- Email: [email protected]
Urease Assay Using a Rapid Radiometric Procedure
NOTES & TIPS 2. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132, 6–13. 3. Feinberg, A. P., and Vogelstein, B. (1984) Anal. Biochem. 137, 266–267. 4. Schowalter, D. B., and Bonner, S. S. (1989) Anal. Biochem. 177, 90–94. 5. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R. (1984) Nucleic Acids Res. 12, 7035–7056. 6. Verbeek, V., and Tijssen, T. (1990) J. Virol. Methods 29, 243– 256. 7. Sturzl, M., and Roth, W. K. (1990) Anal. Biochem. 185, 164–169. 8. Bird, I. M., Mason, J. I., and Rainey, W. E. (1994) Endocrinology 134, 2468–2474. 9. Bradshaw, K. D., Waterman, M. R., Couch, R. T., Simpson, E. R., and Zuber, M. X. (1987) Mol. Endocrinol. 1, 348–354. 10. Bird, I. M., Mason, J. I., and Rainey, W. E. (1995) Endocrinology 136, 5677–5684. 11. Linz, U., Delling, U., and Rubsamen-Waigmann, H. (1990) J. Clin. Chem. Clin. Biochem. 28, 5–13. 12. Millican, D. S., and Bird, I. M. (1997) in Methods in Molecular Bilology: Phospholipids and Cell Signalling Protocols (Bird, I. M., and Graham, J., Eds.), Humana Press, NJ. 13. Scully, S. P., Joyce, M. E., Abidi, N., and Bolander, M. E. (1990) Mol. Probes 4, 485–495. 14. Finckh, U., Lingenfelter, P. A., and Myerson, D. (1991) Biotechniques 10(1), 35–38. 15. Gyllensten, U. B., and Erlich, H. A. (1988) Proc. Natl. Acad. Sci. USA 85, 7652–7656. 16. Mertz, L. M., and Rashtchian, A. (1994) Anal. Biochem. 221, 160–165. 17. Tombline, G., Bellizzi, D., and Sgaramella, V. (1996) Proc. Natl. Acad. Sci. USA 93, 2724–2728.
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tive dyes are incorporated in the system to measure the activity spectrophotometrically (2) or with a pH electrode (3). Urease was also determined by coupling it with NADH-dependent glutamate dehydrogenase (4). In a radiometric technique, the reaction was carried out in specialized metabolic flasks (5). To determine urease, we adopted the Buddemeyer procedure (6). Purified urease from jack bean (Concanavalia ensiformis) and acetohydroxamide were purchased from Sigma Chemical Co. (St. Louis, MO) and [14C]urea (55 mCi/mmol) from American Radiolabelled Chemicals (St. Louis, MO). The reaction was done in 4 1 1.5-cm screw-capped glass vials. The reaction consisted of 1.0 ml 0.2 M phosphate buffer, pH 6.3, appropriate concentrations of [14C]urea, in 5 ml; and appropriate units of enzyme [see figure legends]. The glass vials were loosely capped and placed in poly-Q scintillation vials (Beckman Instruments, Fullerton, CA) containing 4 1 2-cm strips of Whatman No. 1 filter paper, previously dipped in concentrated Liquifluor (DuPont New England Nuclear, Boston, MA) and 2 N NaOH, then air-dried. The scintillation vials were capped tightly and incubated at 377C. (A typical protocol would include measuring the amount of 14CO2 released from 18.0 nmol of substrate, [14C]urea, incubated in the presence of 77.5 units of enzyme from 2 to 4 h at 377C.) To study the effect of a urease inhibitor, acetohydroxamate, it was preincubated with the enzyme for 10 min. At prescribed time intervals, the cumulative CO2 released in each vial was measured in a Beckman Model LS 60001C liquid scintillation counter.
Urease Assay Using a Rapid Radiometric Procedure Eugene B. Harris,1 Baljit Randhawa, and K. Prabhakaran Laboratory Research Branch, GWL HD Center at LSU, U.S. Public Health Service, South Stadium Drive, P.O. Box 25072, Baton Rouge, Louisiana 70894-5072 Received January 13, 1997
Urease has a major role in the nitrogen metabolism of bacteria and plants. When urea is hydrolyzed by urease (EC 3.5.1.5), NH3 and CO2 are released. In the original method of Sumner, described by Gorin et al. (1), ammonia nitrogen was determined by titration. Several other methods have since been developed for urease assay. A few are mentioned below. Since the reaction results in an increase in pH, certain pH-sensi1
To whom correspondence should be addressed. Fax: 504-3465786. E-mail: [email protected]. ANALYTICAL BIOCHEMISTRY ARTICLE NO. AB972173
FIG. 1. Effect of substrate concentration ([14C]urea) on urease activity at different time intervals. Urease concentration: 77.5 units (one unit of enzyme will liberate 1.0 mmol NH3 from urea/min at pH 7.0 and 257C). A, 0.25 h; B, 1.0 h; C, 2.0 h; D, 4.0 h.
249, 117–118 (1997)
0003-2697/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
05-23-97 14:01:54
abnt
118
NOTES & TIPS
Urease activity at different substrate concentrations and time intervals is given in Fig. 1 and for different enzyme concentrations and time intervals in Fig. 2. Figure 3 shows inhibition of the enzyme by a competitive inhibitor, acetohydroxamide. The activity determined by the Buddemeyer method increased with increased concentrations of substrate or enzyme and with incubation time. After completing the reaction, there are no further steps (like titration or Nesslerization) involved before reading the results. Obviously, the technique is quite straightforward and simple. It has been reported (7) that in pathogenic microorganisms like Mycobacterium tuberculosis urease functions as a virulence mechanism. Availability of a direct assay method like the
FIG. 3. Effect of acetohydroxamide (AH) on release of 14CO2 from 1.8 nmol of [14C]urea in the presence of 3.9 units enzyme at different time intervals. A, 1.0 h; B, 2.0 h; C, 3.0 h.
one described here could facilitate further studies on the enzyme in plants and bacteria. REFERENCES
FIG. 2. Effect of enzyme concentration on release of 14CO2 from 1.8 nmol of [14C]urea at different time intervals. Enzyme details are given in the legend to Fig. 1. A, 1.0 h; B, 2.0 h; C, 3.0 h.
1. Gorin, G., Fuchs, E., Butler, L. G., Chopra, S. L., and Hersh, R. T. (1962) Biochemistry 1, 911–916. 2. Ruiz-Herrera, J., and Gonzalez, J. (1969) Anal. Biochem. 31, 366–374. 3. Bibby, J. M., and Hukins, D. W. L. (1992) J. Biochem. Biophys. Methods 25, 231–236. 4. Kaltwasser, H., and Schlegel, H. G. (1966) Anal. Biochem. 16, 132–138. 5. McDonald, J. A., Speeg, K. V., Jr., and Campbell, J. W. (1972) Enzymologia 42, 1–9. 6. Buddemeyer, E., Hutchinson, R., and Cooper, M. (1976) Clin. Chem. 22, 1459–1464. 7. Clemens, D. L., Bai-yu Lee, and Horwitz, M. A. (1995) J. Bacteriol. 177, 5644–5652.
AID
abnt
AB $NAT
/
6m36$$$42x
05-23-97 14:01:54
117
tive dyes are incorporated in the system to measure the activity spectrophotometrically (2) or with a pH electrode (3). Urease was also determined by coupling it with NADH-dependent glutamate dehydrogenase (4). In a radiometric technique, the reaction was carried out in specialized metabolic flasks (5). To determine urease, we adopted the Buddemeyer procedure (6). Purified urease from jack bean (Concanavalia ensiformis) and acetohydroxamide were purchased from Sigma Chemical Co. (St. Louis, MO) and [14C]urea (55 mCi/mmol) from American Radiolabelled Chemicals (St. Louis, MO). The reaction was done in 4 1 1.5-cm screw-capped glass vials. The reaction consisted of 1.0 ml 0.2 M phosphate buffer, pH 6.3, appropriate concentrations of [14C]urea, in 5 ml; and appropriate units of enzyme [see figure legends]. The glass vials were loosely capped and placed in poly-Q scintillation vials (Beckman Instruments, Fullerton, CA) containing 4 1 2-cm strips of Whatman No. 1 filter paper, previously dipped in concentrated Liquifluor (DuPont New England Nuclear, Boston, MA) and 2 N NaOH, then air-dried. The scintillation vials were capped tightly and incubated at 377C. (A typical protocol would include measuring the amount of 14CO2 released from 18.0 nmol of substrate, [14C]urea, incubated in the presence of 77.5 units of enzyme from 2 to 4 h at 377C.) To study the effect of a urease inhibitor, acetohydroxamate, it was preincubated with the enzyme for 10 min. At prescribed time intervals, the cumulative CO2 released in each vial was measured in a Beckman Model LS 60001C liquid scintillation counter.
Urease Assay Using a Rapid Radiometric Procedure Eugene B. Harris,1 Baljit Randhawa, and K. Prabhakaran Laboratory Research Branch, GWL HD Center at LSU, U.S. Public Health Service, South Stadium Drive, P.O. Box 25072, Baton Rouge, Louisiana 70894-5072 Received January 13, 1997
Urease has a major role in the nitrogen metabolism of bacteria and plants. When urea is hydrolyzed by urease (EC 3.5.1.5), NH3 and CO2 are released. In the original method of Sumner, described by Gorin et al. (1), ammonia nitrogen was determined by titration. Several other methods have since been developed for urease assay. A few are mentioned below. Since the reaction results in an increase in pH, certain pH-sensi1
To whom correspondence should be addressed. Fax: 504-3465786. E-mail: [email protected]. ANALYTICAL BIOCHEMISTRY ARTICLE NO. AB972173
FIG. 1. Effect of substrate concentration ([14C]urea) on urease activity at different time intervals. Urease concentration: 77.5 units (one unit of enzyme will liberate 1.0 mmol NH3 from urea/min at pH 7.0 and 257C). A, 0.25 h; B, 1.0 h; C, 2.0 h; D, 4.0 h.
249, 117–118 (1997)
0003-2697/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
05-23-97 14:01:54
abnt
118
NOTES & TIPS
Urease activity at different substrate concentrations and time intervals is given in Fig. 1 and for different enzyme concentrations and time intervals in Fig. 2. Figure 3 shows inhibition of the enzyme by a competitive inhibitor, acetohydroxamide. The activity determined by the Buddemeyer method increased with increased concentrations of substrate or enzyme and with incubation time. After completing the reaction, there are no further steps (like titration or Nesslerization) involved before reading the results. Obviously, the technique is quite straightforward and simple. It has been reported (7) that in pathogenic microorganisms like Mycobacterium tuberculosis urease functions as a virulence mechanism. Availability of a direct assay method like the
FIG. 3. Effect of acetohydroxamide (AH) on release of 14CO2 from 1.8 nmol of [14C]urea in the presence of 3.9 units enzyme at different time intervals. A, 1.0 h; B, 2.0 h; C, 3.0 h.
one described here could facilitate further studies on the enzyme in plants and bacteria. REFERENCES
FIG. 2. Effect of enzyme concentration on release of 14CO2 from 1.8 nmol of [14C]urea at different time intervals. Enzyme details are given in the legend to Fig. 1. A, 1.0 h; B, 2.0 h; C, 3.0 h.
1. Gorin, G., Fuchs, E., Butler, L. G., Chopra, S. L., and Hersh, R. T. (1962) Biochemistry 1, 911–916. 2. Ruiz-Herrera, J., and Gonzalez, J. (1969) Anal. Biochem. 31, 366–374. 3. Bibby, J. M., and Hukins, D. W. L. (1992) J. Biochem. Biophys. Methods 25, 231–236. 4. Kaltwasser, H., and Schlegel, H. G. (1966) Anal. Biochem. 16, 132–138. 5. McDonald, J. A., Speeg, K. V., Jr., and Campbell, J. W. (1972) Enzymologia 42, 1–9. 6. Buddemeyer, E., Hutchinson, R., and Cooper, M. (1976) Clin. Chem. 22, 1459–1464. 7. Clemens, D. L., Bai-yu Lee, and Horwitz, M. A. (1995) J. Bacteriol. 177, 5644–5652.
AID
abnt
AB $NAT
/
6m36$$$42x
05-23-97 14:01:54