A general coupled spectrophotometric assay for decarboxylases

ANALYTlCAL

BIOCHEMISTRY

171, 339-345 (1988)

A General Coupled

Spectrophotometric

DENNISH.BURNSAND Worcester

Foundation

for Experimental

Assay for Decarboxylases’

D. JOHNABERHART' Biology,

Shrewsbury,

Massachusetts

01545

Received February 16, 1988 A rapid, continuous spectrophotometric method has been developed for the assay of decarboxylases. The assay uses a coupled enzyme system in which liberated CO2 is reacted with phosphoenolpyruvate and phosphoenolpyruvate carboxylase to form oxaloacetate, which in turn is reduced by malate dehydrogenase to L-malate concomitantly with the oxidation of NADH to NAD. The resultant decrease in absorbance at 340 nm accurately reflects the activity of the decarboxylase. The method is capable of detecting the liberation of as little as I nmol of C02/min and was tested in assays of lysine decarboxylase, orotidine-5’-phosphate decarboxylase, and 4’-phosphopantothenoyl-L-cysteine decarboxylase. o 1988 Academic press, IIK. KEY WORDS: decarboxylases, assay; spectrophotometry; kinetics, enzyme; detergents (surfactants); lysine decarboxylase; orotidine 5’-monophosphate decarboxylase; 4’-phosphopantothenoyl-L-cysteine decarboxylase.

to follow the formation of the common product, CO:!. We now report the development of such a procedure, based on a published method for measurement of CO2 in blood serum (1 l), which appears (within certain limitations, vide infra) to be generally applicable to decarboxylase assays. The method involves the trapping of liberated CO2 with PEP, catalyzed by PEP carboxylase. The resultant oxaloacetate is in turn reduced by malate dehydrogenase to L-malate, with concomitant oxidation of NADH to NAD. Thus the decarboxylation can be followed continuously by observing the decrease in absorbance at 340 nm.

We recently published the results of a study of the stereochemistry of rat liver PPC3 decarboxylase (1). In that work, the decarboxylase was assayed by trapping 14C02 produced from the substrate (4’-phosphopantothenoylj l-‘4C]-DL-CySteine), a procedure commonly used for assaying a wide variety of decarboxylases (2-4). Such radiometric procedures, as well as related manometric procedures (5,6), have inherent disadvantages, some of which have been summarized elsewhere (7). Several spectrophotometric decarboxylase assays have been developed which use coupling (auxiliary) enzymes to follow the formation of the amine (or other unique) product of the reaction (8- 10). However, to our knowledge, no spectrophotometric procedure for assaying decarboxylases has been reported which uses coupling enzymes

MATERIALS

AND METHODS

Materials. NADH, Nonidet P-40, OMP, PEP, Triton X-100, and Triton X-l 14 were obtained from Sigma Chemical Co. (St. Louis, MO). L-[U-‘4C]Lysine (sp act 329 mCi/mmol) was obtained from DuPont NEN Research Products (Boston, MA). Emulgen 9 11 was a gift from Dr. Karsten Holm. 4’-Phosphopantothenoyl-L-cysteine

’ This work was supported by United States Public Health Service Grant GM24420. 2 To whom correspondence should be addressed. 3 Abbreviations used: MD, malate dehydrogenase; OMP, orotidine-S-monophosphate; PEP, phosophoenolpyruvate; PLP, pyridoxaf-5’-phosphate; FPC, 4’-phosphopantothenoyl+cysteine.

339

0003-2697188 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction m any form resewed.

340

BURNS AND ABERHART

(0.5 ml). The enzyme + buffer were equiliwas available from other work (unpublished) brated to 37°C and, upon addition of lysine, in these laboratories. Enzymex4 Lysine decarboxylase (EC the system closed and the mixture incubated 4.1.1.18) (Bacterium cadaveris, 55 U/mg pro- at 37°C for 15-30 min with gentle oscillation tein), OMP decarboxylase (EC 4.1.1.23) (ca. 60 oscillations/min) while the volume change was monitored. The H2S04 was then (baker’s yeast, 30 U/mg protein), and malic dehydrogenase (EC 1.1.1.37) (porcine heart, tipped into the main flask, and after ca. 15 min, the final volume change was measured. 700 U/mg protein) were obtained from Sigma. Phosphoenolpyruvate carboxylase Radiometric CO2 measurements were per(12) (EC 4.1.1.31) (Zea mays, 0.68 U/mg) formed by using the above apparatus, except that the center well contained 0.2 ml 6 N was obtained from Calbiochem (San Diego, CA). 4’-Phosphopantothenoyl-L-cysteine de- KOH. The same buffers as described above carboxylase (EC 4.1.1.36) (partially purified for manometric runs were used, except that preparations from rat liver, ca. 4-100 U/mg 14C-labeled precursor was included, plus unprotein) was available from other work in this labeled carrier to 5 mM. The addition of laboratory (unpublished). PEP carboxylase MgC12 or detergent did not affect the results. was assayed by a modification of the method After addition of the H2S04 and equilibraof Chen and Jones (13): the assay mixture tion for 15 min, a portion of the solution in contained, per milliliter, 100 mM Tris * HCl the center well was counted. (pH 8.0), 8 mrvt MgC12, 5 U MD, 9.5 mg PEP, Liquid scintillation counting was per0.3 1 mg NADH, 0.01% detergent (Emulgen formed by using a Packard Instrument Co. 9 1 l), and ca. 0.1 U PEP carboxylase. Assays (Downers Grove, IL) Model 3330 instruwere conducted at 37°C and initiated with the ment. Samples (0.15 ml) were dissolved in 10 addition of 10 pmol of NaHC03. Malate de- ml of Ecolite (Wilkem, Ltd., Pawtucket, RI) hydrogenase was assayed by the method of containing 0.85 ml of H20. Wolfe and Neilands ( 14). Spectrophotometric measurements were Manometric measurements of CO;! pro- performed by using a Gilson Model 250 induction were made by using a Gilson Medistrument equipped with a Model 605 1 recal Electronics (Middleton, WI) Model IG 14 corder. Temperatures were controlled by differential respirometer equipped with Gilusing a Thermoset temperature controller. Quartz cells (1 ml, lo-mm path length) were son Model GME 125) Warburg flasks ( 15 ml, with one side arm and center well). In a typiused. cal run with lysine decarboxylase, the reacCoupled spectrophotometric assay of lysine tion flask contained 3 ml of a solution of 100 decarboxylase. The following is typical for an IIIM Tris. HCl (pH 6.0), 10 PM PLP, 5 mM assay of a sample containing ca. 0.05 U of lysine decarboxylase. The buffer was deL-lysine, and ca. 1.2 units of lysine decarboxgassed by vacuum pumping and was stored ylase. Runs with and without added MgCl, (8 mM) and detergent (0.01%) in the buffer under NZ. The buffer contained, per milligave the same results. The center well was liter, 100 mM Tris * HCl (pH 6.0), 8 mM MgC&, 10 PM PLP, 10 U MD, 1.O U PEP empty. The side arm contained 5 N H2S04 carboxylase, 9.5 mg PEP, 0.31 mg NADH, 0.01% detergent (Emulgen 9 11, Triton 4 Enzyme unit definitions: Lysine decarboxylase proX- 100, Triton X- 114, or Nonidet P-40), plus duces 1.O pmol of COI/min; OMP decarboxylase prothe decarboxylase. The recorder was set to duces I.0 pmol of COJmin; PPC decarboxylase produces 1 nmol of COJmin; malate dehydrogenase and ca. 1.0 AU, and a baseline (background) rePEP carboxylase convert 1.0 rmol of their respective corded for a few minutes until stable, while the reaction mixture was equilibrated to substrates to products/min.

GENERAL

COUPLED

SPECTROPHOTOMETRIC

37°C in the thermostatted cell holder. Then 0.5 M L-lysine solution (10 ~1) was added, and the decrease in A340 was recorded. Units of decarboxylase activity (in pmol of COZ produced/min) are calculated by dividing the slope of the trace (aA/min) by 6.22. For assays of OMP decarboxylase, the buffer contained, per milliliter, 50 mM Tris . HCl (pH 8.0), 8 mM M&12, 1 mM Lcysteine, 5 U MD, 1.0 U PEPC, 9.5 mg PEP, 0.3 1 mg NADH, 0.01% detergent, plus the decarboxylase. For assays of PPC decarboxylase, the buffer contained, per milliliter, 100 mM Tris - HCl (pH 8.0) 8 mM MgClz, 12 mM L-cysteine, 1.O U MD, 0.1 U PEP carboxylase, 9.5 mg PEP, 0.31 mg NADH, 0.01% detergent, and the decarboxylase (3.5- 10 U). RESULTS

DECARBOXYLASE

0.1

'

6

37°C 25°C

I

I

I

7

8

9

F+’ FIG. 1. pH-rate profile of PEP carboxylase at 25 and 37”C, using 0.1 unit of PEP carboxylase and 5 units of MD per run.

Development of the auxiliary enzyme system. We first examined the pH and temperhigher protein ature-rate profiles of the coupling enzymes to evaluate the potential generality of the assay. The results revealed useful activities for the enzyme system in the pH range 6-9 (Fig. 1). Assays of PEP carboxylase were performed essentially according to a published procedure ( 13), using MD as coupling enzyme and NaHC03 as CO;? source. In initial runs (at 25°C) using unit ratios of MD:PEP carboxylase of 10: 1, unacceptably long lag times (usually up to 6 min) were observed prior to achieving linear velocities (99% of the steady-state velocity of PEP carboxylase). Furthermore, contrary to expectations based on theoretical treatments of the effects of coupling enzyme ratios on lag times (15-17) increasing the unit ratio (from 1O:l to 25:1, 50:1, or 1OO:l) actually substantially decreased the observed rate of PEP carboxylase and did not shorten the lag time. Such observations were made in runs under a variety of conditions, from pH 6.0 to 8.0. The results were interpreted as resulting from protein aggregation at the

Q +

2

2 0.6 m r

341

ASSAY

concentrations. In an attempt to avoid this problem, a detergent was included at 0.01% in the assay mixtures. Several detergents (Emulgen 9 11, Nonidet P-40, and Tritons X- 100 and X- 114) were tried, all of which produced similar results. With added detergent, lag times were greatly reduced (O-20 s), and variations in the amount of MD showed little difference (l-3%) in the rate of carboxylation of PEP. The protein aggregation effect was less severe in runs at 37°C but we routinely included detergent in all runs, regardless of temperature. Naturally, substantially higher rates were observed for the auxiliary enzymes at 37°C as compared with runs at 25°C (Table 1 and Fig. 1). At pH 5.0 or lower, the auxiliary enzyme system did not function. Assay of lysine decarboxylase. The coupled spectrophotometric assay method was first tested with lysine decarboxylase, since this enzyme has previously been assayed by a variety of alternative procedures and thus our results could be compared with those

342

BURNS AND ABERHART TABLE TEMPERATURE

DEPENDENCE

I

OF RELATIVE

RATES OF COUPLING

Temperature (“C)

Detergent (Emulgen)b

MD MD MD

25 25 37

-

PEP carboxylase’ PEP carboxylas&

25 37

+ +

Enzyme

ENZYMES”

Relative Vmar

L (mW

0.9

0.029d 0.023d 0.048d

1.0 2.4

1.0

0.1751

3.0

0.14f

’ Assays were run at pH 8.0 unless otherwise noted. ’ Emulgen 9 11 (0.0 1W) was used, similar results were obtained with Triton X- 100 or X- 114. c Assay run at pH 1.5. d Km for oxaloacetate. ’ Assayed with MD:PEP carboxylase unit ratios 10: 1. ‘Km for HCOs .

previously published (7,18,19). Assays were conducted at 37°C and at the pH optimum of 6.0. A typical run is shown in Fig. 2. Excellent linearity of CO;! production was

found over an activity range of ca. 0.0030.07 U/ml (Fig. 3, insert). A Km value of 0.4 1 mM was derived from an Eadie-Hofstee plot (Fig. 3) of data obtained by the spectrophotometric method. Excellent agreement was found between lysine decarboxylase rates obtained by the spectrophotometric method

0’ 0

I

I

t

10

20

30

Vo/[S]

0

1

2

3

4

5

MIN FIG. 2. Typical (lysine) decarboxylase assay run using coupled spectrophotometric assay procedure.

FIG. 3. Eadie-Hofstee plot of lysine decarboxylase assay data for runs at pH 6.0, 37°C showing K,,, = 0.41 mM (correlation coefficient = 0.996). Insert: relationship between CO;1 production and lysine decarboxylase concentration, as measured by the coupled spectrophotometric assaymethod at pH 6.0, 37°C (correlation coefficient = 1.00).

GENERAL

COUPLED

SPECTROPHOTOMETRIC

6

2

L d.075

t

I

0.125

0.175

I 0.225

Vo/[S] FIG. 4. Eadie-Hofstee plot of OMP decarboxylase assaydata for runs at pH 7.4, 37°C. showing K,,, = 26 pM for OMP concentrations in the range of IO-50 pM (correlation coefficient = 0.976). Insert: relationship between CO* production and OMP decarboxylase concentration, as measured by the coupled spectrophotometric assay method, pH 8.0, 30°C (correlation coefficient = 0.999).

and by the respirometric method, with the latter assay values averaging 94% (90-99% range) of those obtained by the coupled spectrophotometric assay.5 Assay of orotidine-S-monophosphate decarboxylase. The assay was tested with this enzyme since it appeared to be the only commercially available decarboxylase having a pH optimum (pH 8.0) close to that of the enzyme of our primary interest, PPC decarboxylase. With OMP decarboxylase, good linearity of CO1 production was observed ’ We also compared the spectrophotometric assay results with those obtained by radiometric assays using L-[U-‘4C]lysine. With one batch of L-[U-“‘Cllysine, the radiometric assay rates averaged 75% of those found by spectrophotometry. A second batch of L-[U-?]lysine gave rates which were only 44% of those obtained by the spectrophotometric method. However, the ratio of rates observed by the spectrophotometric vs radiometric procedures was highly reproducible over a wide range of enzyme concentrations (0.005-o. 1 I-l), suggesting that the poor comparison of rates is not the fault of the spectrophotometric assay.

DECARBOXYLASE

343

ASSAY

over a wide range of enzyme concentrations [(ca. 0.33-17) X 10e3 U/ml)] (Fig. 4, insert). The results agreed closely with rate data obtained by the more common direct spectrophotometric method (monitoring at 285 nm) of assaying OMP decarboxylase (20) the latter giving observed rates ranging between 9 1 and 95% of those obtained by using the coupled spectrophotometric assay. In confirmation of a literature report (2 l), K,,, values determined for OMP decarboxylase runs conducted at 37”C, pH 7.5, were substrate concentration dependent. In an OMP concentration range of lo-50 PM, the K,,, = 26 PM (Fig. 4), whereas in a range of 1.O- 10 PM, the K,,, = 11 PM. Assay of 4’-phosphopantothenoyl-L-cysteine decarboxylase. Finally, the coupled spectrophotometric assay was tested with the partially purified enzyme from rat liver (10 U/mg as determined from 14C02 and protein assays). Linearity of CO2 production rate was found with varying PPC decarboxylase concentrations (Fig. 5, insert). A K,,, value of 0.076 tnM was derived from an Eadie-Hofstee plot (Fig. 5) using 6.0 units of PPC decarboxylase per run.

1 Enzyme

0’

0

-

’ 10

(relative

’

’ 20

cont.)

’

’ 30

n

’ 40

.

’ 50

WSI FIG. 5. Eadie-Hofstee plot of PPC decarboxylase assay data showing Km = 0.076 mM for runs at pH = 7.8, 37°C. Insert: relationship between CO2 production and PPC decarboxylase concentration (data for runs with ca. 1.3-14.3 units of PPC decarboxylase).

344

BURNS AND ABERHART

Assay results for PPC decarboxylase obtained by the spectrophotometric method were compared to those obtained by using a radiometric method (trapping 14C02 from 4’-phosphopantothenoyl-DL-[ l-‘4C]cysteine (1)). The radiometric assay rates (for 30-min incubations) ranged between 91 and 98% of rates determined by the coupled spectrophotometric method. DISCUSSION

It became clear during the development of the auxiliary enzyme system that inclusion of a detergent to avoid protein aggregation was essential for achieving maximum rates without substantial lag times and linearity of CO* production from varying amounts of decarboxylase. Assays run using Emulgen 9 11, Nonidet P-40, and Triton X- 100 or X- 114 at 0.01% all gave essentially the same results. With the inclusion of detergent, unit ratios of MD:PEP carboxylase as low as 1: 1 gave adequate results (although unit ratios of 5: 1 were commonly used), but it was necessary to use unit ratios of PEP carboxylase:decarboxylase ofat least 1O:l (15-17). The results obtained with lysine decarboxylase exemplify the utility of the procedure. The assay consistently provided excellent linearity and rates that correlated closely with rates obtained from manometric assays. A K, value (0.4 1 mM) determined for lysine decarboxylase compares well with the K,,, value of 0.37 mM (for pH 5.8, 37°C) determined manometrically by Soda and Moriguchi (18), but not with that (1.9 mM, pH 6.0, 37 “C) determined spectrophotometrically by Phan et al. (7) or manometrically by Gale and Epps (1.2- 1.8 mM, pH 6.0, 30°C) (19). Similarly, with OMP decarboxylase, good linearity of COZ production was observed, and rate comparisons with the traditional spectrophotometric assay for this enzyme (20) were very good. Using the coupled assay, we obtained K,,, values which were substrate concentration dependent, in agree-

ment with Fyfe et al. (21). However, our values were much higher: 26 PM (compared to 2.5 PM) for OMP concentrations of lo-50 PM, and 11 PM (compared to 0.5 PM) for OMP concentrations of 1.O- 10 PM. It should be pointed out that there is little consensus in the literature for K,,, values of OMP decarboxylase, for which values ranging from 0.5 to 8 PM can be found (23,24). The assay was also effective with PPC decarboxylase, both in crude enzyme preparations (ca. 2 U/mg protein) and more highly purified ones (3 100 U/mg protein), although crude enzyme preparation must be clarified before analysis. A K,,, value of 0.076 mM (37°C pH 7.8) determined by the coupled spectrophotometric method is in reasonable agreement with that (Km = 0.13-o. 15 mM, 37°C pH 8.0) reported by Abiko (22). It must be pointed out that the described procedure has certain inherent limitations. Enzyme solutions having substantial absorption at 340 nm cannot be used and systems rapidly consuming (or generating) NADH may produce unacceptably large background absorbance changes. Nevertheless, the assay is clearly a valuable and versatile method of assaying a variety of decarboxylases having activities in the range of pH 6-9 and 20-37°C. REFERENCES 1. Aberhart, D. J., Ghoshal, P. K., Cotting, J.-A., and Russell, D. J. (1985) Biochemistry 24, 1178-7182. Sissons, C. H. (1976) Anal. Biochem. 70,454-462. Jones, R. D., Hampton, J. K., and Preslock, J. P. (1972) Anal. Biochem. 49, 147-154. Morris, D. R., and Pardee, A. B. (1965) Biochem. Biophys. Res. Commun. 20,691-102. Gale, E. F. ( 1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., Ed.), Vol. 3, pp. 1662- 1668, Academic Press, New York. Zeman, G. H., Sobocinski, P. Z., and Chaput, R. L. (1973) Anal. Biochem. 52,63-68. Phan, A. P. H., Ngo, T. T., and Lenhoff, H. M. (1982) Anal. Biochem. 120, 193-197. Racker, E. (1957) Arch. Biochem. Biophys. 69, 300-3 10. Cozzani, I. (1970) Anal. Biochem. 33, 125-131.

GENERAL

COUPLED

SPECTROPHOTOMETRIC

IO. Keeling, D. J., Smith, I. R.. and Tipton, K. F. (1984) NaNnyn-Schmiedeberg’s Arch. Pharmacol. 326, 215-221. 11. Forrester, R. L.. Wataji, L. J.. Silverman, D. A., and Pierre, K. J. (1976) Clin. Chem. 22, 243-245. 12. Uedan, K., and Sugiyama. T. (1976) Planf Physiol. 57,906-9 10. 13. Chen. J. J., and Jones. R. F. (1970) Biochim. Biophys. Acta 214, 3 18-325. 14. Wolfe, R. G.. and Neilands. J. B. (1955) J. Biol. Chem. 221, 61-69. 15. Easterby, J. S. (1973) Biochim. Biophys. Acta 293, 552-558. 16. Cleland, W. W. (1979)Anai. Biochem. 99, 142-145. 17. Brooks, P. J.. Espinola, T., and Suelter, C. H. (1984) Canad. J. Biochem. Cell Biol. 62, 945-955.

DECARBOXYLASE

ASSAY

345

18. Soda. K., and Moriguchi. M. (1969) Biochim. Biophys. Res. Commun. 34, 34-39. 19. Gale, E. F.. and Epps, H. M. R. ( 1944) Biochem. J. 38,232-242. 20. Lieberman, I., Kornberg, A., and Simms, E. S. (1955) J. Biol. Chem. 215,403-415. 21. Fyfe. J. A., Miller, R. L., and Krenitsky. T. A. (1973) J. Biol. Chem. 248, 3801-3809. 22. Abiko, Y. (1967) J. Biochem. (Tokyo) 61, 300-308. 23. Brody, R. S., and Westheimer, F. H. (1979) J. Biol. Chem. 254,4238-4244. 24. Yashimoto, A., Umezu, K., Kobayashi, K., and Tomita, K. (1978) in Methods in Enzymology (Hoffee, P. A., and Jones, M. E., Eds.). Vol. 51, pp. 74-79, Academic Press. New York.