ANALYTICAL BIOCHEMISTRY ARTICLE NO.

234, 912 (1996)

0041

Direct Continuous Fluorometric Assay for Monoamine Oxidase B

Joseph J. P. Zhou, Boyu Zhong, and Richard B. Silverman1
Department of Chemistry and Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208-3113

Received June 2, 1995

The rst direct and continuous uorometric assay for monoamine oxidase B (MAO B) has been developed. E-2,5-Dimethoxycinnamylamine hydrochloride was designed and synthesized and was found to be an excellent substrate for MAO B (Km 218 mM, Kcat 435 min01). This compound has an intense purple uorescence when irradiated at lex 343 nm (lem 393 nm) in Tris buffer, pH 9.0, or sodium phosphate buffer, pH 7.2, but under the same conditions, the corresponding aldehyde, the product of the MAO-catalyzed oxidation of E-,5-dimethoxycinnamylamine hydrochloride, does not uoresce. The activity of MAO B, therefore, can be determined efciently and rapidly by continuously following the decrease in uorescence at 393 nm at enzyme concentrations as low as 100 nM. The change in uorescence is linear up to a substrate concentration of 500 mM. 1996 Academic Press, Inc.

Monoamine oxidase (EC 1.4.3.4; MAO)2 is a avindependent enzyme that catalyzes the oxidation of a variety of amine neurotransmitters and has been shown to be important in the treatment of depression (1) and Parkinsons disease (2). The most commonly used methods for the determination of the activity of MAO are spectrophotometric (3), radiometric (4), uorometric (58), luminometric (9), and ammonia (10) assays; radiometric, uorometric, and luminometric assays are most useful because of their high sensitivity and specicity, but radiometric assays are limited to the labeled compounds commercially available and present safety hazards. In general, uorometric analyses are at least 100- or 1000-fold more sensitive than
1 To whom correspondence should be addressed at the Department of Chemistry, Northwestern University, Evanston, IL 60208-3113. Fax: (708) 491-7713. 2 Abbreviations used: MAO, monoamine oxidase; THF, tetrahydrofuran.

the normal spectrophotometric method, which is only useful when a chromophoric product is produced (6, 11). However, none of the known uorometric or luminometric methods available for assay of MAO is a direct, continuous assay (7, 8, 11, 12). In addition to the serious inconvenience of discontinuous assays, they may fail to measure the true initial rates of the enzyme reaction. Furthermore, deviations from a linear time course immediately following mixing of the enzyme and substrate may go undetected. Fluorometric assays involving the measurement of hydrogen peroxide by means of a coupled uorogenic reaction are continuous, but they require the use of a second enzyme and, therefore, are not direct. A nonzero blank rate is always a problem in these assays (7, 13). Assays that measure ammonia suffer from lack of sensitivity and are only useful for oxidation of primary amines. The disadvantages inherent in these currently used assay methods for MAO prompt us to report the rst direct, continuous uorometric assay for MAO B activity. We have synthesized E-2,5-dimethoxycinnamylamine hydrochloride (1) and have found that it is a good substrate for MAO B. It uoresces maximally at lex 343 nm and lem 393 nm in Tris buffer, pH 9.0, and sodium phosphate buffer, pH 7.2, and under the same conditions, the corresponding aldehyde, the product of the MAO-catalyzed oxidation of 1, does not uoresce. The activity of MAO B, therefore, can be determined efciently and rapidly by continuously following the decrease in uorescence at 393 nm.
MATERIALS AND METHODS

3-Hydroxy-3-(2,5-dimethoxyphenyl)propionitrile (1) was synthesized as previously reported (14). 3-Hydroxy-3-(2,5-dimethoxyphenyl)propylamine (2). Lithium aluminum hydride (0.5 g, 12.5 mmol) was added portionwise to a solution of 3-hydroxy-3-(2,5-dimethoxyphenyl)propionitrile (1.3 g, 6.3 mmol) in THF
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(100 ml) at 0 C under nitrogen. The resulting suspension was reuxed for 14 h. It was cooled to 0 C again and aqueous NaOH (10%, 1 ml) was added dropwise to quench the reaction. The mixture was stirred at room temperature for 1 h; the precipitate was then collected and washed with dichloromethane. The combined ltrate was concentrated in vacuo to give an oily residue which was taken up with dichloromethane and dried (Na2SO4 ). The dichloromethane was removed in vacuo to give the product as a yellow oil, which was used directly without purication (0.92g, 69%); 1H NMR (CDCl3 ) d 7.13 (d, 1 H, J 3 Hz), 6.76 (m, 2 H), 5.18 (dd, 1 H), 3.79 (s, 3 H), 3.78 (s, 3 H), 2.98 (m, 2 H), 1.92 (m, 1 H), 1.70 (m, 1 H) ppm. E-2,5-dimethoxycinnamylamine triuoroacetyl amide (3). Triuoroacetic acid (2 ml) was added to a solution of 3-hydroxy-3-(2,5-dimethoxyphenyl)propylamine (0.8g, 3.8 mmol) in 200 ml of toluene (1% v/v). The resulting red-colored solution was reuxed for 2 h. Aliquot analysis by NMR spectroscopy showed that the reaction was complete. Most of the toluene was removed in vacuo. The residue was dissolved in dichloromethane, washed with water, and the organic layer was dried (Na2SO4 ). Concentration to dryness gave a yellow oil, which was chromatographed with dichloromethane on silica gel to give pale yellow crystals (0.81g, 74%); mp 8385 C; 1H NMR (CDCl3 ) d 6.966.8 (m, 4 H), 6.45 (br, 1 H), 6.18 (dt, 1 H), 4.16 (m, 2 H), 3.81 (s, 3 H), 3.79 (s, 3 H) ppm; IR (KBr) 3309 (br), 2952, 1692, 1553, 1500, 1224, 1181, 1151 cm01; HRMS: calcd for C13H14F3NO3 , 289.0926; found, 289.0936. E-2,5-dimethoxycinnamylamine hydrochloride (4). E-2,5-Dimethoxycinnamylamine triuoroacetyl amide (0.8g, 2.77 mmol) was added to a solution of NaOH in methanol (1 M, 20 ml). The reaction mixture was stirred at room temperature for 6 h at which time TLC showed that no starting material remained. The solvent was removed in vacuo. The residue was partitioned between ether (5 ml) and brine (5 ml); the aqueous layer was extracted with ether (3 1 5 ml); and the combined organic layer was dried over K2CO3 . Concentration to dryness gave a yellow oil which was taken up in dry ether (5 ml) and HCl gas was bubbled into the solution. E-2,5-Dimethoxycinnamylamine hydrochloride was collected as a white solid precipitate (0.36g, 57%); mp 150152 C; 1H NMR (D2O) d 6.956.76 (m, 4 H), 6.13 (dt, 1 H), 3.64 (s, 3 H), 3.62 (s, 3 H), 3.58 (d, 2 H) ppm; IR (KBr) 33662612 (br), 1501, 1245 cm01; MS (EI, m/ z) 193 (38, M/-HCl), 178 (17), 164 (17), 163 (31), 162 (100), 161 (21), 160 (19); anal. calcd for C11H16ClNO2 : C 57.52, H, 7.02, N, 6.10, Cl, 15.43; found: C, 57.48, H, 7.08, N, 5.98, Cl, 15.33. Enzyme. Monoamine oxidase B was puried from beef liver by the method of Salach (15). Assays were carried out at 25 C and pH 9.0 in Tris buffer.

SCHEME 1. Synthetic route to E-2,5-dimethoxycinnamylamine hydrochloride

Assay procedure. A uorescenceconcentration calibration curve was made as follows: Solutions of E-2,5dimethoxycinnamylamine hydrochloride (0, 20, 41, 82, 123, and 164 mM) were made in Tris buffer (100 mM, pH 9.0). The uorescence was measured at lex 343 nm, lem 393 nm (0, 126, 235, 426, 583, and 779 units, respectively). Measurement of initial rates by uorometry at various concentrations of E-2,5-dimethoxycinnamylamine hydrochloride. Solutions of E-2,5-dimethoxycinnamylamine hydrochloride were made in Tris buffer (100 mM, pH 9.0, 2930 ml each) and were preincubated at 25 C. To these solutions was added MAO B (70 ml of a 19.9 mM solution) to give a nal concentration of MAO B of 0.46 mM. The nal concentrations of E-2,5-dimethoxycinnamylamine hydrochloride were 20, 41, 82, 123, 164, and 328 mM, respectively. The initial rates were measured to be 96, 189, 252, 339, 420, and 563 units/min. After conversion using the standard curve, the rates were 16, 36, 50, 69, 87, and 118 mM/min. The assay also was carried out with 100 nM MAO B at pH 7.2 (50 mM sodium phosphate buffer).
RESULTS

E-2,5-Dimethoxycinnamylamine hydrochloride (4) was synthesized as shown in Scheme 1. A linear relationship was observed between the E2,5-dimethoxycinnamylamine hydrochloride concentration and the uorescence up to a concentration of 500 mM (Figs. 1A and 1B). Data for several concentrations of E-2,5-dimethoxycinnamylamine hydrochloride at pH 9.0 and pH 7.2 were tted to a LineweaverBurk plot, which gave Km 218 mM, kcat 435 min01 (pH 9.0) and Km 223 and kcat 55 min01 (pH 7.2) (Fig. 2). When a discontinuous assay (16) was used, the kinetic constants at pH 7.2 were determined to be Km 269 mM and kcat 55 min01, indicating the similarity in

DIRECT CONTINUOUS FLUOROMETRIC ASSAY FOR MONOAMINE OXIDASE B

11

FIG. 1. (A) Linear relationship between substrate concentration and uorescence up to 170 mM substrate concentration (100 mM Tris HCl buffer, pH 9.0). The enzyme concentration is 0.46 mM. (B) Linear relationship between substrate concentration and uorescence to 500 mM substrate concentration and the change beyond 500 mM (100 mM TrisHCl buffer, pH 9.0). The enzyme concentration is 0.46 mM.

results between the continuous and discontinuous methods. Figures 3 and 4 demonstrate the linearity and time dependence of uorescence decrease at a low enzyme concentration (100 nM).
DISCUSSION

Most aldehydes and ketones are not strongly uorescent; therefore, they generally have to be converted to their corresponding carboxylic acids or hydrazones for uorometric analysis (8, 17). It has been reported that 2,5-dimethoxybenzaldehyde has a reasonably strong uorescence (17). Consequently, we attempted to use 2,5-dimethoxybenzylamine to assay MAO B ac-

tivity uorometrically by following the rate of production of 2,5-dimethoxybenzaldehyde. Unfortunately, 2,5-dimethoxybenzylamine is a poor substrate for MAO B (Km 4.5 mM, kcat 5 min01 in sodium phosphate buffer, pH 7.4, at 25 C) (data not shown). Since benzylamine is a good substrate for MAO B, the steric effect of one or both of the 2,5-dimethoxyl substituents is apparently responsible for making 2,5-dimethoxybenzylamine a poor substrate. To improve the binding ability of the substrate with the enzyme, we designed and synthesized E-2,5-dimethoxycinnamylamine hydrochloride from 2,5-dimethoxybenzaldehyde (Scheme 1). We found that E-2,5-dimethoxycinnamylamine hydrochloride is indeed an excellent substrate for MAO B (Km 218 mM, kcat 435 min01 in TrisHCl buffer,

FIG. 2. Inverse plots of velocity versus substrate concentration at pH 7.2 (50 mM sodium phosphate; enzyme concentration 100 nM) and at pH 9.0 (100 mM TrisHCl; enzyme concentration 0.46 mM).

FIG. 3. Time dependence of uorescence. The enzyme concentration is 100 nM and the E-2,5-dimethoxycinnamylamine concentration is 200 mM in 50 mM sodium phosphate buffer, pH 7.2, 25 C.

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REFERENCES
1. Ives, J. L., and Heym, J. (1989) Annu. Rep. Med. Chem. 24, 21 29. 2. Tetrud, V. W., and Langston, J. W. (1989) Science 245, 519522. 3. (a) Tabor, C. W., Tabor, H., and Rosenthal, S. M. (1954) J. Biol. Chem. 208, 645661. (b) Weissbach, H., Smith, T. E., Daly, J. W., Wipkop, B., and Udenfriend, S. A. (1960) J. Biol. Chem. 235, 11601163. (c) Houslay, M. D., and Tipton, K. F. (1974) Biochem. J. 139, 645652. (d) Kalgutkar, A. S., Castagnoli, K., Hall, A., and Castagnoli, N., Jr. (1994) J. Med. Chem. 37, 944 949. 4. (a) Wurtman, R. J., and Axelrod, J. (1963) Biochem. Pharmacol. 12, 14391441. (b) Otsuka, S., and Kobayashi, K. (1964) Biochem. Pharmacol. 13, 9951006. (c) Tipton, K. F. (1985) Methods Find. Exp. Clin. Pharmacol. 7, 361367. (d) Da Prada, M., Kettler, R., Keller, H. H., Burkard, W. P., Muggli-Maniglio, D., and Haefely, W. E. (1989) J. Pharmacol. Exp. Ther. 248, 400414. 5. (a) Snyder, S. H., and Hendley, E. D. (1968) J. Pharmacol. Exp. Ther. 163, 386392. (b) Tipton, K. F. (1969) Anal. Biochem. 28, 318325. 6. (a) K. F. Tipton (1990) in Enzyme Assays: A Practical Approach (Eisenthal, M. J., Ed.), Oxford Univ. Press, Oxford. (b) Tipton, K. F., and Youdim, M. B. H. (1983) in Methods in Biogenic Amine Research (Parvez, S., Nagatsu, T., Nagatsu, I., and Parvez, H., Eds.), pp. 441465, Elsevier, Amsterdam. 7. Tipton, K. F., and Singer, T. P. (1993) Biochem. Pharmcol. 46, 13111316. 8. Morinan, A., and Garratt, H. M. (1985) J. Pharmacol. Methods 13, 213223. 9. (a) OBrien, E. M., Kiely, K. A., Tipton, K. F. (1993) Biochem. Pharmacol. 46, 13011306. (b) Tenne, M., Finberg, J. P. M., Youdim, M. B. H., and Ulitzur, S. (1985) J. Neurochem. 44, 1378 1384. 10. (a) Nagatsu, R., and Yagi, K. (1966) J. Biochem. 60, 219221. (b) Meyerson, L. R., McMurtrey, K. D., and Davis, V. E. (1978) Anal. Biochem. 86, 287297. 11. G. G. Guilbault (Ed.) (1990) Practical Fluorescence, Vol. 2, Dekker, New York. 12. Krajl, M. (1965) Biochem. Pharmacol. 14, 16841686. 13. Tipton, K. F. (1969) Anal. Biochem. 28, 318325. 14. Zhou, J. J. P., Zhong, B., and Silverman, R. B. (1995) J. Org. Chem. 60, 22612262. 15. Salach, J. I. (1979) Arch. Biochem. Biophys. 192, 128137. 16. (a) Szutowicz, A., Kobes, R. D., and Orsulak, P. H. (1984) Anal. Biochem. 138, 8694. (b) Silverman, R. B., and Hawe, W. P. (1995) J. Enzyme Inhib. 9, 203215. 17. Crowell, E. P., and Varsel, C. J. (1963) Anal. Chem. 35, 189 192.

FIG. 4. Time dependence of uorescence. The enzyme concentration is 100 nM and the E-2,5-dimethoxycinnamylamine concentration is 200 mM in 100 mM TrisHCl buffer, pH 9.0, 25 C.

pH 9.0, 25 C). A comparison between this method and a discontinuous assay (16) indicated that similar kinetic constants are obtained. Interestingly, whereas E-2,5dimethoxycinnamylamine hydrochloride has an intense purple uorescence when irradiated at l 343 nm, the corresponding aldehyde product of the enzymatic reaction, E-2,5-dimethoxycinnamaldehyde, has no uorescence under the same conditions, thereby allowing a continuous decrease in the uorescence of the substrate with time, even at enzyme concentrations as low as 100 nM (Figs. 3 and 4). We have not determined the substrate activity of this compound with MAO A, so it is not yet known if this is an isozymeselective substrate. The important criteria for any enzyme assay include speed, accuracy, and ease of use. The utilization of E-2,5-dimethoxycinnamylamine to assay MAO B meets all of the above-mentioned requirements.
ACKNOWLEDGMENT
The authors gratefully acknowledge the National Institutes of Health (grant GM32634) for nancial support of this work.