12o BIOCHIMICA ET BIOPHYSICA ACTA

BBA 4 5 0 4 7

E L E C T R O N - P A R A M A G N E T I C - R E S O N A N C E S T U D I E S

O F T H E C H L O R P R O M A Z I N E F R E E R A D I C A L F O R M E D

D U R I N G E N Z Y M I C O X I D A T I O N

B Y P E R O X I D A S E - H Y D R O G E N P E R O X I D E

L. H. P I E T T E AND G. BULOVV

Instrument Division, Varian Associates, Palo Alto, Calif. (U.S.A.)

1SAO Y A M A Z A K I

Hokkaido University, Sapporo (Japan)

(Received December 23rd, 1963)

S U M M A R Y

I . The t ranqui l iz ing drug ch lorpromazine was enzymica l ly oxid ized to the free- rad ica l i n t e rmed ia t e which was ident ica l wi th the red in t e rmed ia t e observed op t ica l ly

at 53o mtt. 2. The s to ich iomet ry for the pe rox idase -H202 reac t ion was de t e rmined to be

2 CI)Z ~- H202 horse radish peroxidase . . . . . . . . . . . . . . > 2 CpZ- + (2 H20 ) (~)

3. The d i smuta t ion reac t ion ra te cons tan t , ka, at p H 4.8 was measured to be I5.O M - l ' s e c -1. This ex t reme s t ab i l i t y of the free radical al lowed fur ther ox ida t ion of the free rad ica l b y the enzyme in the subs t ra t e - l imi ted case; the ra te cons tan t for th is reac t ion was found to be 3 .9 .1o 4 M- l - s ec 1, approx. IO t imes slower than k4, the first ox ida t ion .

4. I t is suggested t ha t the s t ab i l i t y of the free radica l m a y be responsible for the psycho t rop ic a c t i v i t y of the drug.

INTRODUCTION

The un iva len t ox ida t ion of CPZ in vitro to a f ree-radical i n t e rmed ia t e has been clear ly d e m o n s t r a t e d i n a va r i e ty of E P R exper imen t s 1, 2. In each case ox ida t ion was p roduced b y e i ther a me ta l ion (Fe s+, Ce4+), concen t r a t ed acid, or cont ro l led electrolysis. The un iva len t ox ida t ion produced a colored in t e rmed ia t e wi th an absorp t ion m a x i m u m

at 255 and 530 mtt. CAVANAUGH 3 repor ted CPZ to be a subs t ra te for bo th the peroxidase (donor : H20 ~

oxidoreductase , EC I . I I . i . 7 ) and cata lase (H202:H202 oxidoreductase , EC 1.11.1.6) sys tem. He repor ted the fo rmat ion of a colored p roduc t (53 ° m/z) in the reac t ion of CPZ wi th bo th p e r o x i d a s e - H 2 0 2 and cata lase over a p H range of 3 to 6.3. GILLETTE

Abbrev i a t i ons : CPZ, ch lo rp romaz ine ; E P R , electron p a r a m a g n e t i c resonance.

Biochim. Biophys. Acla, 88 (1964) 12o 129

EPR STUDY OF THE CHLORPROMAZINE F R E E RADICAL 1 2 1

AND KAMM 4 have shown that CPZ is completely oxidized to both the sulfoxide and sulfone by the enzymic action of liver microsomes.

We have observed the formation of a free radical from CPZ during the enzymic oxidation by peroxidase-H202 (Fig. i). The free radical is responsible for the ab- sorption at 530 m#. Complete kinetic studies on the formation and decay of these free radicals in the peroxidase-H202 reaction have been made and are reported in this paper.

g=2.005

3.4 GAUSS

Fig. i . C P Z free r a d i c a l o b s e r v e d in t h e s t e a d y s t a t e d u r i n g f low of 6. lO -4 M H 2 0 2 a n d 4" IO-I M C P Z w i t h lO -7 M hor se r a d i s h p e r o x i d a s e a t p H 4.8.

MATERIALS AND METHODS

Horse radish peroxidase (RZ ~-- 3.0)* was purified and crystallized by the method of KENTEN AND MANN 5. CPZ-HC1 was obtained from SKF of (USP) grade and not purified further. The potassium salt of nitrosyl disulfonate (Fremy's salt) was obtained from Aldrich and recrystallized using the method of HARVEY AND HOLLINGSHEAD 6.

The EPR spectrometer used was a Varian V-45oo X-band instrument, utilizing IOO kcycles field modulation and Fieldial control of the magnetic field. The flow apparatus was a modified version of that described previously 7. The important modifi- cations were a reduced volume from mixer to the center of the cavity of o.135 ± o.oo3 ml and the incorporation of three Skinner V-5 solenoid valves for stopping the flow with closing times of 4 msec.

All free-radical determinations were made by a comparison of the calculated first moment of the CPZ radical in question with the first moment of a standard nitrosyl disulfonate sample. The nitrosyl disulfonate standards were made up in buffered solutions of pH 8.o and concentrations were determined optically from the molar extinction coefficient at 545 m/~ = 2o.8 reported by MURIB AND RITTER s. Reproduci- bility of the moment calculations was better than IO %. The proportionality between signal amplitude and the first moment was verified for the standard over the entire concentration range encountered.

Simultaneous EPR and optical absorption measurements were made using a

* T h e R Z i R e i n h e i t s z a h l is a d e g r e e of p u r i t y of e n z y m e as d e f i n e d b y T. THEORELL, A cta Chem. Scan& 4 (195o) 442. I t i s a r a t i o of e n z y m e a b s o r b a n c i e s m e a s u r e d a t t w o d i f f e r e n t w a v e l e n g t h s :

A a t 403 m # _ R Z A a t 275 rn#

Biochim. Biophys. Acta, 88 ( i964) 12o 129

122 L.H. PIETTE, G. BULOW, I. YAMAZAKI

Var ian V-4534 opt ica l - t ransmiss ion cavi ty . Light from a tungs ten l amp was f i l tered b y means of a Corning in terference fi!ter a t 542 m/~ and beamed th rough the sample flow cuve t te and collected on four series solar cells. The ou tpu t s of the solar cells and the E P R spec t romete r were pu t in to a dual channel recorder.

RESULTS AND K I N E T I C S

Stoichiometry of the reaction

In order to de te rmine the s to ich iomet ry of a reac t ion involv ing a free-radical i n t e rmed ia t e i t is necessary to measure the concen t ra t ion of the i n t e rmed ia t e quan t i - t a t ive ly . Fo r most in t e rmed ia te s involv ing free radicals , the life t ime of the radica l is genera l ly too shor t to allow accura te concen t ra t ion measurements b y s t a n d a r d techniques. E P R , however, which de tec ts only free radicals , when used in con junc t ion wi th l iquid flow systems, does allow q u a n t i t a t i v e measurements to be made. The s to ich iomet rv of the reac t ion

2 C P Z + H202 horse radish peroxidase + 2 C P Z . + (2 H 2 0 ) ( t )

was de te rmined by p lo t t i ng the f ree-radical (CPZ.) concen t ra t ion as a funct ion of the CPZ concen t ra t ion (Fig. 2). A t a cer ta in CPZ concen t ra t ion a m a x i m u m free- rad ica l concen t ra t ion (spin concentra t ion) is reached which does not increase fur ther upon increase of CPZ, nor does i t increase wi th increas ing enzyme concen t ra t ion

Z

x 1.0

,I

~o.5 8 c "~_ t/)

1 2 3 4 5 6 CPZ concentration (raN)

Fig. 2. A plot of maximum free-radical concentration obtained as a function of substrate (CPZ) concentration. 5" io 5 M H202, 2.0. IO 7 M horse radish peroxidase at pH 4-7. Temperature 23 °.

above 10 -7 M. At lower levels of enzyme concent ra t ion , and therefore slower fo rmat ion rates , the m a x i m u m spin concen t ra t ion is decreased because of a decay of the free radicals b y d i smuta t ion before the t rue s to ichiometr ic m a x i m u m is reached. Fig. 2 i l lus t ra tes t h a t under o p t i m u m condi t ions , the accumula ted spin concen t ra t ion is ve ry close to twice the concent ra t ion of H202 used. This resul t is consis tent wi th previous observa t ions of the peroxidase reac t ion 9 t h a t I mole of H202 gives 2 moles of subs t r a t e free radical . In most of the previous cases, however, the subs t r a t e free radicals were too uns tab le and decayed very rapid ly , al lowing only a s t eady - s t a t e concen t ra t ion to be measured. W i t h CPZ as the subs t ra te the q u a n t i t a t i v e accumu- la t ion of CPZ. was observed direct ly .

Biochim. Biophys. Acta, 88 (i964) i2o-i29

EPR STUDY OF THE CHLORPROMAZINE FREE RADICAL 123

Estimation of kl and k 4

According to the theory of CHANCE 10 the following mechanism was proposed:

Horse rad i sh peroxidase + H202 ht--+ Complex I (2)

Complex I + CPZ h7 > Complex II + CPZ. (3)

Complex II + CPZ k4--+ horse rad i sh peroxidase + CPZ. (4)

We have been able to measure bo th k 1 and k 4 dire,ct!y from measurements of the velocity of formation of the CPZ radical as a function of H202 concentration and CPZ concentration. The measurements are made by adjusting the EPR spectrometer magnetic field by means of the calibrated Fieldial to the field expected for the free radical. The flow system is activated so that the flow rate through the EPR cavity is faster than reaction can occur, then the flow is stopped rapidly, and a complete trace is recorded on a fast recorder of the formation of the free radical as a function of time. The experiments are repeated with different H202 concentrations and CPZ concen- trations.

20

z~

~o

<l 10 J

I k 0 ' 0.5 1.0

Ha02 concentration (raM) Fig. 3- A plot of CPZ free-radical reac t ion ve loc i ty as a func t ion of H202 concent ra t ion . 4.0" lo s M

horse rad i sh peroxidase , lO -3 M CPZ a t p H 4.8. T e m p e r a t u r e 23 °.

Fig. 3 illustrates a plot of the rate of formation of CPZ- as a function of H202 concentrations. At low H20~ concentrations and therefore a H202-1imited reaction rate, k 1 is determined directly from the expression

V = 2 k l ~ E ~ [H202] (5)

in which [E l means enzyme concentration; k 1 was measured to be 4.5" ~o6 M-l" see-1. Fig. 4 illustrates a plot of the rate of formation of CPZ- as a function of CPZ

concentration. In the two-step reduction of peroxidase-H202 complex (Reactions 3 and 4), kl is usually much greater than k~ as CHANCE 10 has confirmed by using nitrous acid as the electron donor. If we assume the same mechanism here, at low CPZ concentrations and, therefore, a CPZ-limited reaction rate, k4 is determined directly from the expression

v = 2 k 4 [ E ~ [CPZI (6)

k~ was measured to be 3.2-lO 5 M-l.sec -1.

B i o c h i m . B i o p h y s . A c t a , 88 (1964) 12o-129

I 2 4 L . H . PIETTE, G. BULOW, I. YAMAZAKI

Dismutation reaction rate constant k~l

Previous results on the formation of substrate free radicals in the peroxidase reaction indicated that the dominant mechanism for disappearance of the free radical was a simple bimolecular dismutation reaction.

2 CPZ. I¢a ~ CPZ + CPZ: (7)

In the peroxidase-CPZ reaction it was observed that only under H202-1imited con- ditions was a simple dismutation reaction observed. Fig. 5 illustrates that at different enzyme concentrations only the rate of accumulation of free radical is affected, and not the decay rate. kd therefore, can be determined directly from these curves. Table I is a tabulation of kd measurements.

As the pH is gradually increased, the CPZ free radical becomes more unstable and ka increases. Close to neutral pH's the decay of the free radical is so fast the

3 °

CPZ concentro£ion (raM)

Fig. 4. A p lo t of CPZ free-radical reac t ion ve loc i ty as a func t ion of s u b s t r a t e (CPZ) concent ra t ion . 4 . o - i o -s M horse r ad i sh peroxidase , 1.2. i o a M H202 a t p H 4.8. T e m p e r a t u r e 23 °.

A

- j

TIME (SEC)

TIME (SEC)

Fig. 5. Di rec t record ing of f ree-radical decay as a func t ion of t ime, ka. Spec t rome te r magne t i c field is pos i t ioned a t m a x i m u m signal a m p l i t u d e and flow is s t a r t ed t h e n s topped. Signal rise is fo rma t ion curve and drop is decay. A, 4 ' Io - s IVI horse rad i sh peroxidase , i o -a M CPZ, 2- lO -4 M

H202 a t p H 4.8; B, same excep t horse rad i sh pe rox idase concen t ra t ion = 3.2' i o T M.

Biochim. Biophys. Acta, 88 (i964) 12o - i 29

EPR STUDY OF THE CHLORPROMAZINE F R E E RADICAL 1 2 5

TABLE I

E S T I M A T I O N O F k d A T D I F F E R E N T E N Z Y M E C O N C E N T R A T I O N S

C a l c u l a t i o n w a s d o n e f r o m t h e d e c a y v e l o c i t y of C P Z r a d i c a l a t t h e s p i n c o n c e n t r a t i o n of 2.2. lO -4 M, w h e r e t h e d e c a y is d u e o n l y t o t h e d i s m u t a t i o n r e a c t i o n . See F ig . 5- lO-3 M CPZ, 2- i 0 - I M H s O 2,

o.o 5 M a c e t a t e a t p H 4.8.

Peroxida~se concentration (M) ka(M-] "sec-X)

2" 10 -8 14.6 16.1

4" IO--S 14'5 14.5

8" 10 -8 16.O

14.5

1.6" 10 -7 14. 3 15.7

3.2" 10 -7 15.O 14.6

steady-state free-radical accumulation is observed in less than a few seconds after the reaction starts. The feature of appearance and disappearance of the free radical depends on the enzyme concentration as illustrated in Fig. 6. Here, the duration of the free radical is inversely proportional to the enzyme concentration and the steady-state concentration of free radical is proportional to the square root of the enzyme concen- tration as reported previously 9.

kd can be measured directly by mixing the CPZ free radical accumulated by the enzyme reaction at pH 4.8 where the radical is stable, with a neutral buffer solution where the radical is unstable. The rapid flee-radical decay is observed directly. These experiments were done under stopped-flow conditions with a rapid recorder. Under

A

7 q m 3_

TIME

*tm~u.a • . . . . . . . . . .

TIME

Fig . 6. D i r e c t r e c o r d i n g of f r e e - r a d i c a l f o r m a t i o n a n d d e c a y as a f u n c t i o n of t i m e a t p H 6.43. A, 8. IO -s M h o r s e r a d i s h p e r o x i d a s e , 6. lO -4 M C P Z , 2. lO -4 M H202 . B, s a m e e x c e p t h o r s e r a d i s h

p e r o x i d a s e c o n c e n t r a t i o n = 32.o" lO - s M.

Biochim. Biophys. Acta, 88 (1964) 1 2 o - 1 2 9

126 L. H. PIETTE, G. BULOW, I. YAMAZAKI

these conditions, the decay depends only on the dismutation of the free radical. Fig. 7 illustrates two decay curves as measured directly with the E P R spectrometer under the conditions illustrated above.

r

' ' ' 'TIME

B

w ,

TIME

Fig. 7. Direct record ing of free-radical decay as a func t ion of t ime. F low s y s t e m con ta ins CPZ free radical a t p H 4.8 on one side and s t rong buffer a t p H 8.6 (A) and p H 6.0 (B) on o the r side. Radica l decays when flow is s topped. R e s u l t a n t p H ' s are 7.8 and 5-4, respect ively, ka =

2. 5. lO 4 M - l . s e c -1 a t p H 7.8 and 7.6- lO S M - l . s e c -1 a t p H 5.4.

k 4' estimation

I t might be expected tha t another mechanism for the disappearance of the free radical is possible, namely the reaction of the free radical directly with the unreacted enzyme.

CPZ" + Complex I kv' ~ CPZ: + Complex II (8)

CPZ. + Complex II k4" --> CPZ: + e n z y m e (free) (9)

In the previous study of peroxidase-substra te free radicals ° no such reaction between the substrate free radical and the enzyme was detected and the only decay mechanism observed was dismutation. In the peroxidase-CPZ reaction, however, the dismutation reaction is quite slow and we were able to detect the enzyme-catalyzed disappearance of the CPZ free radical. Fig. 8 shows tha t under CPZ concentration-limited conditions, the decay of the CPZ free radical greatly depends upon the enzyme concentration. The decay reaction is a combination of the reaction of CPZ. with the enzyme as well as dismutation.

If we plot the decay velocity against enzyme concentration, the extrapolated velocity at zero enzyme concentration should correspond to the dismutation velocity. Such a plot is illustrated in Fig. 9. Providing that the same argument for the esti- mation of k 4 applies here, we can calculate k 4' the reaction rate constant between the enzyme (Complex II) and free radical directly from Fig. 9 using the expression

va = 2k4"[E? [CPZ ' ] + z k a [ C P Z ' ] z (lO)

B i o c h i m . B i o p h y s . Ac ta , 88 (1964) 12o 129

E P R STUDY OF THE CHLORPROMAZINE FREE RADICAL 12 7

Vd

[CPZ- - - = 2k4'[E ] + 2hd[CPZ-] ( I I )

zJ Vd

2k4, [CPZ" ] A E

in which Vd is the velocityof decay. This is the first t ime such a reaction rate has been measured directly, k 4' was measured to be 3.9" lO4 M-l" sec-1-

TIME (SEC)

/ I I'

TIME (SEC)

Fig. 8. D i rec t measurement of f ree-radical react ion w i t h enzyme. Subst ra te- l imi ted react ion. A, 4.0. lO -4 IV[ CPZ, 6.0.1o -4 M H~O2, 4.0. lO -8 M horse radish peroxidase, a t p H 6.43. B, same

except home radish peroxidase concent ra t ion = 32.0 • IO -7 M.

2 3 × C

o

8

C .~_

_~c ¢

O

I I I I 2 3

Horse radish peroxidase concentration x I0-7M

Fig. 9. P lo t of t he change in t he free-radical decay ra t e per free-radical concen t r a t ion as a f unc t i on of e n z y m e concen t ra t ion . E x t r a p o l a t i o n to zero e n z y m e concen t ra t ion yields ka.

DISCUSSION

The kinetic results certainly suggest that tranquilizers such as CPZ are catalytically oxidized to free-radical intermediates by oxidative enzymes such as pe rox idase-H 202- The rate of oxidation of CPZ is fast and is comparable to substrates such as ascorbic acid, hydroquinone, etc.

Biochim. Biophys. Acta, 88 (1964) 12o-129

128 L . H . PIETTE, G. BULOW, I. YAMAZAKI

The kinetic results definitely establish the stoichiometry of the reaction as that of Eqn. i and the complete mechanism of reaction as that illustrated in Eqns. 2- 4.

The unique feature of the reaction is the extreme stability of the CPZ radical. The rate constant of dismutation, kd, is 15.o M 1.sec-1 at pH 4.8 as compared to lO 8 M -1.sec -1 for the ascorbate radical at the same pH. At biological pH's the CPZ radical becomes less stable but still much more stable than the ascorbate and non- heterocyclic semiquinones and other free radicals encountered in nature. I t is suggested that this extreme stability of the intermediate reactant in the ult imate oxidation of tranquilizers of this type could be responsible for their psychotropic activity.

The kinetic results indicate that, due to the stability of the intermediate, further oxidation of the radical by the enzyme becomes an important mechanism for its disappearance. This second enzymic electron transfer does not mean the successive two-electron transfer from CPZ on the surface of the enzyme. The CPZ radical once formed on the surface of the enzyme comes off and reacts with another enzyme com- peting with flesh CPZ. Removal of an electron from the CPZ radical results in the formation of the thionium ion which has been shown 1~ to be capable of rearrangement and further addition to form substituted phenothiazines.

The colored intermediate observed during oxidation of CPZ is identified as the free radical by means of simultaneous EPR-opt ica l absorption measurements. Fig. IO illustrates the rise and decay of both the free-radical spectrum as measured by E P R and the change in absorption at 542 m/~ as measured optically during a dynamic enzymic oxidation.

I t has been suggested that ascorbic acid inhibits the formation of the red inter- mediate (530 m#) and therefore, the free radical. Preliminary experiments in con- junction with the enzymic oxidation of CPZ indicate that ascorbic acid does not inhibit formation of the free radical but instead acts as a radical t rap once the free

OPTICAL ABSORPTION

~-- STOP FLOW

I L i ~ I I D I I V I S I O N = = 2 0 S E C =

, I , J i i I D I I V I S I O N ~ 2 0 S E C J L , I l l

Fig. io. Simultaneous EPR-op t i ca l absorpt ion formation and decay of CPZ free radical. Signal from photo detector and EPIZ spect rometer pu t into separate channels of recorder. 4" i o -a M CPZ,

6. lO -4 M H202, io -7 M horse radish peroxidase at p H 4.8. Tempera ture 23 °.

Biochim. Biophys. Acta, 88 (1964) I2o-12:9

E P R STUDY OF THE CHLORPROMAZINE FREE RADICAL 12 9

r a d i c a l is f o r m e d . T h e r e a c t i o n of t h e C P Z r ad i ca l w i t h t h e a s co rb i c ac id is f a s t e r t h a n

t h e e n z y m i c o x i d a t i o n of t h e C P Z rad ica l . F u r t h e r s t u d y is in p r o g r e s s on t h e a s co rb i c

ac id r e a c t i o n .

ACKNOWLEDGEMENT

T h e a u t h o r s are i n d e b t e d to N a t i o n a l I n s t i t u t e of M e n t a l H e a l t h , C o n t r a c t No.

P H 43-63-53, for f inanc ia l s u p p o r t of t h i s s t u d y .

REFERENCES

1 L. H. PIETTE AND I. S. FORREST, Biochim. Biophys. Acta, 57 (1962) 419. 2 D. C. BORe AND A. C. COTZlAS, Proc. Natl. Acad. Sci., U.S. 48 (1962) 623. 3 D. J. CAVANAUGH, Science, 125 (1957) lO4O. 4 j . R. GILLETTE AND J. J. KAMM, J. Pharmacol., 13o (196o) 262. 5 R. H. KENTEN AND P. J. A. MANN, Biochem. J., 57 (1954) 347. e G. HARVEY AND R. G. W. HOLLINGSHEAD, Chem. Ind. (London), (1953) 244. 7 I. YAMAZAKI, H. S. MASON AND L. H. PIETTE, J. Biol. Chem., 235 (196o) 2444. s j . H. MURIB AND D. M. RITTER, J. Am. Chem. Soc., 74 (1952) 3394. $ I. YAMAZAKI AND L. H. PIETTE, Biochim. Biophys. Acta, 41 (1952) 416.

10 13. CHANCE, Arch. Biochem. Biophys., 41 (1952) 416. 11 j . C. CRAIG, M. E. TATE, F. W. DONOVAN AND W. P. ROGERS, J. Med. Pharm. Chem., 2 (196o) 669.

Biochim. Biophys. Acta, 88 (1964) 12o-129