Evidence of a specific copper(II) in human ceruloplasmin as a binding site for inhibitory anions

BIOCHIMICAET BIOPHYSICAACTA

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BBA 35502 E V I D E N C E OF A SPECIFIC COPPER(II) IN HUMAN CERULOPLASMIN AS A B I N D I N G SITE FOR I N H I B I T O R Y ANIONS

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Department of l~iochemistry, Umversztv qf (;6teborg and Chalmers Institute q[ Tcchnolog.t~, Gdteborg (Sweden) (b~eccived A u g u s t z,:nd, 19~)9)

SUMMARY

I. Studies on the nature of the copper components of human ceruloplasmin were made with the electron paramagnetie resonance (EPR) technique. This method and optical absorption spectra gave information on the binding of azide and fluoride to the protein. The inhibition with azide was studied by E P R measurements of radical formation from tetramethyl-p-phenylenediamine. 2. The 9 GHz E P R spectrum was analyzed in terms of Type I ("blue") Cu 2and Type 2 ("non-blue") Cu 2., two of each. For sinmlated spectra, the two Type 2 (;u 2. were given slithtly different parameters. The essential features of the lessresolved 35 GHz spectrum could also be reproduced by simulations. 3. E P R spectra showed that azide, in low concentration, and fluoride bind to the Type 2 Cu z- without changing the properties of the Type I CuZ-( An absorption band centered at 3qo nm is associated with the azide complex. The data are consistent with the binding of one azide ion per protein molecule. 4. The inhibition with azide closely paralleled the formation of the, Type Cu"'--azide complex, suggesting that the Type 2 Cu"- is the site of inhibition. The formation of product at zero time was unaffected bv azide, indicating that some centers are reduced even with azide bound to tile enzyme.

INTRODUCTION

Ceruloplasmin is an intensely blue copper-containing protein which is obtained from plasma and which shows oxidase activity. Most workers report that there are 8 copper atoms in the moleculO, but some results indicate 2 that this number should be 7. The difference may be due to variability in tile preparations or to insufficient accuracy in the determination of molecular weight and copper content of the protein. In several laboratories quantitative E P R (refs. 3-6) and magnetic susceptibilitv 23 studies have been performed. Although tile results vary, thev indicate that A b b r e v i a t i o n : "I'MPI), ,V,N,N',N'-tetr',mu~thyl-p-phenylenediaminv.

l~iochim. Biophys..qcta, 200 (I97 o) 247-257

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3 4 copper ions per molecule arc d e t e c t e d by these techniques and thus reside in the C u ( l l ) s t a t e in the oxidized protein. It has l)een suggested t h a t the renmining c~q~per ions are ('u(I) a ", but recenth" MAI.K1N AND .~IAI.3ISTRi)M8 have indicated t h a t l h e v might exist in pairs of coupled ('u(l I) ions as was prop~sed earlier for fungal Iaccase"'. The blue color of the oxidized ceruloplasmin has been shown to be due to the copper ions having the narrow hypertine structt, re in the E P R spectrunfL This kind of COl)per is referred to as "l'ylw I ('u
concentrated against deionized water. The ceruloplasmin solutions were stored in liquid nitrogen until used. Working solutions we.re prepared by addition of buffer.

Reagents R e a g e n t g r a d e chemicals were used w i t h o u t further purification. T M P I ) dih y d r o c h l o r i d e (TMP1) - : V , N , - V ' , A " - t e t r a m e t h y l - p - p h e n y l e n e d i a m i n e ) was purchased from E a s t m a n Organic Chemicals, Rochester, N.Y., a n d was stored as a Biochzm. 13iophys. Acta, zoo (197o) e47-e57

C O P P E R ( I I ) AND ANION BINDING IN CERULOPLASMIN

249

o.I M solution at --25 °. Solutions used ff)r experiments were prepared by dilution of the stock solution with buffer and were kept at o ° until used, to limit the autoxidation. Sodium azide and sodium fluoride were dissolved directly in buffer and used immediately. All solutions contained o.r mM of the disodium salt of EDTA.

optical absorption measurements Spectra were recorded at 25 ° in I-cm cells in a Zeiss PMQ II or a Cary 15 spectrophotometer.

E PR measurements l.ow-temperature measurements were made at 77°K and about q GHz in a Varian E-3 spectrometer and at about 9o°K and 35 GHz in a Varian V-45o 3 spectrometer. Kinetic E P R measurements were made in the E-3 spectrometer at 25 °. When TMPD is oxidized by ceruloplasmin, an intensely colored product is formed. Because of its radical nature 24, it is possible to follow the formation of the product with the E P R technique. The method used was essentially identical to that described by BRo.~IA.~ et al. '2. Befi~re each series of experiments the flat quartz cell in the cavity of the spectrometer was calibrated with a solution of a stable nitroxide radical of known concentration. The ceruloplasmin concentration chosen was low so that a distinct steady state could be observed before the oxygen was exhausted. At the same time it had to be high enough that small changes in the copper E P R signal in the parallel low-temperature experiments could be detected. A protein concentration of5o/~M after mixing was found suitable at the substrate concentrations used (2 raM). Various amounts of azide were added to ceruh)plasmin solutions about 5 rain bef~re mixing with substrate.

RESULTS

E P R spectrum of ceruloplasmin Figs. i and 2 show E P R spectra of ceruloplasmin at 9 and 35 GHz, respectively. The 0 GHz spectrum (Fig. I, a) agrees in all essential features with an earlier-published spectrum 6 and consists of one component with a narrow hyperfine splitting (Type I Cu 2,) and one component with a broader hyperfine splitting (Type 2 C,u-°-). Integrations e of the low-field line in spectra from several preparations all gave a Type 2 Cu 2~ content of 45-5o°,',, of the total EPR-detectable Cu. In an earlier publication 25 a ¢~ GHz spectrum of ceruloplasmin was simulated without consideration of the presence of Type 2 Cu "¢ . Therefore, new simulations were tried as shown in Fig. x(b). The simulated spectrum was obtained by the superposition of three component spectra as indicated. ()ne component accounts for 50% of the total intensity and has parameters characteristic of Type I Cu 2+, whereas the two others each contribute 25 % of the intensity and have Type 2 like parameters. The 35 GHz spectrum (Fig. 2,a) is much less resolved than the corresponding spectrum from fungal laccase 9, although the 9 GHz spectra of the two proteins are very similar. For example, the hyperfine splitting of the Type I Cu 2+ is not resolved Biochim. Biophys. Acta, 200 (i97 o) 247--~'57

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Fig. 2. Experimental s p e c t r u m (a) an(t low-fiel(t part of a simulated s p e c t r u m (b) of ceruloplasmin at a b o u t 35 G t t z and 9o°K. The experinlental s p e c t r u m (a) was obtained from the same sample its in Fig. i (a). S p e c t r u m (b) was simulated with the same p a r a m e t e r s as in Fig. l (b) except t h a t the line widths were, for T y p e I, 15o gauss, and for the two T y p e 2 components, 3o0 and x2o gauss, respectively. The hyperfine peaks of the three c o m p o n e n t s are indicated as in Fig. x. The high-field p a r t (a') of the experimental s p e c t r u m is shown with lo times lower gain..Microwave frequency, 34.655 (;llz. lt*ochzm, ltioph~,s. .4cta. zoo (z97 o) -'-t7-'57

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Fig. 3. I(ttect of azide on the absorption s p e c t r u m of ceruloplasmin. Difference spectra were recorded between protein treated with azide and protein alone. The ceruloplasmin concentration was 48 tiM, in o.I M acetate buffer (ptt 5.5). The azide concentration was in (a) o, in (b) 49/iM, in (c) 98 tt.M, and in (d) 49o t~M. The small peak seen at 6 t o nm in (a), (b), and (c) is caused by a slight excess of protein in the sample relative to the reference. Fig. 4- Effect of azide on some peaks in the optical absorption and E P R s p e c t r u m of ceruloplasrain. (), absorbance at 39o n m ; O, absorbance at 6 I o nm: [--, E P R intensity at z76o gauss and q.2t G H z (at the arrow in Fig. 5, b') in a r b i t r a r y units, corrected for the absorption of the untreated protein at this field. The full line is calculated from liqn. I with a stability c o n s t a n t of 15 m M - L As drawn, the line corresponds to an increase of 4.-' m M - ~ ' c m ~ in the extinction coefficient at 39o nm on formation of the azide complex. The ceruloplasmin was 57/tM in o.x M acetate buffer (pH 5.5)-

in Fig. 2(a). The low-field p a r t of this s p e c t r u m was s i m u l a t e d (Fig. 2,b) with the same p a r a m e t e r s as in Fig. z.

Effect of azide and fluoride on the optical and EPR absorption of ceruloplasmin A d d i t i o n of r a t h e r low c o n c e n t r a t i o n s of azide to ceruloplasmin causes an a b s o r p t i o n b a n d centered a r o u n d 39o nm to a p p e a r w i t h o u t a n y sigmificant effect on the b a n d a r o u n d 61o nm. This is i l l u s t r a t e d in Fig. 3 which shows difference s p e c t r a between protein plus azide a n d p r o t e i n ahme. No t i m e d e p e n d e n c e of the reaction has been observed. W h e n the azide c o n c e n t r a t i o n is increased, the a b s o r b a n c e at 300 nm levels off, as shown in Fig. 4. The full line in Fig. 4 is a t h e o r e t i c a l curve o b t a i n e d u n d e r the a s s u m p t i o n t h a t one azide ion binds to ceruloplasmin (Cp) in a reversible m a n n e r according to the relation N:,- , ( ' p ~ C p N 3-

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The plot is c a l c u l a t e d with a b i n d i n g c o n s t a n t of x5 mM -1 a n d a difference of 4.2 mM -1 .cm -1 between the e x t i n c t i o n coefficients of the complex a n d of the protein ahme. Fig. 4 also c o n t a i n s some of the E P R d a t a discussed below. EPR

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Fig. 5. E I ' R spectra recorded at 77 ~l,Z and a b o u t 9 GI tz of native ceruloplasinin (a), ccruloplasmin treated with azide (b), and ceruloplasmin treated with both azide and T M P D (c). The ceruloplasmin concentration was in (a), 250 tt3I, and in (b) and (c), 2 to tzM, in all cases in o. I 31 acetate buffer (pH 5.5)- The sample for s p e c t r u m (b) contained 3 mM sodium azide and this sample gaw~ spectruin (c) when frozen 15-3o sec after the addition of 3 mM "rMI-~I). The spectra in (a) and (b) are recorded with the same gain, and their low-field p a r t s are shown with 1o times increased gain (a' and b'). The gain in (c) is 4 ° times higher t h a n in (a) and (b). At s p e c t r u m (a') the lmvfield hyperfine peaks of the two Type 2 c o m p o n e n t s used for the simulation in l:ig. ~ are indicated. The arrow at s p e c t r u m (b') shows the position of the amplitude m e a s u r e m e n t s used for Figs. 4 and 7- Microwave frequency, 9.2o (;I tz. In (c) the base line slopes as indicated.

as a measure of the relative concentration of the ceruloplasmin-azide complex. The samples used for the optical absorption data in Fig. 4 were analyzed in this way and the amplitude values are plotted in the same figure. The change in the E P R spectrunl levels off at higher azide concentrations in the same way as does tile absorbance at 39o nm. The spectrum in Fig. 5((:) is obtained from the sample of Fig. 5(b) to which the reducing substrate T M P I ) has been added. T M P D is present in excess over oxygen in the solution, but the sample was frozen 15 3o sec after mixing, when some oxygen remains, as judged from the kinetic experiments described below. The hype.rfine peaks from the Type r Cu" ~ have disappeared, but the low-field peaks attributed to Type 2 C u " in Fig. 5(b') remain. The interpretation of the spectrum in Fig. 5(c) is not obvious, hut at least two components must be present. Addition of fluoride to ceruloplasmin leads to changes in the Type 2 Cu z, E P R signal with no changes in the spectrum from T y p e I Cu 2~. However, the resulting spectrum is less resolved than the one from fluoride-treated fungal laccase u. If 17 mM fluoride is added to 2o ffM ceruloplasmin having an increased absorpti(m at 3!1o nm due to t r e a t m e n t with IOO ffM azide, this increase is reduced by, 5o%. Further addition of fluoride causes additional reduction of the absorption. A sample treated first with azide and then with a large excess of fluoride gives the same E P R spectrum as the one obtained on fluoride addition only. l:Hochim. Hiophys. /lcla, 200 (197o) 2.{.7 -z57

COPPER(II) AND ANION BINDING IN CERI'LOPLASMIN

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Effect of azide on the oxidase activity of ceruloplasmin Azide is known to have a strong inhibiting effect on the activity of ceruloplasmin ~5. In the present work this effect was studied by room-temperature E P R measurements of the formation of free radicals from TMPD as described in MATt'-'RIALS A N t ) .~I~:THOI)S. Chloride ions are known to affect the activity of ceruloplasminaL ~9, but calculations show that this influence can be neglected in our experiments. Under conditions rather diffcrent froln ours, the free radical fi)rmed from TMPI) has been reported to modi~" the kinetic behavior of ceruloplasmin ~6. No evidence of such an interaction has been observed in the present work. Fig. 6 gives the radical concentration as recorded in the E P R spectrometer. With azide present (Fig. 6, b-d) the initial rate of radical formation is as high as in absence of azide (Fig. 6,a). After an initial period there fi)llows a phase with a reduced and constant rate up to the point where the oxygen is exhausted. Extrapolation of the linear part of the curve in Fig. 5(b-d) to zero time indicates that 3 5 moles of product is formed per mole of enzyme befiwe the onset of inhibition. '

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Fig. 7- F o r m a t i o n of the azide complex with ceruloplasmin and inhibition of the oxictase activity. ' ;, F.PR intensity at 276o gauss (see l:ig. 5, b') of samples used ff~r kinetic e x p e r i m e n t s as in Fig. 6, frozen after incubation with azide b u t before nfixing with substrate. ",?, inhibition of the activity obtained from the slope of the linear p a r t of curves as in Fig. 6. The full line is the inhibition calculated from Eqn. i, a s s u m i n g no activity of the azide complex and using tho same stability c o n s t a n t as in I:ig. 4. Concentrations of reagents and other conditions as in Fig. e).

The decrease in the rate of radical formation obtained from the linear part of the curves in Fig. 6 relative to the rate without azide is plotted in Fig. 7 as a function of tile azide concentration. Also shown in this figure is tile amplitude at 276o gauss of the E P R spectra of aliquots of the samples in Fig. 6, frozen after the addition of azide. These amplitudes should be a measure of the concentration of the ceruloplasmin-azide complex (cf. Fig. 4). The solid line in Fig. 7 is obtained under the assumption that the binding of one azide molecule, according to Eqn. I, completely inhibits the activity. The same binding constant has been used for this curve as for the curve in Fig. 4Biochim. Bioph.vs. ,:Icta, 2o0 (I07 o) 247-.257

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..ks a basis for an u n d e r s t a n d i n g of the effect of anions on ceruloplasmin ,me would like to have a coinplete i n t e r p r e t a t i o n of its E P R spectrum. This requires a knowledge of the n u m b e r of ('u a t o m s in the ceruloplasmin molecule which, however, is not known with c e r t a i n t y , and it has been suggested t h a t 4 out of a total of 8 (ref. 3) c,r 3 out of 7 (ref. z) coppers are p a r a m a g n e t i c . We find equal a m o u n t s of "['ype i and T y p e 2 ('u 2 ~ in ceruloplasmin and this indicates t h a t an even numher, i.e. 4, of I'IPRd e t e c t a b l e ('u'-" are present in the molecule, 2 of each type. The azide e x p e r i m e n t s f u r t h e r confirm the presence ~f two T y p e 2 ('u ') reacting differently with azide as seen in Fig. 5(t.)') and also in Fig. 5(c) where no signal from T y p e I ('u 'a-' interferes. Based on a molecular weight "6 of ~6o ooo a n d .4 ~10 r:¢, n ,~,,, = o.b8 (ref. 27) the tJl e x t i n c t i o n coefficient per " b l u e " T y p e ~ Cu '-'~ becomes 5.4 m M - ~ - c m 1 which is quite close, to the corresponding value r e p o r t e d for laccase la. The present d a t a s o m e w h a t c o n t r a d i c t an earlier ret)ort 6 t h a t a purilied ceruloplasmin gave an i n t e n s i t y of the Tyt)c 2 (-fie- signal of 34'Io of the t o t a l E P R signal'. The reason for t]ie difference is not ('lear, a n d more experinlents on salllplcs prepared in various ways are required to resolve this probleln. Also, in the same pat)er a T y p e 2 C u '~" was said to be absent in a serum sample, as j u d g e d fronl the low-field line in E P I C ' . This would seem r a t h e r surprising in view of the i m p o r t a n c e of the Ty'pe 2 Cu efl)r the enzymic a c t i v i t y discussed below. However, in the present work it is shown t h a t the position a n d shape of this line d e p e n d on the presence of certain anions. Also, we know (L.-E. ,'\X~)R1::.~SSOX,untmblished studies) t h a t the p l t of the solution a n d some buffer anions have an effect on the low-field line. Therefore, it is very likely t h a t the various c o m p l e x i n g agents present in serum could cause a broadening of this line to m a k e it difficult to observe at the low signal-to-noise ratio o b t a i n e d from such a sample. A l t h o u g h the ceruloplasmin used for this work and the two laccases studied in this l a b o r a t o r y l°,~a all have a ~ : i ratio of T y p e I to T y p e 2 ('u 2' , the spectra of the. laccases are much more readily i n t e r p r e t e d ~t,~a t h a n t h a t of ceruloplasmin. This difference most likely arises because the laccases have only one Cu 'a- of each t.vpe in the molecule, whereas ceruloplasmin has two. Thus, for the s m m l a t i o n s of the ceruloplasmin 9 G H z s p e c t r u m (Fig. 1,b) it was found useful to ascribe slightly different E P R p a r a m e t e r s to the two T y p e 2 Cff',. A suitable choice of these p a r a m e t e r s makes the shoulder at 2830 gauss a p p e a r s h a r p e r t h a n the line at 2655 gauss, as found in the e x p e r i i n e n t a l s p e c t r u m . F u r t h e r n m r e , from a comparison of Fig. 5(a' a n d b') it is seen t h a t the effect of azi(te can be described as a shift of the signal from one of the T y p e a C u " ' with the o t h e r one left unaffected. The s i n m l a t i o n of the 35 G H z s p e c t r u m using t h e same values of the p a r a m e t e r s * One sample obtained froln fresh blood by a nfininmln amount of handling was stated n to have the corresponding value as low as z 5 %. l towever, due to a numerical error m the integ r a t i o n s t h i s v a l u e is t o o l o w a n d s h o u k l , m f a c t , b e a s h i g h a s t h a t o f t h e p u r i f i e d p r o t e i n . "" PE1SACH AND ]~LI.J.~II~ERG28 r e p o r t t h a t C O l I c e n t r R t e d s e r u m , t a k e l l f r o n l a p r e g l l a l l t woman, had precisely the same spectrum as purified material. We have repeated this experiment w i t h s u c h s e r u m b u t w i t h o u t c o n c e n t r a t i n g it (I..-E. ANDR~;ASSON, u n p u b l i s h e d e x p e r i m e n t s ) . T h e r e is s o m e l : l q ¢ a b s o r p t i o n o n t h e l o w - l i e l d s i d e o f t h e T y p e t ( ' u 2~ s i g n a l , b u t it is d i s t i n c t l y d i f f e r e n t from t h a t o f t h e p u r i t i e d p r o t e i n . T h e r e a s o n f o r t h i s d i s c r e p a n c y is n o t c l e a r .

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COPPER(II)

A N D A N I O N B I N D I N G IN C E R U L O t ' L A S M 1 N

255

(Fig. 2,b) is not as successful, although it reproduces the essential features of the experimental spectrum around g:, fairly well. It would not be surprising if it turns out that all four Cu 2,- giving rise to the E P R spectrum of ceruloplasmin have different parameters so that our analysis of this spectrum must be somewhat modified.

Binding of azide and fluoride to ceruloplasmin There are three main types of copper in ceruloplasmin, Type I Cu 2+, Type 2 Cu 2~, and copper that is not detected by EPR. No inff~rmation is presented here on tile possible binding of anions to the EPR-nondetectable copper. Under our conditions, azide and fluoride do not bind to tile Type I Cu 2f as its optical and E P R absorption are unchanged on the addition of these ions. However, our E P R data do show that they bind to tile Type 2 Cu in its divalent state. The close correlation, illustrated in Fig. 4, between the changes in the E P R spectrum and the appearance of an absorption band around 39 ° n m is strong evidence that this band is due to the Type 2 Cu2+--azide complex. The fair agreement in Fig. 4 between the experimental points and tile curve calculated on the basis of Eqn. I indicates that the binding of only one azide ion produces changes in the spectroscopic properties. This view is also supported by the changes in the E P R spectra which could be considered as a modification of the signal from a single Type 2 ('u" ~ (see the preceding section). The effect of anions on tile optical absorption of ceruloplasmin has also been studied by KaSVF.R"L With low concentrations of azide, he got a large increase in the absorbance at 375 nm without any change at 61o nm (Fig. 2A of ref. ai). Higher concentrations of azide than the ones used in the present work caused a reduction of the absorbance at 61o nm accompanied by an additional increase at 375 nm. KASeEIt concludes that the spectral transitions resulting from the interaction of the anions with ceruloplasmin Cu 2+ involve predominantly "blue" copper. However, his results, as well as ours, are best interpreted as a strong binding of azide to "non-blue" Type z ('u 2-~ already at low concentrations of azide. The interaction with Type I Cu 2~ occurs only at higher concentrations. Thus, the extinction coefficient for the 375 nm band (IO mM-~.cm -~ at IO mM azide) reported by KASPER21 contains contributions from azide interactions with both Type I and Type z Cu 2' . It is interesting to compare anion binding to ceruloplasmin and to fungal laccase. For the latter protein, ligand hyperfine structure in E P R has shown H that fluoride and cyanide bind directly to Type 2 Cu 2+. Also, azide additions ~° produced an absorption band centered slightly above 4oo nm without an}' change in the 01o nm absorption. At the same time the Type 2 Cu 2- EPR signal was modified'. Other experiments performed in our laboratory (B. REI.NHM,L~IAR, personal communication) have shown that high concentrations of azide will cause a decrease in the absorption at 61o nm accompanied by a further increase at 4oo nm. Thus, there exists a great similarity between the two proteins in their reactions with anions. From the results discussed above one can conclude that in ceruloplasmin, as in fungal laccase t°, the Type 2 Cu"' is more readily available for modifications, such as binding of anions, than is the Type ~ Cu"*. The low accessibility of Type I Cu '¢ • To avoid misunderstanding w e w o u M l i k e t o p o i n t o u t t h a t t h e s t a t e l n e n t in ref. l o a b o u t r e d u c t i o n o f t h e s i g n a l f r o i n T y p e 2 C u =* o n a z i d e a d d i t i o n d o e s n o t i m p l y r e d u c t i o n o f t h i s copper to its Cu(I) state.

Biochtm. Bu~phys. ..1eta, ,~oo (Iq7o) z47 2 5 7

25()

1..--I.i. ANI)I?I~'ASS~)N, "f. \'.7{,NN(;:{RI~

seems t~ be a general prol)ert y ~f t he proteins studied up till now. Thus, a recent eh.ct r~mnuclear double resonance s t u d y ~f lhc ~me-copper protein stella%'anin '-''~, in which the Cu e. is of T y p e i c h a r a c t e r , shows that the copper ion is at least () ,~ from the solvent w a t e r . ( h l t z o x ~r' found t h a t the binding of one azide molecule to cerulophtsnfin c . m pletely inhit)its the oxidase a c t i v i t y , and he suggested t h a t this inhibition occurs through complex fi~rmation between azide and ceruloplasmin copper. F r o m our e x p e r i m e n t s , the degree of inhibition can be v e r y well correlated to the formation of the specific "l'Ytw 2 ( ' u " azidc conq)lex discussed al)ove (see Fig. 7). Consequently, the inhibition probat)ly o c c u r s t h r o u g h the formation of an inactive complex according to Eqn. i. W i t h a stabilit\" c o n s t a n t of 15 nT.~t ~ this equation quite well a c c o u n t s G,r all our d a t a (of. the fllll line in Figs. 4 anti 7). T h u s this suggestion lw (;URZ()N is confirmed. Also, ('L'RZON AND SI'EYI'~R l'q found through kinetic e x p e r i m e n t s t h a t azidc a n d fluoride have a c o m m o n i n h i b i t o r y site, although t h e y max" b m d in a s o m e w h a t different manner. ()ur tindings t h a t both azide and fluoride bind t - the T y p e 2 (~u"-' and t h a t , in fact, a large excess of fluoride can rephtce an a l r e a d y bound azide ion, s u p p o r t the view that the T y p e 2 Cu 2~ is the inhibition site fiw several anions. Thus, it al)pears t h a t T y p e 2 ('u ~ is required fi}r the enzx'mic a c t i v i t y of cerulol)lasmin as it is for fungal laccase ~. T h e kinetic curves in Fig. ~ show no effect of azide on the rate of radical form a t i o n at the beginning of the reaction. Ct'Rzox o b t a i n e d similar curves ~:' and proposed t h a t azide inhibits by binding to a r e d u c e d form of the enzyme, later specified to be the valence-changiilg copl)er in its Cut1) s t a t e "s°. In a s o m e w h a t more e l a b o r a t e model given t)v BLt~II~t.:I¢¢;a'', azide inhibits bv b i n d i n g to the E l ~ R - n o n d e t e c t a b l e c o p p e r ions. In c o n t r a s t to both these ideas, we find a correlation between the inhibition a n d the f o r m a t i o n of an azide complex with the fully oxidized protein. The copt)er inw}lved in this complex remains C u ( l l ) even when the ceruloplasnfin has been reduced to a s t e a d y s t a t e (Fig. 5,c). The. simplest e x p l a n a t i o n of the shapes of the kinetic curves in Fig. () is t h a t the r e d u c t i o n of some centers in cerulol)laslnin is not affected by the binding of azidc to "I'ype 2 Cu" ~. Also, the correlation between the inhibition and the binding of azide to the oxidized protein implies t h a t the reduction of these centers does not significantly a l t e r the degree of binding of the inhibiting azide ion to the protein. At the time when the two models described above~'~,a°,a° were prc.sented, it was considered likely t h a t ceruloplasnfin contained only two kinds of Cu, four of each kind. Therefore, it was reasonable to give the models a high degree of s x m m e t r v with respect to the function of the copper ions, p a r t i c u l a r l y as four electrons are required to reduce one oxygen molecule to water. As discussed b y MAI.MSTRiJbl a~, high sx.'mmetries cannot exist in fungal laccase which has at least three kinds of ('u a m o n g its four copper per molecule, and yet this e n z y m e has a much higher oxidasc a c t i v i t y than ceruloplasmin. Our present results which s t r o n g l y suggest t h a t azide inhil)its the a c t i v i t y of ceruloplasmin b y b i n d i n g to one specific copper ion indicate t h a t a low degree of s y m m e t r y is present also in this protein. E v i d e n t l y , nmch m o r t work is needed before a realistic model of ceruloplasmin can bc trot forward which could explain all its spectroscopic and c a t a l y t i c properties. However, it is interesting to observe t h a t our kinetic d a t a s t r o n g l y resemble those o b t a i n e d h\' MAI.M.WI'R()Mct al. a2 l(w ftlngal l~tccase. T h e y find t h a t althollglt fluoride Hio~him. tCi,/~hys. .Iota, z o o

(rq7c,) 247 -'57

COPPER(II)

AND ANION B I N D I N G IN CI':tiVI.OPLASMIN

257

inhibits enzyme action, it does not affect tile initial burst of product formation and the reduction of the Type I Cu 2" With these results in mind, we would like to suggest that reduction of Type i Cu 2+ in ceruloplasmin gives rise to initial product formation that is unaffected by azide binding to Type 2 Cu 2+, and that azide reduces the activity of the enzyme by inhibiting some step later in the reaction sequence. This step could be tile binding of oxygen, as indicated by (~t:RZONl~ and BLt'.XIBER(;'-'°, but other possibilities should also be considered. More detailed studies are required to confirm our explanation of the kinetic results, particularly as our simple extrapolation procedure indicates the formation of 3 5 moles of product in the initial phase, whereas tile protein has only two Type I Cu in the molecule. ACKNOWLEDG.',II';NTS We are indebted to Mr. H. Bj6rling, AB Kabi, Stockholm, for the generous gift of ceruloplasmin. We wish to thank I)r. R. Malkin and Dr. B. G. Malmstr6nl for man,,, helpful discussions and comments. This work was supported by grants from the Swedish Natural Science Research Council and tile U.S. Public Health Service (GM I228o-o4). REFEI~ENCES I C. G. HOL.~BERG AXr~ C.-B. LAVRELL, Acta Chem. Scand., 2 (1948) 55 ° . 2 P..'\\SEN, S. H. I{OENIG AND H. R. LILIEN rH.~L, J. Mol. Baol., 28 (r967) 225. 3 L. BROMAN, ]aI. G. MALMSTR(SM, R. AASA AN'I) T. \'ANNGARD, J . 3Iol. Biol., 5 (19{12) 3 OI4 C. B. KASPER, H. F. DFUTSCH AND tt. BEIrVERT, J. Biol. Chem., 238 (1963) 2338. 5 V~,'. E. BLIYMBERG, J. EISINGER, P. AISEN, A. (;. *IORELL AND I. H..~,ClII~:INBI';RG, J. Biol. Chem., 238 (1963) 1675. 6 T. VXNNGARD, in A. EIIRENBERG, B. (~..MAI.MSTR{JM AND T. V~XNNG.~RI), .XIagnetic Resonance in Bu,log,cal Systems, P e r g a m o n , O x f o r d , 1967, p. 213. 7 A. EIIRENBERG, B. (;. ~IALMSTRiSM, L. BROMAN AND R. MOSBACIt, J. 3Iol. Biol., 5 (19t)2) 45 °. 8 R. MALKIN AN'I) g . G. MALMSTR/-3M, Advan. Enzymol., in the press. 9 J. A. FEE, R. MALKIN, la;. G. MALMSTRiSM AND T. V;~,N.','G.~RD, .]. Biol. Chem., 244 (I9091 4"oo. 1o ]a). (;. MALMSTR(JM, [{. REINtIAMMAR AND Z. V.:i,NNGARI), Biochim. Ihophys. Acta, 15~) (t0~,8) 67. i i It..'~IALKIN, 11. (;..MALMS'rRL)M AND T. V.~NNG.~,RI), F E B S Letters, t (1968) 5 O. 12 L. BROMAN, }~}.G..X, IAI.MSTRi-iM, R. AASA AND T. VXNN(;,~.RI.), Biochiw. Biophys. Acta, 75 (I963) 365 • 13 l:{. REINHAMMAR, B. (;..~IALMS'I'R{JM AND "l'. \'XNNG,~.RI), tO be p u b l i s h e d . 14 1¢.. MALKIN, B. (;..XlAL.~ISTR6M ANI~ T. VX~'X<;.~RD, F.uropea~* .I. Bioct, cm., 7 (1969) 253" x5 (3. ( ' t ' ~ z o x , Bu~chem. J., i o o (1900) 295. I~ G. CuRzo.x', Biochem. J., lO 3 (1967) 280. 17 (;. C t ' R z o x AND I'~. I~.. ~I'I':YP2R, Biochem. J., t o 5 (x967) 243. i s B. 1'2. Sv/-'vl.:R AND (;'. ('t:RZON, lqiochem. J., 1o6 (I968) tlo 5. 19 (;. CI'RZON ANt) B. ['~. SPEYER, Biochcm. J . , I o 9 (1968) 25. 2o W. I-. HLU.',tr~ERCL in J. PI-:ISACH, P. AISEX AND W. F. BLI;MI¢I-;R6, "l'he Biochemistry of Copper, A c a d e m i c P r e s s , N e w V o r k , 1966, p. 57~. 2I C. B. I{ASPER, .]. Biol. Chem., 243 (r968) 3218. 22 S. HJERT~N, Biochim. t3iophys. Acta, 31 (1959) 216. 23 L. BROMAN, Acta Soc. 3Ied. Upsalien., 69 (I964) s u p p l . 7. 24 L..\[ICHAE;LIS, .~I. P. ScHtru~:R'r ANt) ,q. (;RANICK, J. Anl. Chem. 5oc., ()I (I939) ~08~. 25 T. VXNNG:~.RI) AND R. AASA, in W . L o w , Paramagnetic Resonance, A c a d e m i c Press, N e w Y o r k , I 9 6 3 , p. 5(×). 26 C. B. K.~,SPER AND H. F. DI'CUTSCH, J. Biol. Chem., 238 ( i 9 6 3 ) 2325. 27 H. F. I)Eb'TSCH, Arch. Biochem. Biophys., 89 (I96o) 225. 28 J. PE~SACH AX~ W . I g. BLU.~tUERG, Federation Proc., 26 (I,467) 834. 29 (}. H. RIST, J. S. IIYDE AND T. VXr,'N(;:~R~L Proc. Natl. Acad. Sci. U.S., in the press. 3 ° (;. (.'IrRZON, in J. PEISACH, t'..'\ISF.N ANII '~\:. l';. BLUMI~I,:RG, The Biochemistry of copper, A c a d e m i c Press, N e w Y o r k , I966, p. 577. 31 B. (;. MALMSTR{}M, in A. |':NGSTR(JM ANt) ]~. S'rRANI)BFR(;, Symmetry and Function of Biological Systems at the 3lacromolecular Level, Nobel Symposium ~2, W i l e y , N e w Y o r k , I969, p. ~53. 32 B. G. MALMSTR{}M, A. FINAZZI AGR~) AND E. ANTONINI, European .]. Biochem., 9 (I969) 383 •

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