Spectrophotometric method for the estimation of arginine decarboxylase

4NAI

k’TICAI

92. 331-337

HlOCHtMlSTRY

(1979)

Spectrophotometric Method for the Estimation Arginine Decarboxylase A. SMITH

TERENCE

Received A sensitive has

method

for

the

developed,

which

is based

prctduct and the

of arginine. hydrogen

In the peroxide

presence generated

the peroxidative other amino

acid

May

determination

been

oxidation of guaiacol. decarboxylases.

upon

of

I?.

1978

of arginine the

of pea seedling is estimated The

Amino (acid decarboxylases are widespread in animals. plants, and bacteria. They are concerned in the formation of various amines, some of which, notably the catecholamines, histamine, and the polyamines are of considerable biochemical significance. Methods employed for the estimation of the activity of these decarboxylases have depended primarily on the formation of CO,. which has been measured by manom’etry or more usually as ‘“CO, evolved from carboxyl labelled amino acids. Methods for estimating amino acid decarboxylases lbased on the formation of the amine have been used less frequently. In the present paper, a rapid method is evolved to estimate the amine produced, which depends on its oxidation by amine oxidase with the production of hydrogen peroxide. A very active amine oxidase occurs in pea seedlings (I). The substrate range is quite wide but the enzyme is particularly active with the diamines, cadaverine and putrestine. which may be formed as decarboxylation products of lysine and ornithine. respectively. .4gmatine, tyramine. histamine, and tryptamine. the decarboxylation products of arginine, tyrosine. histidine. and tryptophan. are also attacked. This method for the determination of decarboxylase

decarboxylase

estimation

method

of agmatine,

amine oxidase, as a red-brown may

be

applied

from the

oat

\eedling\

decarboxylation

the agmatine chromogen to the

is oxidised formed on estimation

of

activity is based upon the consecutive oxidation of the amine product by pea seedling amine oxidase. diamine:oxidoreductase (deaminating) (EC 1.4.3.6) and the utilization of the hydrogen peroxide generated to oxidise guaiacol in the presence of peroxidase to a red-brown chromogen. This chromogen is apparently complex and contains quinones (2). A similar peroxidase system has been used to determine the activity of di- and polyamine oxidases (3). A method is described for the estimation of arginine decarboxylase (I.-arginine carboxy-lyase, EC 4. I. 1.19). from the leaves of oat seedlings based on these principles. This technique has much greater sensitivity than the standard manometric procedures and approaches that of the radioactive tracer techniques. METHODS

This was purified from the leaves of 21. day-old oat seedlings (A \YFZN.votirw L. cv Black Supreme) grown in sand in diurnal illumination (16-h day. IO klx, 24’72, light, 19°C. dark). A potassium-deficient nutrient medium was used. which increased the yield of the enzyme (4). The plants were watered

332

TERENCE NH

COOH

NH 2 !NHCH CH CH 222 Arginine

~HNH

2

4--

- - ---Argtnine docorboxylase

co2 tNH 2 !EHCH 7CH 2CH z CH 2 NH 2 +-------Dinmine Agmotine 02

oxidase

NH NH2!NHCH,cH,CH,CH0

A. SMITH

One international unit (micromole per minute) equals 16.7 nkat. The final preparation (about 20-fold purified) normally had 8 to 16 nkatiml (about 0.7- 1.3 nkatimg). The optimum pH in Tris and phosphate buffer, determined by release of ‘YYO, from [CT-‘“Clarginine was 7.0. Stoichiometry for CO, formation by Warburg manometry at pH 6.3 was 96 and 98% (two estimations).

NHs 3 Guo~acot

+

H,O,

Pea seedlings (Pisum sati\‘trttl L. cv Meteor) were grown for 11 days in sand in the J dark ( 19°C) and watered daily with a nutrient Red brown oxidation products medium containing 2 mM K,SO,, 1.5 mM MgSO,, 4 mM CaCI,, 0.33 mM Na,HPO,, Xmax 470nm 4 mM NaNO,,, and 4 mM (NH&SO,, with SCH~MF. I FeEDTA and micronutrients. The method of McGowan and Muir (5) was used to purify daily with a nutrient medium containing the DAO. After protamine sulphate treat2 mM Na&S04, I .5 mM MgSO,, 4 mM CaCl,, ment and (NH,),SO, and ethanol fractiona0.33 mM Na,HPO,, 4 mM NaNO:,, and 4 mM tion, the preparation was dissolved in 50 mM phosphate buffer (pH 6.3). Final activity (NHJZS04, with FeEDTA and micronutriwas 89 nkatiml(26 nkatimg) with putrescine ents. The leaves were macerated in 2 vol of 50 mM Na,HPO,, and the extract was as substrate in 0.1 M Tris buffer, pH 7.5. using the spectrophotometric procedure filtered under pressure through nylon cloth for assay. No loss of activity could be deand frozen for 18 h. On thawing, (NH,),SO, tected on storage for 6 weeks at ~ 10°C. was added (200 g/liter) and the precipitate was discarded after centrifuging at 2OOOg By comparison with the preparation of for 15 min. (NH,),SO, was then added to McGowan and Muir (5) and correcting for raise the concentration to 500 g/liter, the the lower activity of DA0 with benzylamine, precipitate (2OOO‘e, 15 min) taken up in 10 mM the substrate they used (5.7% of the activity phosphate buffer (pH 6.3) and dialysed with putrescine). the specific activity of their preparation was about three times greater against this buffer. Acetone at - 15°C (2 vol) was then added with stirring at 0°C and the than that of the present study. Two attempts extract centrifuged for 5 min at 5000&r. On to purify the DA0 by the method of Hill (6) redissolving in 10 mM phosphate buffer gave very low yields, due to denaturation (pH 6.3), the extract was dialyzed against this by the chloroform-ethanol mixture used. buffer and centrifuged at 15.000~ for 15 min. Horseradish peroxidase (Sigma type II, Enzyme activity is expressed in terms of 195 purpurogallin unitsimg). bovine liver the katal (kat).’ the amount ofactivity which catalase (Sigma, 2000 unitsimg). and superconverts I mol of substrate per second. oxide dismutase (Sigma, 2700 unitsimg) were dissolved in water (1 mgiml). Protein was estimated by the method of Lowry et ’ Abbreviations used: kat, katal; DAO. diamine oxida$e. I/l. (7). *-------Peroxidase

ARGININE DECARBOXYLASE

Standard Warburg flasks were used, with enzyme (normally I-5 nkat) and 0.1 M phosphate buffer (pH 6.3) in the main compartment, and 0.5 ml of 23 mM r.-arginine was added from the side arm. On incubating at 3O”C, CO, formation was almost linear with time for the first 30 min. and for the subsequent 30 min, it was 18% below the expected value for linearity (mean of three estimations). Due to CO, retention at high pH values. reliable estimates of activity can be obtained only at pH 6.3 and less.

The assay was conducted in Conway units. Enzyme (up to 1 nkat) in 2 ml of buffer, normally 0.1 M Tris, pH 7.5 (correct at 30°C). was placed in the outer compartment. The centre well contained 0.2 ml of 0.1 M NaOH. Arginine (0.5 ml, 20 mM. containing 0. I &i [(/-“Clarginine) was added to the outer compartment. The units were then sealed with Vaseline and incubated at the surface of a water bath for I h at 30°C covered with a sheet of expanded polystyrene. HCI (0.5 ml, I M) was added to the outer compartment, the units were resealed, and the CO, was distilled at room temperature for 45 min. CaCI, (0.1 ml, 0.2 M) was then added to the centre well. and the suspension was transferred quantitatively to planchettes. After drying at 100°C for I5 min, the radioactivity was estimated as the mean of three replicate 1000-s counts. Blanks were used to estimate the volatile “C impurities distilling from the arginine without enzyme addition.

In an optically prematched glass tube (I. i.d., x 12 cm). 2.4 ml of0. I M Tris buffer

ESTIMATION

333

(pH 7.5) (correct at 30°C). 0.1 ml of DA0 (9 nkat), 0. I ml of peroxidase. 0.1 ml of guaiacol (25 mM), and 0.1 ml of extract, containing SO to 250 pkat of arginine decarboxylase, were placed. Greater dilutions of enzyme can be accommodated by reducing the volume of the Tris buffer and increasing the volume of extract to a total volume of 2.8 ml. The tube was preincubated in a water bath at 30°C for 2 min and then placed in a spectrophotometer with a thermostatted cuvette holder at 30°C. On addition of substrate (0.1 ml of 25 mM t.-arginine) and mixing. increase in A d7,1was determined on a IO-mV recorder with a log converter (full scale normally 0.2 A unit).

The spectrophotometric method was used. which depends on the guaiacollperoxidase system to estimate the hydrogen peroxide (3,8). For the estimation of the K,n for oxygen. the oxygen electrode was used according to the method of Smith and Bickley (9). Both assays were conducted at 30°C in 0.1 M Tris buffer (pH 7.5). RESULTS

AND DISCUSSION

The main structural requirement for the substrate of pea seedling DA0 appears to be the possession of the -CH,-NH, moiety. but activity varies with structure and some compounds bearing this grouping are quite resistant to oxidation (1). In the present work, oxidation of neither 4-aminobutyric acid nor ornithine by the DA0 could be detected. Neither is the guanidino group oxidised and arginine is not a substrate. However, lysine is attacked at about 5%~ of the rate of cadaverine with a pH optimum at 8.5 (1). Agmatine was oxidised by the DA0 with a pH optimum at about 7.5. Activity in Tris buffer was about twofold greater than in phosphate buffer (Fig. 1) at the same optimum pH (7.5). In both Tris. pH 7.5, and phosphate buffer. pH 6.8, using the spectrophotometric assay,

TEKENCE

334

5

6

7

8

9

10

PH FIG. I. pH activity curves for the oxidation of agmatine by pea seedling diamine oxidase using the spectrophotometric procedure. Phosphate buffer. 0: Tris buffer. a.

putrescine was oxidised 4.6 times faster than agmatine by the pea seedling DAO. Using the oxygen electrode in Tris buffer. pH 7.5. putrescine was oxidised 4.8 times faster than agmatine. Hill and Mann (I) found this ratio to he 2.9:l in phosphate buffer, pH 7. Good agreement was found between the activity for arginine decarboxylase, estimated by the manometric. isotopic, and spectrophotometric methods. Correcting the value obtained by manometry at pH 6.3 to the expected value (=lOO%) at pH 7.5 by reference to the pH activity curve for arginine decarboxylase (Fig. 2), the activity determined by the ld’c method was 101% and for the spectrophotometric method, 102%. Addition of equivalent amounts of the DA0 substrates, agmatine or putrescine, or of arginine in the presence and absence of the arginine decarboxylase, showed increments of absorbance which were in good agreement (Table 1). This indicates that the conversion of arginine to agmatine is stoichiometric. At pH 7.5, the K,,, for agmatine oxidation

A. SMITH

by the DA0 in Tris buffer was lower than the K,,, for putrescine. McGowan and Muir (5) give the K,,, for putrescine as 8 x IO-” M (Tris buffer. pH 7). while Yamasaki ct rrl. (10) give 7.4 x IO-” M (phosphate/borate buffer, pH 8). The K,,, for arginine for the decarboxylase system, 8 x IO-” M (Table 2). was considerably less than the concentration of arginine provided in the standard assay (normally about IO-:{ M). Increase in absorbance was not normally detected prior to the addition of arginine. However, on adding large amounts of the arginine decarboxylase preparation (I nkat), A-I70increased by 0.04 over a period of 5 min before becoming constant. This may be attributed either to traces of agmatine derived from arginine or of another DA0 substrate (e.g., putrescine, spermidine, or spermine) in the preparation of arginine decarboxylase. Prolonged dialysis reduced this increase in absorbance, though it did not eliminate it. The new spectrophotometric method may be used for the assay of t--arginine in the

A

I 5

’

I ti

’

I 7

’

I 8

’

I 9

PH

FIG. 2. pH activity curve for oat leaf arginine decarhoxylase determined by measurement of “C02 liberated from I.-[U-‘“Clarginine. Phosphate buffer. (2: Tris buffer. ~5.

ARGININE

DECARBOXYLASE

ESTIMATION

TABLE STOIC HIOMF

rRy

OF rHt

SPFUKOPHOI-OM~

I RIG Mt

335

I

I HOD

FOR I HE ESTIMATION

OF ARGINI~L

D~(ARBOXII.L

ASP”

Substrate Putrescine

Increase in absorbance Time tahen to reach

peak

Agmatine

-AD

+AD

-AD

-t AD

-AD

0.224

0.180 Cl

0.w- -

0.190 8

0

(min)

cl

7

solution

was

” Duplicate

stirred

and

the

increased

absorbance

was

0.190.0.186 9.9

measured

(recorder

f.s.d.

A ,?,, = 0.4).

estimations.

presence of D-arginine. which is ineffective as a substrate of the oat leaf arginine decarboxylase. However. large concentrations (5 mM) of D-arginine inhibit the arginine decarboxylase. The spectrophotometric method of arginine decarboxylase assay may be particularly useful in the rapid monitoring of column effluents for amino acid decarboxylases. since each assay takes only 3 to 4 min by the new method. Artefacts may arise when polyphenolases are present, which could oxidise the guaiacol, and the preparations should be free of DA0 substrates. Neither catalase (200 units) nor superoxide #dismutase (270 units) added to the system had any apparent effect on the increase in absorbance, possibly for kinetic reasons. The oxidative decarboxylation of amino

acids by pyridoxal phosphate in the presence of peroxidase requires Mn”+ ions. and this reaction is inhibited by H,O, (1 I). It is therefore unlikely that this decarboxylation reaction would interfere in the spectrophotometric method described in the present work. even if exogenous pyridoxal phosphate is needed for activity of an amino acid decarboxylase to be assayed. No loss of activity was found on incubating the mixture of peroxidase. DAO. guaiacol. arginine decarboxylase, and buffer for 60 min at 30°C prior to the addition of the arginine. The apparent activity was linear with increasing arginine decarboxylase. up to 250 pkat (Fig. 3). However at 1 I50 pkat. activity was only 54% of that expected. This nonlinearity is apparently dependent on the limitation imposed by the activity

TABLE MI~HAFI

+ AD”

pH 7.5, 0. I ml diamine oxidase. 0.1 ml decarboxylase (AD) ( 1.4 &at, in 0. I ml). 5 ~1 of 15 mhr substrate was added.

” Reaction mixture consisted of 2.4 ml of 0. I hl Tris buffer, peroxidase. and 0.1 ml of 25 rnhl guaiacol. with or without arginine to a total volume of 2.8 ml. After establishing the blank absorbance, the

Arginine

I\ CONSTANIS

F-OR rHr

OXIIIAIION

OF ACMATINF

2 AND

PurRr-scrhE

OVIDASL 4%~ FOR THE: Dee ARR~XYI 41-10~ OF ARr+ININr. Dr (‘ARROxyl ASF USING I HI Spt-c TRoPHoroLItTRI~ Buffer (0.1 hl) Agmatine Agmatine

Phosphate. Phosphate.

Agmatine Putrescinr Arginine

Tris. Tris. Tris.

pH pH pH

pH pH 7.5 7.5 7.5

BY OAT METHOD

Michaelis

6.8

7.5

constant

BI

PLA

SFLL)LIN;

DI,.WINI

LLAF. ARGININL OF ASSAM SE

I hl)

(\I)

4 x IO ’ 9 * 10 i

8 x IO ; I * IO-’

3 x 10 ,-’ 4, IO T 8 x IO-’

3 x IO-” 6 x lo-” 2 Y IO i

TERENCE

336

0

.2

.4

.6

.8

I.0

1.2

n kat FIG. 3. Increase in the rate of absorbance at 470 nm with increasing amounts of arginine decarboxylase in the spectrophotometric assay.

of the DAO. With arginine decarboxylase assays containing 115 pkat of arginine decarboxylase. increasing the diamine oxidase by IO-fold made no difference to the ultimate rate of colour formation, though this increased DA0 did eliminate the slight initial lag period of about 2 min before maximum rate was achieved, found with the normal amount of DAO. With greater (nonlinear) activities of arginine decarboxylase (I.5 nkat). increasing the DA0 IO-fold increased the apparent activity of the arginine decarboxylase by about three times. Lower activities of arginine decarboxylase could be detected by incubation at 30°C. and measuring absorbance at intervals of 0. 15, 30. and 60 min. Activity was almost linear to at least 60 min at pH 7.5. unlike the Warburg assay where activity was nonlinear over this period at pH 6.3. Oxygen. dissolved in the water and necessary for the oxidation of the amine by the DAO, is unlikely to be limiting with low DA0 activities, as already noted by Mann (12). No effect of saturating the assay solution with oxygen was found on the rates of increase of absorbance with 100 or 1000 pkat of arginine decarboxylase. Oxygen dissolved in water at 30°C (2.5 x IO-” M)

A.

SMITH

was sufficient for the oxidation of about 0.7 pmol of substrate (absorbance at 470 nm = 0.5). Without additional aeration. measurements approaching this value may give nonlinear assessments of decarboxylase activity due to oxygen depletion. The K,,, values for oxygen for the oxidation by the pea seedling DA0 of putrescine and agmatine ( 10PJ M each) were about 2 x IO-’ M and 0.7 x IOP M. respectively. Yamasaki ct rrl. (IO) gave K,,, for oxygen with tryptamine as substrate as 4 x IO-” M. By comparison with other methods, the spectrophotometric procedure described here has advantages of rapidity, simplicity, and sensitivity. For IO pmol of substrate. the A,,,, found in the present method is 7.2 units, corresponding to a theoretical deflection of 720 cm on the recorder chart. Assuming a difference of 2 mm to be significant. sensitivity is about 1 in 3600. For the Warburg method, IO pmol of arginine at pH 6.3 gives 224 PI of CO,. measurable as 330 mm (flask constant about I .5) on the manometer scale. Sensitivity is therefore about 1 in 150. assuming a difference of 3 mm to be significant. Using [I/-‘“Clarginine (0. I @IX/ assay) with arginine at a final concentration of 5 x IO--’ M (K,,, of arginine decarboxylase = IO-” M) planchette counting of ‘aCO, from 10 pmol of arginine gave 92 cps. Assuming a difference of 0.1 cps to be significant, sensitivity is about I in 920. On this basis, sensitivity of the spectrophotometric method is 4 times and 25 times that of the 14C and Warburg methods, respectively. Sensitivity of the present method could be further increased using fluorometry with scopoletin (5), or leuco-2’,7’-dichlorofluorescein ( 13) in place of guaiacol. The K,,, for the oxidation of agmatine (2.2 x IO-: M) by the pea seedling DA0 in Tris buffer was much less than in the phosphate buffer, and activity was also about twice as great in Tris as in phosphate buffer at the optimum pH (Fig. 1). A similar effect of phosphate buffers has been found with

ARGININE

DECARBOXYLASE

putrescine oxidation (3). whereas benzylamine oxidation by the pea seedling DA0 is tlrpc~ntlcnt on the presence of phosphate in glycine or borate buffers at pH 8.6 (5). Presumably hog kidney DA0 could be used in place of the pea seedling DAO. though the specific activity of crude material and final product of the pea DA0 (6) are higher (105 times and 58 times, respectively) than for the hog kidney DA0 (14). The present method may be applied to the estimation of other amino acid decarboxylases and it may be especially useful in monitoring ornithine decarboxylase though the sulphydry1 compounds added for stabilization may interfere. Unlike the corresponding enzymes ftom plants and animals, bacterial amino acid decarboxylases generally have low pH optima (5-6) and might not be compatible with the pea seedling DAO.

REFERENCES Hill. 91, 2.

1 am grateful to Mr. assay. analysis.

to Mr.

G. R. Best

C. P. Lloyd-Jones and to Miss Gillian

for

with for

the plants. the the

carbon-14 statistical

Mann.

P. J. G. ( 1964)

3. 4.

Smith,

T. A. ( 1963)

McGowan. f’l~wi~~l. 6.

Hill.

hc,wi.vrry Muir.

Rosebrough, R. J. (19.51)

265-275. Smith. T. A. ( 1977)PI1~r~,c,hc,r,ri.\tr~

9.

Smith.

T.

A..

~~lr~wrisfr~ IO. Yamasahi. ! I.

( 1070) Mazelis.

and

Bickley.

./. C/w,t~. 108 I.

R.

M.

(1971)

Pltrllr

in

W.,

Enzymology eds.). Vol. 17. Press, New York.

D.

Farr. A. L., (‘lr<,~>~. 193, 16, 1647-

1649.

1 lY74)

P/r)~/~~-

A.

13, 2437-2443. E.

F..

Swindell.

Ritw/lctnibfry M. (1971)

York. Mann, P. .I. G. (1955) Kochli. H., and van Ri~~~~/f<'ll/. 84, 127Mondovi, B.. Rotilio. A. F. (1971)iu

Methods

and Tabor. C. W.. 735-740. Academic

R..

9, 1206iu Method&

(Tabor. H.. and Tabor. Part B. pp. 606-608.

14.

.I.

2, 241-352.

N. J.. ./. Bit)/.

8.

1tcv1.

13, 107%

Ph~to~,/~l,,rrivtrl\‘

R. E.. and 47, 644-648.

0. H.. Randall,

Biro

B. C. ( 1956)

J. M. (1971) irr Methods (Tabor. H.. and Tabor. C. Part B. pp. 730-735. Academic

7. Lowry. and

13.

for growing

help Arnold

J. M.. and 171-182.

Booth. H.. and Saunders. SfW.. Y40-948. Smith. T. A. (1974)f%~t~~c

I?.

ACKNOWLEDGMENTS

337

ESTIMATION

and 1210. in

C. W.. Academic

Bi~~~.h<~u/. Wartburg, 135. G.. Costa.

D.

J.

Enzymology

eds.), Vol. 17. Pres\. Neu J. 59,

609-630.

J. P. (1978)

Atrtrl.

M. T., and

in Enzymology eds.). Press.

Reed.

Agro.

(Tabor.

Vol. 17. Part New York.

H.

B, pp.