 M phosphate buffer, pH 7.0, to provide a stock solution of about 2 units/ml. Purified Dactyfium dendroides galactose oxidase was purchased from Worthington Biochemical Co. It had a specific activity of 440 o-tolidine units/mg, or 98 o-dianisidine unitsimg when determined by the Worthington’s Galactostat assay. It was dissolved in 1% BSA in 0.1 M phosphate buffer, pH 7.0, to yield a stock enzyme solution of about 0.5 mg/ml. If they were required, all further dilutions of NADH peroxidase and of galactose oxidase stock solutions were done in the buffered 1% BSA solution. NADH disodium salt was obtained from Boehringer-Mannheim, serum acid glycoprotein was obtained from Miles Laboratories, Inc., and o-tolidine and o-dianisidine chromogens were obtained from Worthington Biochemical Co. All other biochemical reagents used were purchased from Sigma Chemical Co. Spectrophotometry was conducted in Gilford Model 240 or Cary Model
472
GAD
AVIGAD
118 recording spectrophotometers using quartz cuvettes with a 1.O-cm light path. An A,,,’ cm value of 6.22 x lo3 M-km-’ for NADH was used to calculate molar concentrations and specific activities. A unit of enzyme activity was that amount which oxidized 1.0 pmol of NADH/min at standard assay conditions at pH 7.0. Calorimetric assays of galactose oxidase in the coupled horseradish peroxidase system with o-dianisidine or o-tolidine as the chromogens were carried out using standard procedures (4,8). Units of activity in these assays was equivalent to an increase in optical density of 1.O per minute at 425 nm. Procedures Assay of NADH peroxiduse activity. The reaction mixture (1.0 ml) contained, in micromoles: sodium phosphate buffer, pH 7.0, 60; sodium acetate, 60; NADH, 0.14; and HzOz, 25. NADH peroxidase in the range of 0.002 to 0.06 units was added to start the reaction. Decrease in absorption at 340 nm was followed spectrophotometrically. A control system without H,O, was examined to obtain a measure of NADH-oxidase activity exhibited by the NADH-peroxidase. For the enzyme preparations used in this study, the value for NADH oxidase was 1.3% of that of NADH peroxidase activity. Assay of galactose oxiduse activity. The reaction mixture (1.0 ml) was similar in composition to the system described above for NADHperoxidase with these exceptions: H,O, was excluded, and 40 mM of galactose and 0.05 to 0.08 units of NADH peroxidase were added. Inclusion of a larger excess of peroxidase will unnecessarily elevate the contribution of NADH oxidase activity to the overall observed rate of NADH consumption. Galactose oxidase, 0.002 to 0.05 units, was added to start the reaction. HzO, produced by the action of galactose oxidase on galactose is used to oxidize NADH by the peroxidase, and the rate of decline in absorption at 340 nm will therefore provide a measure of galactose oxidase activity. It is important for this assay that the amount of NADH peroxidase units in the system exceeds that of galactose oxidase. The baseline control value observed for the low rate of NADH oxidase activity should be deducted from the initial rates observed in the coupled system, particularly when assaying very low levels of galactose oxidase activity. The same coupled system described above for galactose can be used to determine galactose oxidase activity on other substrates (1,4,8). When the maximum rate of oxidation of a tested substrate is significantly higher or lower than that of galactose, the amount of galactose oxidase added to the assay solution should be increased or decreased accordingly, in order to yield measureable rates of NADH oxidation (Table 1).
GALACTOSE
OXIDASE
ASSAY
473
Use of the coupled system for the end-point determination of galactose and other galactose oxidase substrates in solution. The reaction system (1.0 ml) contained, in micromoles: sodium phosphate buffer, pH 7.0, 60; sodium acetate, 60; NADH, 0.15 or 0.30 (according to the selected initial setting of the spectrophotometer); a sample of galactose to be assayed, 0.005-0.30: NADH peroxidase, 0.06-0.15 units; and galactose oxidase, added last, 0.10-0.50 units. The rapid drop in absorption at 340 nm is followed until it reaches the rate of that of the NADH oxidase baseline. Similar to galactose, other galactose oxidase substrates (1) could be determined by the same procedure. RESULTS
AND DISCUSSION
The K, values for NADH and H,O, for NADH peroxidase were found to be 2.5 x 10-j and 1.2 x lo-” M, respectively, when determined at the test conditions described in this paper. These values indicate that NADH peroxidase can effectively be used to trap H202 at a concentration range suitable for a standard spectrophotometric assay of reduced pyridine nucleotide. One international unit of galactose oxidase in the coupled NADH peroxidase system (when measured at pH 7.0) was found in the present test to be equivalent to 6.4o-dianisidine units or 28.8 o-tolidine units of activity based on the arbitrary change in absorption value (4,7,8) per minute. These relative values for units of activity are close to those which were obtained for galactose oxidase activity in a coulometric assay procedure and compared to the calorimetric methods (21). In other studies it was estimated that 1.O international unit of galactose oxidase was equivalent to 3 o-dianisidine units when assayed in the horseradish peroxidase coupled assay (22). This may indicate differences in the efficiency of H20Z removal from the galactose oxidase active site. It could also reflect varying degrees of activation of galactose oxidase by different types of peroxidases (19). These differences may also reflect uncertainties about the exact specific absorption coefficient of the o-dianisidine chromogen formed by peroxidase action. Under the assay conditions described here. NADH oxidation in the coupled system commenced with an insignificant lag period of 10 to 15 sec. The horseradish peroxidase coupled assay system for galactose oxidase often starts with a significant lag period before attaining zero-order kinetics to allow the calculation of initial rates of oxidation. The present method was found to be useful for the determination of amounts of galactose and simple galactosides in the lOed to 10-j M range of concentration. The procedure described can most probably be adapted for different ranges of sensitivity by modifications in reaction mixture volumes and in the light path of the cuvette used. A complete molar stoichiometry was observed between the amount of NADH oxidized and the amount of
474
GAD
AVIGAD
substrate added to the coupled system in the concentration range described. This stoichiometry was obtained for galactose, raffinose, melibiose, and methyl-P-D-galactoside when tested in detail as model substrates. Similar stoichiometry is expected for the oxidation of any other galactose oxidase substrate. As an example of the use of the present NADH peroxidase coupled procedure for the assay ofgalactose oxidase activity, several kinetic values TABLE OXIDATION
OF VARIOUS
Compound D-galactose Dihydroxyacetone N-Acetyko-galactosamine Methyl-P-o-galactoside O-Nitrophenyl-P-thiogalactoside Lactose Melibiose Raffinose Stachyose Guar gum Locust bean gum Thyroglobuline. porcine desialylated thryoglobuline Transfer+ human desialylated transferrin Acid glycoprotein. bovine plasma desialylated acid glycoprotein Mucin, bovine submaxillary desialylated mucin Fetuin, calf serum desialylated fetuin
SUBSTRATES
Highest concentration in assay 40 rnM 40 rnM 8 mM 10 rnM IrnM 20 rnM 16 mM 20 rnM 4rnM 0.2% 0.2% 0.35% 0.35% 0.35% 0.35% 0.35% 0.35% 0.35% 0.35% 0.35% 0.35%
I BY GALACTOSE
Sugar contents” (%)
OXIDASE"
Sialic acid contents” (%)
9
1.02
6
1.45
10
3.97
42
9.30
20
5.42
Relative oxidation rate 100 430 93 260 12 12 84 150 210 37 44 co.4 I.1 0.6 1.1 co.4 2.5 <0.4 6.3 0.5 2.1
K, (mM) 14.3 13.3 2.0 6.7 5.5 25.0 2.0 2.5 0.5” 0.7”
0.3’ 0.4’ 0.5’
I’ Standard NADH-coupled spectrophotometric assay system included 0.02-0.50 units of galactose oxidase according to the relative activity observed for different substrates. Under these assay conditions the lowest level of detection was equivalent to 0.4% of the rate observed for galactose. !’ Assayed by the phenol-sulfuric acid procedure (23). o Sialic acid was determined by the thiobarbiturate assay (24). Glycoproteins were desialylated in 0.1 N HCI at 80°C for 1 hr then neutralized before enzyme assay. d Calculated as described previously (4) assuming a galactosyl content of 33% for guar gum and 20% for locust bean gum (25). I’ Concentrations of the available terminal galactosyl and N-acetyl-o-galactosylamine residues were roughly estimated from the sialic acid contents determined for these glycoproteins and from literature values (26, 27).
GALACTOSE
OXIDASE
ASSAY
475
for a selected list of various substrates were obtained using this method. These findings are summarized in Table 1. The values reported here are generally comparable to those found in previous studies (1,4,8,16). They are particularly close to those derived in studies which used oxygen uptake monitoring procedures for the analysis of galactose oxidase, and the K, values are lower than those obtained by the horseradish peroxidase coupled system. The results in Table 1 also show that the present assay procedure could be used to follow the oxidation of galactoside-containing polysaccharides and glycoproteins which are susceptible to oxidation by galactose oxidase. Removal from the glycoproteins of the terminal N-acetylneuraminic acid (sialic acid) residues, which usually mask penultimate D-galactosyl or N-acetyl-D-galactosylamine moieties in the oligosaccharide structure (26,2J), rendered the glycoprotein more susceptible to attack by galactose oxidase (28). Preliminary qualitative tests have indicated that a similar coupledenzyme system may be employed for the measurement of other H,O,-producing oxidases. For example, coupling NADH peroxidase to glucose oxidase (EC 1.1.3.4), uricase (EC 1.J.3.3), cholesterol oxidase (EC 1.1.3.6), D-aminoacidoxidase(EC 1.4.3.3),diamineoxidase(EC 1.4.3.6), or xanthine oxidase (EC 1.2.3.2) could provide a method for evaluating enzyme activity as well as for an end-point assay of the oxidase’s substrate. The feasibility of these assays was successfully tested with commercial purified enzyme preparations. The procedure most probably will not be useful if NADH or H202 are to be consumed by interfering contaminating activities present in crude enzyme solutions or biological fluids. ACKNOWLEDGMENTS I thank Mrs. Martha grant from the Rutgers
Strizzi for competent technical help. This work was supported Medical School General Research Support Fund.
by a
REFERENCES 1. Malstrom, B. G., Andreasson. L. E., and Reinhammar, B. (1975)in The Enzymes (Bayer. P. D., ed.) 3rd ed. Vol. 12B. pp. 507-533, Academic Press, New York. 2. Gahmberg, C. G.. Itaya, K.. and Hakomori. S. I. (1976) Methods Membr. Biol. 7, 179-210. 3. Avigad, G., Asensio. C.. Amaral, D., and Horecker. B. L. (1961)Eiochem. Biophys. Res. Commun. 6, 474-477. 4. Avigad, G., Amaral. D.. Asensio, C., and Horecker, B. L. (1962) J. Bid/. Chern. 237, 2736-2743. 5. Fischer, W., and Zapf, J. (1964) Hoppe-Seyler’s Z. Phgsiol. Chem. 337, 186-195. 6. Roth, H., Segal. S., and Bertoli, D. (1965) Anal. Biochem. 10, 32-52. 7. Forster, H., and Haslbeck, M. (1967) Z. K/in. Chem. K/in. Biochem. 5, 198-202. 8. Schlegel. R. A., Gerbeck. C. M., and Montgomery, R. (1968)Carhohyd. Res. 7, 193-199. 9. Guilbault. G. G., Brignac, P. J., and Juneau. M. (1968) A&. C/rem. 40, 1256-1263. IO. Cooper. J. A. D., Smith, W., Bacila, M., and Medina, H. (1959) J. Bjol. C/rem. 234, 445-448.
476
GAD AVIGAD
1 I. Hamilton, G. A., DeJersey, J., and Adolf, P. K. (1973) in Oxidases and Related Redox Systems (King, T., Mason, H. S., and Morrison, M., eds.) Vol. I, pp. 103-124, University Press, Baltimore, Md. 12. Pardue, H. L., and Frings, C. S. (1964) J. Elecfroanal. Chem. 7, 398-402. 13. McGlothlin, C. D., and Purdy, W. C. (1977) Anal. Chim. Acta 88, 33-39. 14. Amaral, D.. and Bacila, M. (1967) Arquiv. Biologia Technol. Brazil 12, 179-186. 15. Zancan, G. T., and Amaral, D. (1970) Biochim. Biophys. Acta 198, 146-147. 16. Cleveland, L., Coffman, R. E., Coon, P., and Davis, L. (1975) Biochemistry 14, 1108-1115. 17. Kwiatkowski, L. D., and Kosman. D. J. (1973) Biochem. Biophys. Res. Commun. 53, 715-721. 18. Hamilton, G. A., Libby, L. R., and Harzell, C. R. (1973) Biochem. Biophys. Res. Commun.
55, 333-340.
19. Olsen, J. (1976) Ph.D. Thesis, University of Iowa; Dissert. Abst. In{. B 37, 3934 (1977). 20. Dolin. M. I. (1957) J. Biol. Chern. 225, 557-573. 21. McGlothlin, C. D. (1973) Ph.D. Thesis, University of Maryland;Disserr. Abst. Int. B 35, 66 (1974). 22. Kosman, D. J., Ettinger, M. J., Weiner, R. E., and Massaro, E. J. (1974)Arch. Biochem. Biophys. 165, 456-467. 23. Ashwell, G. (1966) in Methods in Enzymology, Vol. 8, pp. 93-95, Academic Press, New York. 24. Spiro, R. G. (1966)in Methods in Enzymology, Vol. 8, pp. 16-18, Academic Press, New York. 25. Davis, S. E., and Lewis, B. A. (1975) ACS Symposium Series (Jeans, A., and Hodge. J., eds.), Vol. 15, pp. 296-311. Amer. Chem. Sot.. Washington, D. C. 26. Spiro. R. G. (1973) Adv. Protein Chem. 27, 349-467. 27. Kornfeld, R., and Kornfeld, S. (1976) Ann. Rev. Biochem. 45, 217-237. 28. Morel], A. G., and Ashwell, G. (1972) in Methods in Enzymology, Vol. 28, pp. 205-208, Academic Press, New York.
BIOCHEMISTRY
86,
An NADH Coupled
470-476 (1978)
Assay System for Galactose
Oxidase
GAD AVIGAD Department
of Biochemistry, College of Medicine and Dentistry of New Medical School, Piscataway. N. J. 08854
Jersey,
Rutgers
Received August 1, 1977; accepted December 16, 1977 A galactose oxidase (EC 1.1.3.9):NADH-peroxidase (EC 1.11.1.1) coupled assay system is used for the estimation of galactose oxidase activity. Spectrophotometric measurement of NADH consumption yields direct quantitative value of enzymic activity or can be used for the end-point determination of the amount of galactose oxidase substrate present in test solutions. Use of similar coupled systems is suggested for the assay of other H,O,-producing enzymes and their substrates.
Galactose oxidase (EC 1.1.3.9) from Dactylium dendroides is a useful tool for the detection and labeling of glycoconjugates and also is often employed for the specific quantitative analysis of galactose and galactosides (1,2). The common procedure for the quantitative assay of galactose oxidase activity is a spectrophotometric method which involves coupling of the enzyme with a horseradish peroxidase-chromogen system (3,4). Among the chromogens employed, o-dianisidine (3,4) is probably the most popular. Use of different chromogens such as o-cresol(5), benzidine (6), and o-tolidine (7,8) as well as others, has also been recommended. Alternative procedures introduced for the estimation of galactose oxidase activity were fluorimetric (9), manometric (10,l l), amperometric (12), coulometric (13), and polarographic using the Clark oxygen electrode (14- 16). These methods were useful for specific analytical purposes or for the study of enzyme mechanisms, but failed to be adopted for standard routine assays of enzyme activity or for the quantitative determination of substrates. This failure was due to several reasons, such as: a requirement for highly specialized equipment or for a relatively large volume of reaction mixture; the disadvantages of a discontinuous assay; in some cases, by a lower degree of sensitivity compared to the peroxidase-chromogen assay procedure: an inhibition of the enzyme by the H,O, produced in the reaction or by other reagents introduced into the system in order to carry out the titration. The peroxidase-coupled system was found to have several disadvantages. An important one is the difficulty in exactly estimating the specific molar extinction coefficient of the chromogen obtained in the peroxidase 0003.2697/78/0862-0470$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
470
GALACTOSE
OXIDASE
ASSAY
471
reaction (several values can be found in the literature). As a consequence, enzyme activities are usually recorded in arbitrary optical density units rather than in moles of substrate oxidized. In addition, the chromogen was often found to inhibit galactose oxidase, to be sensitive to light or to sulfhydryl reagents, to tend to precipitate, to require the introduction of organic solvent into the reaction mixture, and to be effected by pH. Many of these chromogens are known to be carcinogenic and their use is now severely restricted. It has also been noted that horseradish peroxidase itself can have an activating effect on galactose oxidase (16- 19). It is obvious that establishment of an alternative simple, sensitive, and inexpensive method for the quantitative determination of galactose oxidase activity would be of a significant benefit to the biochemist. In the present communication, the assay of galactose oxidase using a coupled system with NADH peroxidase (EC 1.11.1.1; Ref. 20) is recommended. The reaction is straightforward, involves the use of a relatively stable, commercially available NADH peroxidase, and employs standard uv-spectrophotometric procedures which are simple to quantify. The method is adaptable both for the estimation of rates of enzymic activity and for the quantitative determination of the amount of substrate oxidized. MATERIALS
AND METHODS
Streptococcus fuecalis NADH peroxidase was obtained from Boehringer-Mannheim Biochemicals as a suspension in ammonium sulfate solution. When assayed in 0.05 M acetate buffer, pH 5.5, it had a specific activity of 48 units/mg of protein. In 0.05 M phosphate buffer, pH 7.0, its specific activity was 9 units/mg, whereas in the same pH 7.0 buffer supplemented with 0.05 M sodium acetate its specific activity was 26 units/mg of protein (20). The enzyme was diluted in 1% bovine serum albumin (BSA) in 0.1 M phosphate buffer, pH 7.0, to provide a stock solution of about 2 units/ml. Purified Dactyfium dendroides galactose oxidase was purchased from Worthington Biochemical Co. It had a specific activity of 440 o-tolidine units/mg, or 98 o-dianisidine unitsimg when determined by the Worthington’s Galactostat assay. It was dissolved in 1% BSA in 0.1 M phosphate buffer, pH 7.0, to yield a stock enzyme solution of about 0.5 mg/ml. If they were required, all further dilutions of NADH peroxidase and of galactose oxidase stock solutions were done in the buffered 1% BSA solution. NADH disodium salt was obtained from Boehringer-Mannheim, serum acid glycoprotein was obtained from Miles Laboratories, Inc., and o-tolidine and o-dianisidine chromogens were obtained from Worthington Biochemical Co. All other biochemical reagents used were purchased from Sigma Chemical Co. Spectrophotometry was conducted in Gilford Model 240 or Cary Model
472
GAD
AVIGAD
118 recording spectrophotometers using quartz cuvettes with a 1.O-cm light path. An A,,,’ cm value of 6.22 x lo3 M-km-’ for NADH was used to calculate molar concentrations and specific activities. A unit of enzyme activity was that amount which oxidized 1.0 pmol of NADH/min at standard assay conditions at pH 7.0. Calorimetric assays of galactose oxidase in the coupled horseradish peroxidase system with o-dianisidine or o-tolidine as the chromogens were carried out using standard procedures (4,8). Units of activity in these assays was equivalent to an increase in optical density of 1.O per minute at 425 nm. Procedures Assay of NADH peroxiduse activity. The reaction mixture (1.0 ml) contained, in micromoles: sodium phosphate buffer, pH 7.0, 60; sodium acetate, 60; NADH, 0.14; and HzOz, 25. NADH peroxidase in the range of 0.002 to 0.06 units was added to start the reaction. Decrease in absorption at 340 nm was followed spectrophotometrically. A control system without H,O, was examined to obtain a measure of NADH-oxidase activity exhibited by the NADH-peroxidase. For the enzyme preparations used in this study, the value for NADH oxidase was 1.3% of that of NADH peroxidase activity. Assay of galactose oxiduse activity. The reaction mixture (1.0 ml) was similar in composition to the system described above for NADHperoxidase with these exceptions: H,O, was excluded, and 40 mM of galactose and 0.05 to 0.08 units of NADH peroxidase were added. Inclusion of a larger excess of peroxidase will unnecessarily elevate the contribution of NADH oxidase activity to the overall observed rate of NADH consumption. Galactose oxidase, 0.002 to 0.05 units, was added to start the reaction. HzO, produced by the action of galactose oxidase on galactose is used to oxidize NADH by the peroxidase, and the rate of decline in absorption at 340 nm will therefore provide a measure of galactose oxidase activity. It is important for this assay that the amount of NADH peroxidase units in the system exceeds that of galactose oxidase. The baseline control value observed for the low rate of NADH oxidase activity should be deducted from the initial rates observed in the coupled system, particularly when assaying very low levels of galactose oxidase activity. The same coupled system described above for galactose can be used to determine galactose oxidase activity on other substrates (1,4,8). When the maximum rate of oxidation of a tested substrate is significantly higher or lower than that of galactose, the amount of galactose oxidase added to the assay solution should be increased or decreased accordingly, in order to yield measureable rates of NADH oxidation (Table 1).
GALACTOSE
OXIDASE
ASSAY
473
Use of the coupled system for the end-point determination of galactose and other galactose oxidase substrates in solution. The reaction system (1.0 ml) contained, in micromoles: sodium phosphate buffer, pH 7.0, 60; sodium acetate, 60; NADH, 0.15 or 0.30 (according to the selected initial setting of the spectrophotometer); a sample of galactose to be assayed, 0.005-0.30: NADH peroxidase, 0.06-0.15 units; and galactose oxidase, added last, 0.10-0.50 units. The rapid drop in absorption at 340 nm is followed until it reaches the rate of that of the NADH oxidase baseline. Similar to galactose, other galactose oxidase substrates (1) could be determined by the same procedure. RESULTS
AND DISCUSSION
The K, values for NADH and H,O, for NADH peroxidase were found to be 2.5 x 10-j and 1.2 x lo-” M, respectively, when determined at the test conditions described in this paper. These values indicate that NADH peroxidase can effectively be used to trap H202 at a concentration range suitable for a standard spectrophotometric assay of reduced pyridine nucleotide. One international unit of galactose oxidase in the coupled NADH peroxidase system (when measured at pH 7.0) was found in the present test to be equivalent to 6.4o-dianisidine units or 28.8 o-tolidine units of activity based on the arbitrary change in absorption value (4,7,8) per minute. These relative values for units of activity are close to those which were obtained for galactose oxidase activity in a coulometric assay procedure and compared to the calorimetric methods (21). In other studies it was estimated that 1.O international unit of galactose oxidase was equivalent to 3 o-dianisidine units when assayed in the horseradish peroxidase coupled assay (22). This may indicate differences in the efficiency of H20Z removal from the galactose oxidase active site. It could also reflect varying degrees of activation of galactose oxidase by different types of peroxidases (19). These differences may also reflect uncertainties about the exact specific absorption coefficient of the o-dianisidine chromogen formed by peroxidase action. Under the assay conditions described here. NADH oxidation in the coupled system commenced with an insignificant lag period of 10 to 15 sec. The horseradish peroxidase coupled assay system for galactose oxidase often starts with a significant lag period before attaining zero-order kinetics to allow the calculation of initial rates of oxidation. The present method was found to be useful for the determination of amounts of galactose and simple galactosides in the lOed to 10-j M range of concentration. The procedure described can most probably be adapted for different ranges of sensitivity by modifications in reaction mixture volumes and in the light path of the cuvette used. A complete molar stoichiometry was observed between the amount of NADH oxidized and the amount of
474
GAD
AVIGAD
substrate added to the coupled system in the concentration range described. This stoichiometry was obtained for galactose, raffinose, melibiose, and methyl-P-D-galactoside when tested in detail as model substrates. Similar stoichiometry is expected for the oxidation of any other galactose oxidase substrate. As an example of the use of the present NADH peroxidase coupled procedure for the assay ofgalactose oxidase activity, several kinetic values TABLE OXIDATION
OF VARIOUS
Compound D-galactose Dihydroxyacetone N-Acetyko-galactosamine Methyl-P-o-galactoside O-Nitrophenyl-P-thiogalactoside Lactose Melibiose Raffinose Stachyose Guar gum Locust bean gum Thyroglobuline. porcine desialylated thryoglobuline Transfer+ human desialylated transferrin Acid glycoprotein. bovine plasma desialylated acid glycoprotein Mucin, bovine submaxillary desialylated mucin Fetuin, calf serum desialylated fetuin
SUBSTRATES
Highest concentration in assay 40 rnM 40 rnM 8 mM 10 rnM IrnM 20 rnM 16 mM 20 rnM 4rnM 0.2% 0.2% 0.35% 0.35% 0.35% 0.35% 0.35% 0.35% 0.35% 0.35% 0.35% 0.35%
I BY GALACTOSE
Sugar contents” (%)
OXIDASE"
Sialic acid contents” (%)
9
1.02
6
1.45
10
3.97
42
9.30
20
5.42
Relative oxidation rate 100 430 93 260 12 12 84 150 210 37 44 co.4 I.1 0.6 1.1 co.4 2.5 <0.4 6.3 0.5 2.1
K, (mM) 14.3 13.3 2.0 6.7 5.5 25.0 2.0 2.5 0.5” 0.7”
0.3’ 0.4’ 0.5’
I’ Standard NADH-coupled spectrophotometric assay system included 0.02-0.50 units of galactose oxidase according to the relative activity observed for different substrates. Under these assay conditions the lowest level of detection was equivalent to 0.4% of the rate observed for galactose. !’ Assayed by the phenol-sulfuric acid procedure (23). o Sialic acid was determined by the thiobarbiturate assay (24). Glycoproteins were desialylated in 0.1 N HCI at 80°C for 1 hr then neutralized before enzyme assay. d Calculated as described previously (4) assuming a galactosyl content of 33% for guar gum and 20% for locust bean gum (25). I’ Concentrations of the available terminal galactosyl and N-acetyl-o-galactosylamine residues were roughly estimated from the sialic acid contents determined for these glycoproteins and from literature values (26, 27).
GALACTOSE
OXIDASE
ASSAY
475
for a selected list of various substrates were obtained using this method. These findings are summarized in Table 1. The values reported here are generally comparable to those found in previous studies (1,4,8,16). They are particularly close to those derived in studies which used oxygen uptake monitoring procedures for the analysis of galactose oxidase, and the K, values are lower than those obtained by the horseradish peroxidase coupled system. The results in Table 1 also show that the present assay procedure could be used to follow the oxidation of galactoside-containing polysaccharides and glycoproteins which are susceptible to oxidation by galactose oxidase. Removal from the glycoproteins of the terminal N-acetylneuraminic acid (sialic acid) residues, which usually mask penultimate D-galactosyl or N-acetyl-D-galactosylamine moieties in the oligosaccharide structure (26,2J), rendered the glycoprotein more susceptible to attack by galactose oxidase (28). Preliminary qualitative tests have indicated that a similar coupledenzyme system may be employed for the measurement of other H,O,-producing oxidases. For example, coupling NADH peroxidase to glucose oxidase (EC 1.1.3.4), uricase (EC 1.J.3.3), cholesterol oxidase (EC 1.1.3.6), D-aminoacidoxidase(EC 1.4.3.3),diamineoxidase(EC 1.4.3.6), or xanthine oxidase (EC 1.2.3.2) could provide a method for evaluating enzyme activity as well as for an end-point assay of the oxidase’s substrate. The feasibility of these assays was successfully tested with commercial purified enzyme preparations. The procedure most probably will not be useful if NADH or H202 are to be consumed by interfering contaminating activities present in crude enzyme solutions or biological fluids. ACKNOWLEDGMENTS I thank Mrs. Martha grant from the Rutgers
Strizzi for competent technical help. This work was supported Medical School General Research Support Fund.
by a
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