328

Biochimica et Biophysica Acta, 421 (1976)328--333

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27824 AN EPR STUDY OF MYELOPEROXIDASE IN HUMAN G R A N U L O C Y T E S

R. WEVER a, D. ROOS b, R.S. WEENING c, T. VULSMA a and B.F. VAN GELDER a a Laboratory of Biochemistry, B.C.P. Jansen Institute, University of Amsterdam, Plantage Muidergracht 12, Amsterdam, b Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, Amsterdam, and c Pediatric Clinic, University of Amsterdam, Binnengasthuis, Grimburgwal 10, Amsterdam (The Netherlands)

(Received September 25th, 1975)

Summary 1. EPR spectra of human granulocytes (4 • 10 8 cells per ml) show an intense high-spin ferric heme signal with rhombic s y m m e t r y (gx = 6.90 and gy = 5.07) for the h eme group. These g-values are identical to those of partially purified myeloperoxidase and thus the signal is derived from ferric myeloperoxidase. In chicken granulocytes, which contain little or no myeloperoxidase, only an axial t y p e o f heme iron signal, weak in intensity, can be detected at g = 6.0. 2. Upon phagocytosis of latex particles by human granulocytes the high-spin heme signal with rhombic s y m m e t r y is slowly converted into a signal with axial s y m m e t r y (gx = gy = 6.0), showing that the EPR signals of myeloperoxidase in the intact cell can be used to study the involvement of the e n z y m e in metabolic changes during phagocytosis.

In t ro d u ctio n P o l y m o r p h o n u c l e a r leucocytes (granulocytes) are able to phagocytose and destroy microorganisms. Current interest in granulocytes is focussed on the antimicrobial mo de of action. Recently, it has been dem onst rat ed by several groups [1--6] that during phagocytosis of latex particles and bacteria, human granulocytes generate singlet oxygen ( 1 0 2) and superoxide anion radicals (O~). These oxygen species are found to be p o t e n t bactericidal agents [2,7--10] and thus it has been proposed that in granulocytes 0 2 and/or IO2 play a role in the killing o f microorganisms. Several lines of evidence suggest t hat in these processes also myeloperoxidase is involved. Klebanoff [11,12] has reported that even the isolated enzyme has antimicrobial activity in the presence of H 202 and an halide. Allen [13,14] has demonstrated that myeloperoxidase catalyzes the oxidation of an halide which may further react with a second molecule of

329

hydrogen peroxide to generate singlet oxygen. However, it has also been suggested by Rotilio et al. [15] that the oxygen radical formation during the phagocytic process is caused by a decay of the oxygen adduct of myeloperoxidase (compound III). Direct evidence for the participation of myeloperoxidase in these reactions in the intact cell is still lacking, and thus it is important to study the role of the enzyme in granulocytes. Since myeloperoxidase accounts for 5% of the dry weight of a granulocyte [16[ it seems possible to study the enzyme in the intact cell by using EPR at low temperature. This technique has already proven to be a powerful tool in the study of the mechanism of action in whole tissue of many metalloproteins with paramagnetic centres. This paper describes the EPR characteristics of isolated myeloperoxidase and of the enzyme in resting and phagocytosing granulocytes. Materials and Methods Polystyrene latex as 10% solution, containing 3 • 10' ' particles per ml with a diameter of 0.82 pm was purchased from Dow Chemical Company (Midland, Mich.}. Human and chicken granulocytes were isolated from fresh acid citrate dextrose (U.S.P. Formula A ) b l o o d as described previously [17,18] and suspended in Krebs-Ringer Tris buffer (pH 7.4, at 37°C), containing 10% human AB serum and 5.5 mM glucose. The granulocytes were counted electronically with a Coulter counter, Model ZF. Phagocytosis was initiated by addition of latex particles to the prewarmed cells (37°C). During the incubations the granulocyte suspensions were shaken in a waterbath (37°C) and oxygen was bubbled through the suspension in order to avoid anaerobiosis. Phagocytosis was terminated by transferring a sample (0.25 ml} of the suspension to a cold (0°C) EPR tube and subsequent freezing of the tube in liquid nitrogen. Myeloperoxidase was partially purified from granulocytes essentially according to Takanaka and O'Brien [19]. For the determination of the concentration of myeloperoxidase, an absorption coefficient of 89 mM -~ • cm -I at 430 nm was used [20,21]. EPR experiments were carried out on a Varian E-9 spectrometer equipped with a helium transfer system (Air Products Inc. Model LTD-3-100) with automatic temperature controller or with a low-temperature device as described by Lundin and Aasa [22] and Albracht [23]. Magnetic field and microwave power were measured as previously reported [24]. Results

Fig. 1A shows the EPR spectrum of resting human granulocytes (4 • 108 cells per ml} at 15°K. The EPR spectrum shows a well-resolved signal near g = 6, characteristic of high-spin ferric heme iron in an environment of rhombic symmetry (gx = 6.90 and gy = 5.07}. In addition resonances are observed around g = 2 (not shown) and at g = 4.3. The rhombic high-spin heme signal can be assigned to myeloperoxidase since the g values are identical to those of

330 I

I

6.90 5.0

I

300

I

1100

I

5.07

I

Z..3

I

H ( gauss )

I

1900

Fig. I . E P R s p e c t r a o f r e s t i n g g r a n u l o c y t e s a n d i s o l a t e d m y e l o p e r o x i d a s e . A , h u m a n g r a n u l o c y t e s , 4 • 108 cells per m l : B, i s o l a t e d m y e l o p e r o x i d a s e ( 2 7 p M ) in 50 m M s o d i u m a c e t a t e ( p H 5.5) a n d 2 M NaCl; C, c h i c k e n g r a n u l o c y t e s , 7.5 • 108 cells p e r ml. C o n d i t i o n s o f E P R s p e c t r o s c o p y w e r e : f r e q u e n c y , 9 . 3 0 4 G H z , m i c r o w a v e p o w e r , 10 roW; m o d u l a t i o n a m p l i t u d e , 16 G; s c a n n i n g rate, 5 0 0 G • r a i n - l : t i m e c o n s t a n t 1.0 s; t e m p e r a t u r e , 15OK.

isolated myeloperoxidase (Fig. 1B). The concentration of myeloperoxidase as determined from direct comparison of the intensity of the g = 6 signal with that of the isolated enzyme (27 pM) was found to be 7.4 #M (4 • 10 s cells per ml). Since it has been reported [25] that chicken leucocytes contain little or no myeloperoxidase it was of interest to study the properties of these cells. A comparison of the enzymic activity of myeloperoxidase [26] in broken chicken granulocytes with human granulocytes shows that the former contain only 1.7% of the enzymic activity of the latter. In agreement with these data the EPR spectra of chicken granulocytes (7.5 • l 0 s cells per ml) only show a weak axial type of heme-iron signal (Fig. 1C) whereas the rhombic high-spin signal of myeloperoxidase is absent. Fig. 2 illustrates the changes observed in the EPR spectrum of granulocytes under phagocytosing conditions. A and C are spectra of resting granulocytes in the absence of latex before and after an incubation period of 30 min at 37°C, respectively. It is evident that this treatment has little effect on the shape of the signal. After addition of latex to resting granulocytes (spectrum B) and incubation at 37°C for 30 min (spectrum D) a signal appears a t g = 6 indicative of high-spin iron with axial symmetry for the heme group. It should be noted that the apparent enhancement in the intensity of the signals in time is due to evaporation of water from the incubation medium which consequently increases +,he number of cells per ml. This evaporation is caused by flushing of the incubation medium at 37°C with oxygen gas, in order to avoid anaerobiosis of the samples.

332 Discussion Our results have demonstrated that both the partially purified enzyme and the enzyme in intact granulocytes have g-values characteristic for rhombic (gx = 6.90 and gy = 5.07) high-spin heme iron. These signals are similar in shape to those reported by Ehrenberg [27] and Schultz et al. [28] at liquid nitrogen temperature for the isolated enzyme, but differ in their g-values (ref. 27, gx = 6.3 and gy = 5.3}. At low temperature (15 ° K) the signal of myeloperoxidase is so distinct that the concentration of the enzyme in the intact white blood cell can be determined. According to Schultz and Kaminker [16] the mean concentration of myeloperoxidase per leucocyte as derived from activity measurements is 7.1 • 10-12 g. Since we work with 4 • 108 cells per ml (cf. Fig. 1A) the concentration of the enzyme in the EPR tube on the basis of a molecular weight of 149 000 [29] is estimated (cf. ref. 16) to be 20 pM. This value agrees reasonably well with the values of 7.4 pM obtained by us from direct comparison of the intensity of the EPR signal of the enzyme in granulocytes with that of the isolated enzyme. The changes in the EPR spectrum of granulocytes observed after phagocytosis of latex suggest that the rhombic signal of myeloperoxidase is converted into an intense signal with axial symmetry for the heme group. Thus, under phagocytosing conditions, the observed high-spin ferric heme iron in granulocytes may exist in two environments with different symmetry. In general, these changes in ferric heme high-spin EPR signals are due to changes in the conformation of the protein molecule wrapped around the heme group. It has been reported [30,31] that the EPR spectra of high-spin ferric hemoproteins are easily perturbated by solvents, addition of ligands or pH changes. It is, therefore, tempting to speculate that the changes in the EPR spectrum of myeloperoxidase during phagocytosis reflect the drop in pH, known [32] to occur within the phagocytic vacuole.

Acknowledgements We wish to thank Mr. J.J.Fr.M. de Witt, Mrs. J.W.T. Homan-Miiller and Mrs. M. van der Steen-Kamerbeek for their excellent technical assistance. This investigation was in part supported by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). References 1 B a b i o r , B.M., K i p n e s , R.S. a n d C u r n u t t e , J . T . ( 1 9 7 3 ) J. CUn. Invest. 52, 7 4 1 - - 7 4 4 2 Keele, J r . , B.B., J o h n s t o n , R., R a j a g o p a l a n , K . V . and Kessler, D. ( 1 9 7 3 ) Abstr. 9 t h I n t e r n . C o n g r . B i o c h e m . , S t o c k h o l m , A k t i e b o l a g e t E g n e l l s k a B o k t r y c k e r i e t , S t o c k h o l m . p. 3 4 3 3 Allen, R . C . . S t j e r n h o l m , R . L . and Steele. R . H . ( 1 9 7 2 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 4 7 , 679--684 4 W e e n t n g , R . S . , Wever, R. a n d R o c k , D. ( 1 9 7 5 ) J. L a b . Clin. Med. 8 5 , 2 4 5 - - 2 5 2 5 C u r n u t t e , J . T . and Babior, B.M. ( 1 9 7 4 ) J. Clin. Invest, 5 3 . 1 6 6 2 - - 1 6 7 2 6 N a k a g a w a r a . A. a n d M i n a k a m L S. ( 1 9 7 5 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 6 4 , 7 8 0 - - 7 6 7 7 G r e g o r y , E.M., Y o s t , F . J . , J r . a n d F r i d o v i c h , I. ( 1 9 7 3 ) J. B a c t e r i o l . 1 1 5 , 9 8 7 - - 9 9 1

331 I

I

1

I

I

I

I

i

..< R

I

b-90 I GO0

2,00

I 1000

&90 ~A5 5O7

5.O7 I 1,400 H { gauss i

I 1830

I COO

I 1000

I 1"00

I 18~

H { gauss',

Fig. 2. E f f e c t o f l a t e x o n t h e E P R s p e c t r u m o f m y e l o p e r o x i d a s e in h u m a n g r a n u l o c y t e s . A , r e s t i n g g r a n u l o c y t e s , 4 • 1 0 8 cells p e r m l ; B, a f t e r a d d i t i o n o f l a t e x ( f i n a l c o n c e n t r a t i o n , 6 • 1 0 1 0 p a r t i c l e s p e r m l ) a t 0 c C ; C, a f t e r i n c u b a t i o n o f r e s t i n g g r a n u l o c y t e s , 3 0 r a i n a t 3 7 ° C in t h e a b s e n c e o f l a t e x ; D , a f t e r i n c u b a t i o n o f g r a n u l o c y t e s , 3 0 r a i n a t 3 7 ° C in t h e p r e s e n c e o f l a t e x . F o r p r o c e d u r e o f i n c u b a t i o n cf. M a t e r i a l s a n d M e t h o d s . C o n d i t i o n s o f E P R s p e c t r o s c o p y as i n F i g . 1.

The time dependency of the ratio of the intensities of the axial to the rhombic signal is given in Fig. 3. Apparently under phagocytosing conditions in the presence of latex (,9 ©) the rhombic high-spin iron signal is converted into an axial signal. It is noteworthy here that the observed changes can only be induced by particles and are not due to a nonspecific disruption of the granulocytes, since it was found that disruption of resting granulocytes by repeatedly freezing and thawing does not affect the EPR spectrum.

1.2

l

I

,

i

u~ .~ 0.8

22

-/

~0.~ o

°o /

O.O~ ~ 0

i I0

I

I

I

I

20

30

&O

50 hme

60

', m , n }

Fig. 3. Ratio of the intensity of the axial a n d r h o m b i c high-spin E P R signal at g = 6 in granulocytes u n d e r phagocytosing conditions. Phagocytosis w a s started b y addition of latex particles (final concentration, 6 • 1010 per ml) to granuloeytes (4 " 108 per ml); temperature of the incubation, 37~C..'> c), i n t h e presence of latex; • • , in t h e a b s e n c e o f l a t e x . C o n d i t i o n s o f E P R s p e c t r o s c o p y a s in F i g . 1.

333 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

25 26 27 28 29 30 31 32

B a b i o r , B.M., C u r n u t t e , J . T . a n d Kipnes0 R.S. ( 1 9 7 5 ) J. L a b . Clin. Med. 8 5 , 2 3 5 - - 2 4 3 Y o s t , F.J.0 J r . a n d F r i d o v i c h , I. ( 1 9 7 4 ) A r c h . B i o c h e m . B i o p h y s . 1 6 1 , 3 9 5 - - 4 0 1 K r i n s k y , N.I. ( 1 9 7 4 ) S c i e n c e 1 8 6 , 3 6 3 - - 3 6 5 K l e h a n o f f , S.J. ( 1 9 6 8 ) J. B a c t e r i o l . 9 5 , 2 i 3 1 - - 2 1 3 8 K l e b a n o f f , S.J. ( 1 9 7 0 ) in B i o c h e m i s t r y o f t h e P h a g o c y t i c P r o c e s s ( S c h u l t z , J., ed.), p p . 8 9 - - 1 1 0 , North-Holland, Amsterdam ALien R . C . ( 1 9 7 5 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 6 3 , 6 7 5 - - 6 8 3 AUen, R . C . ( 1 9 7 5 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 6 3 , 6 8 4 - - 6 9 1 R o t i l i o , G., F a l c i o n i , G., F i o r e t t i , E. a n d B r u n o r i , M. ( 1 9 7 5 ) B i o c h e m . J. 1 4 5 , 4 0 5 - - 4 0 7 S c h u l t z , J. a n d K a m i n k e r , K. ( 1 9 6 2 ) A r c h . B i o c h e m . B i o p h y s . 9 6 , 4 6 5 - - 4 6 7 W e e n i n g , R.S., R o o s , D. a n d L o o s , J . A . ( 1 9 7 4 ) J. Lab. Ctin. Meal. 8 3 , 5 7 0 - - 5 7 6 De Wit, J . J . F r . M . , H e n r i c h s , H.J.P., O d i n k , J. a n d Prins, H . K . ( 1 9 7 5 ) V o x S a n g , 2 9 , 3 5 2 - - 3 6 2 T a k a n a k a , K. a n d O ' B r i e n , P.J. ( 1 9 7 5 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 6 2 , 9 6 6 - - 9 7 1 A g n e r , K., ( 1 9 5 8 ) A c t a C h e m . S c a n d . 1 2 , 8 9 - - 9 4 A g n e r , K. ( 1 9 6 3 ) A c t a C h e m . S c a n d . 17, $ 3 3 2 - - 3 3 8 L u n d i n , A. a n d Aasa, R. ( 1 9 7 2 ) J. M a g n . R e s o n a n c e 8, 7 0 - - 7 3 A l b r a c h t , S.P.J. ( 1 9 7 4 ) J. M a g n . R e s o n a n c e 1 3 , 2 9 9 - - 3 0 3 Wever, R., V a n D r o o g e , J . H . , V a n A r k , G. a n d V a n G e l d e r , B.F. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 4 7 , 215--223 Brune, K., Schmid, L., Glatt, M. and Minder, B. (1973) Nature 245, 209--210 Klebanoff, S.J. (1965) Endocrinology 76, 301--311 E h r e n b e r g , A. ( 1 9 6 2 ) A r k . K e m i 1 9 , 1 1 9 - - 1 2 8 S c h u l t z , J., S n y d e r , H., Wu, N.-C., Berger, N. a n d B o n n e r , M.J. ( 1 9 7 2 ) in T h e M o l e c u l a r Basis o f E l e c t r o n T r a n s p o r t ( S c h u l t z , J. a n d C a m e r o n , B . F . , eds), p p . 3 0 1 - - 3 2 5 , A c a d e m i c Press, N e w Y o r k E h r e n h e r g , A. a n d A g n e r , K. ( 1 9 5 8 ) A c t a C h e m . S c a n d . 12, 9 5 - - 1 0 0 B l u m b e r g , W.E. a n d Peisach, J. ( 1 9 7 1 ) in O x i d a s e s a n d R e l a t e d R e d o x S y s t e m s ( K i n g , T . E . , M a s o n , H.S. a n d M o r r i s o n , M., eds), Vol. 1, p p . 2 9 9 - - 3 1 0 Univ. P a r k Press, B a l t i m o r e T a m u r a , M. a n d H o r i , H. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 8 4 , 2 0 - - 2 9 J e n s e n , M.S. a n d B a i n t o n , D . F . ( 1 9 7 3 ) J. Cell Biol. 56, 3 7 9 - - 3 8 8