The stoichiometry of the paramagnetic copper and the oxidation-reduction potentials of type I copper in human ceruloplasmin

Biochimica et Biophysica Acta, 31o (1973) 321-33o © Elsevier Scientific Publishing Company, A m s t e r d a m - Printed in The Netherlands

BBA

36420

T H E S T O I C H I O M E T R Y OF T H E P A R A M A G N E T I C C O P P E R AND T H E O X I D A T I O N - R E D U C T I O N P O T E N T I A L S OF T Y P E I C O P P E R IN HUMAN C E R U L O P L A S M I N

J O K E D E I N U M AND T O R E V A N N G A R D

Institutionen f6r biokemi, G6teborgs Universitet och Chalmers Tekniska H6gskola, Fack, S-4o2 20 G6teborg 5 (Sweden) (Received N o v e m b e r 9th, 1972)

SUMMARY

I. H u m a n ceruloplasmin was prepared from two different Cohn fractions and from fresh serum. The E P R spectrum, the total electron-accepting capacity and the optical and potentiometric titration behavior of the preparations were studied and compared. 2. Although the preparations had undergone hydrolytic attack to a very different degree, no significant difference in the properties mentioned was found. Only the rate, at which electrons were taken up by the protein in anaerobic experiments, was found to vary with the preparation. 3. The E P R spectra indicated that in addition to two Type I Cu 2+, the protein contained only one Type 2 ion, with an E P R spectrum being affected by the presence of azide. 4. In agreement with previous reports, the number of electrons the protein can accept equals the total number of copper atoms. 5. The titrations of the 6Io-nm absorbance band are interpreted in terms of two non-identical Type I Cu 2+ having oxidation-reduction potentials of 49 ° and 580 mV, respectively, in acetate buffer (pH 5.5)- Both Type I ions have the same extinction coefficient at 61o nm, 5.5 mM-l"cm-1. Reductive titrations with Fe 2+ or ascorbate or oxidative titrations with 02 all produced the same titration curves, which also were independent of mediator concentration in a rather large range. 6. The titration behavior of the 33o-nm chromophore was found to be complicated and no simple interpretation directly associating this absorption with the redox state of a one- or two-electron aeceptor was found possible.

INTRODUCTION

Ceruloplasmin is a blue copper-containing plasma protein, which catalyses the oxidation of Fe 2+ and certain diamines 1 with concomitant reduction of 02 to water z.

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In the oxidized form 4o-5o% of the total copper is EPR-detectablea,% and can be separated into two classes, Type I and Type 2 (refs 4, 5)- Type I Cu 2+ has a strong optical absorption band at 61o nm and a typical narrow hyperfine splitting in the E P R spectrum. Type 2 Cu 2+ has more normal E P R parameters and no detectable optical absorption bands in the visible or ultraviolet region. Ceruloplasnfin has also an absorbance band at 33o-34 o n m disappearing upon reduction. Recent reports have suggested that ceruloplasmin from fresh blood, isolated in the presence of an inhibitor of the hydrolytic enzyme plasmin, consists of one polypeptide chain 6. Preparations from Cohn fractions v, or outdated blood have been shown to give rise to ceruloplasmin molecules apparently consisting of a number of smaller peptides. In view of these findings and some diverging reports in the literature s,9 we have re-examined the stoichiometry of the copper and the number of reducing equivalents accepted by ceruloplasmin. To obtain further information on the individual electron acceptors potentiometric titrations were performed. It was found that, independent of the source of the protein, one electron equivalent per copper atom could be taken up. Also, the data are consistent with the presence of two Type 1 Cu 2~ with different oxidation-reduction potentials and one Type 2 ion per molecule. MATERIALS AND METHODS

Proteins and chemicals Three different ceruloplasmin preparations were used, starting from a Cohn IV-a fraction (a gift from the Dutch Red Cross), from a Cohn I I I fraction (AB Kabi, Stockholm) 1° and from flesh blood, respectively. The blood was collected in presence of e-aminocaproic acid to a final concentration of 0.02 M (ref. 7), to inhibit the hydrolytic action of plasminogen and plasmin n. The purification was a modification of the procedure of Deutsch 12 with no (NH4)2SO 4 precipitation or crystallisation. The anion exchangers employed were DEAE-Sephadex A-5o (Pharmacia, Uppsala) and DEAE-cellulose WE-32 (Whatman, England). Sephadex G-2oo was used in the final purification step. For desalting or exchange of anions Sephadex G-25 was used instead of dialysis. The ratio of the optical absorption at 61o and 280 nm was 0.047 and 0.048 for the preparations from Cohn fractions and flesh blood, respectively. The preparation from the Cohn IV-a fraction was tested immunologically and found to be pure 13. Upon polyacrylamide gel electrophoresis in 8 M urea at p H lO.2 of reduced and cyanoethylated protein% both preparations from Cohn fractions showed a multiplicity of bands, all considerably faster than the two closely spaced bands obtained from the preparation from fresh blood. The protein was stored in 0.05 M NaC1 and 0.05 M sodium acetate at p H 7 in liquid N 2 and thawed directly before use. Before the titrations the C1-was removed by passing the protein through a Sephadex (;-25 column. The oxidase activity of ceruloplasmin towards N,N,N',N'-tetramethyl-pphenylenediamine at pH 5.5 in o.3 M sodium acetate and IOO/zM E D T A was measured as the initial velocity of the appearance of the absorbance at 563 nm 14 corrected for non-enzymic oxidation. In the substrate concentration range o.3 2.5 mM the activity

REDOX POTENTIAL OF TYPE I C u IN CERULOPLASMIN

323

was found to be the same for all preparations used when related to the absorbance of ceruloplasmin at 6Io nm. EPR-detectable copper was determined by double integration of the E P R spectrum, using a copper standard solution in o.oi M HC1 and 2 M NaC104. Total copper was measured as the 2,2'-biquinoline complex in hexano115. The concentration of solutions of ascorbate and NADH was measured from the absorbance at 265 and 34 ° n m , respectivelyl6,17. The Fe 2+ concentration was determined with I,Io-phenanthroline TM. The potassium octacyanotungstate(IV) was a gift from Dr James A. Fee and was prepared according to the procedure of Heintz 19. All other chemicals were analytical grade and used without further purification. Solutions were prepared from deionised distilled water.

Spectral measurements E P R spectra were recorded at 77 °K and about 9 GHz in a Varian E-3 spectrometer. Absorbance measurements were made at 25 °C in a Zeiss PMQ 20 A spectrophotometer.

Anaerobic oxidation-reduction titration techniques For the reductive titrations without potentiometric measurements Thunberg cells were used. The cell was made anaerobic" and the reduction was started by addition of the reductant from the side bulb to the protein solution in the euvette part. The reaction was completed under the conditions used within I 11 and the absorbances remained constant at least four more hours. Correction for dilution and evaporation was made by weighing the complete assembly before and after evacuation and by measuring the protein concentration from the 28o-nm absorbance after the experiment. The final value for the absorbance at 61o nm after reoxidation indicated that no denaturation had taken place. For the oxidation-reduction titrations with potentiometric measurements the same technique was applied as used by Reinhammar 2°. The glass assembly was redesigned to reduce its volmne. RESULTS

EPR spectrum and copper content The ceruloplasmin preparations prepared from Cohn fractions or f o m flesh blood, though different with respect to the protein part, showed the same E P R spectrum. In Fig. I is given the spectrum of ceruloplasmin from flesh blood in 0.05 M NaCI and 0.05 M sodium acetate at pH 7. From integration and copper determination it followed that 43% of the total copper was EPR-detectable for all preparations. Type 2 Cu ~+ makes up 33 ~/o of the EPR-detectable copper as calculated from integration of the low-field line 5 and the remaining 67% is accounted for by Type i Cu 2+. The E P R spectrum taken from the crude preparation, after the first DEAESephadex treatment, could also be resolved into 33% Type 2 and 67% Type I Cu 2+. On addition of IO mM azide to I mM ceruloplasmin in 0.3 M sodium acetate at pH 5.5 all of the low-field E P R peak associated with Type 2 Cu 2+ was shifted. The extinction coefficient of ceruloplasmin, based on two Type I Cu 2+ per

324

j. DEINUM, T. VANNGARD EPR at

I

spectrum 9 GHz

I

I

and I

of

ceruloplasmin

at

pH

7

77°K I

I

I

I

I

I

I

I

I

j-

20

I

I 2600

X

I

I

I

2800

MAG.ET,C

I 3000

F,E,o

l

l 3200

3400

[GAuss]

Fig. I. E P R s p e c t r u m of ceruloplasmin f r o m fresh blood at a b o u t 9 G H z and 77 °K. The protein, 28o #M, was in o.o 5 M NaC1 and o.o 5 M s o d i u m acetate at p H 7. The low-field line, a t t r i b u t e d to T y p e 2 Cu 2~ is also s h o w n with 2o times higher gain.

molecule, was c a l c u l a t e d to be II.O m M - l . c m -1 for the 6 I o - n m optical a b s o r p t i o n b a n d . The a b s o r b a n c e at 6 I o n m per t o t a l copper was 1.6 m M - l . c m 1. T r e a t m e n t with Chelex d i d n o t change this value.

Electron-accepting capacity The t o t a l e l e c t r o n - a c c e p t i n g c a p a c i t y of different p r e p a r a t i o n s was tested. In a T h u n b e r g cell a slight excess of electron e q u i v a l e n t s of N A D H per t o t a l copper (1.4) was a n a e r o b i c a l l y a d d e d to 60/~M c e r u l o p l a s m i n in 0.3 M s o d i u m acetate, at p H 7, in presence of 2 # M p h e n a z i n e m e t h o s u l f a t e . The r e d u c t i o n of c e r u p l a s m i n a n d the o x i d a t i o n of N A D H was followed at 6 I o a n d 340 nm, respectively. F o r t h e Cohn fractions t h e r e a c t i o n was c o m p l e t e d in a b o u t I h, b u t for the p r e p a r a t i o n from fresh b l o o d c o n s t a n t values were reached w i t h i n 15 min. The final values r e m a i n e d cons t a n t for at least 16 h. F r o m the r e m a i n i n g a b s o r b a n c e at 34 ° n m one can calculate how m a n y e q u i v a l e n t s of N A D H are oxidized p e r t o t a l c e r u l o p l a s m i n - b o u n d copper. I t was f o u n d t h a t o . 9 - 1 . i electrons per copper were t a k e n up. Consequently, more electrons can be t a k e n up t h a n r e q u i r e d for r e d u c t i o n of only t h e p a r a m a g n e t i c copper.

REDOX POTENTIAL OF TYPE I Cu IN CERULOPLASMIN

325

Anaerobic reductive titrations at pH 5.5 and pH 7 Ceruloplasmin from t h e Cohn I V - a fraction was a n a e r o b i c a l l y t i t r a t e d with either a s c o r b a t e or N A D H in presence of trace a m o u n t s of p h e n a z i n e methosulfate. The r e d u c t i o n with a s c o r b a t e was p e r f o r m e d a t p H 5.5 a n d p H 7. W i t h N A D H o n l y the higher p H value was used, because at lower p H a u t o - o x i d a t i o n of N A D H is too rapid. U p o n r e d u c t i o n of eeruloplasmin with more t h a n 0.6 electron e q u i v a l e n t of N A D H p e r t o t a l c o p p e r the d i s a p p e a r a n c e of the 6 I o - n m a b s o r b a n c e is t y p i c a l l y b i p h a s i c ; the first 5 0 % of the t o t a l a b s o r b a n c e is lost within 3 min, b u t t h e f u r t h e r r e d u c t i o n proceeds m u c h m o r e slowly. T h e results for the t i t r a t i o n were f o u n d to be i n d e p e n d e n t of p H or r e d u c t a u t . Fig. 2 gives the t i t r a t i o n curves at 61o a n d 34 ° n m o b t a i n e d w i t h a s c o r b a t e at p H 7. 100 q

g

d o 0.5

o Electrons

1 per

copper

Fig. 2. Anaerobic reductive titration of ceruloplasmin with ascorbic acid. Each point represents a single Thunberg cell experiment. The ceruloplasmin was prepared from a Cohn IV-a fraction. The protein concentration was 38~M. The reaction conditions were: 0. 3 M sodium acetate at pH 7, 25 °C. The remaining optical absorption at 61o (0) and 34° (©) nm were plotted with corrections for the background absorbance of the completely reduced protein, being 4% for the absorbance at 61o nm.

Potentiometric titrations F o r the three different ceruloplasmin p r e p a r a t i o n s t h e direct relation of the p e r c e n t a g e r e m a i n i n g a b s o r b a n c e at 61o n m a n d the o x i d a t i o n - r e d u c t i o n p o t e n t i a l was m e a s u r e d . I t was f o u n d t h a t o c t a c y a n o t u n g s t a t e ( m i d p o i n t p o t e n t i a l 51o mV) 21 a n d F e 3+ or f e r r i h e x a c y a n i d e (430 mV) 22 were r e q u i r e d to m e d i a t e t h e p r o t e i n to the electrode. A change in the m e d i a t o r c o n c e n t r a t i o n from 0.5 to 4 t i m e s the cerulop l a s m i n c o n c e n t r a t i o n d i d n o t influence t h e final values. O t h e r m e d i a t o r s tried, which d i d n o t i m p r o v e t h e r e a c t i o n rate, were m o l y b d e n u m o c t a c y a n i d e (800 mV),

326

j. DEINUM, T. VXNNG~RD

porphyrexide (725 mV), porphyrindine (565 mV) and diphenylaminosulfonic acid (4oo mV) ~2. The mediators were added in their oxidized form. The protein in presence of mediators in o.3 M sodium acetate buffer at pH 5.5 was reductively titrated by stepwise anaerobic addition of the reductant. The reaction was over in o.5 h, when the first electron per molecule was added to the completely oxidized protein. With further addition of tile reductant to the protein, the reaction was complete in about IO min. Addition of the last electron per molecule of ceruloplasmin, required to reduce the protein completely, resulted in a slow decrease of both the absorbance at 61o nm and the electrode potential that lasted for several hours. The intermediate values, however, were consistent with the final values, obtained on addition of smaller amounts. The oxidation of the reduced protein was done stepwise by introducing into the solution small volumes of air with a gas-tight syringe equipped with a very thin needle. Final values were reached within 2 5 min. The results from three titrations are shown in Fig. 3 in the form of a Nernst plot. It is clear that the different ceruloplasmin preparations are the same with

i

I

i

I

+I

0 IO

I

_o oIO

-1 I

I

500 Oxidation-reduction

I

I

600 potential

[mV]

Fig. 3. Potentiometric titration of ceruloplasmin at p H 5.5 and 25 °C in o. 3 M s o d i u m acetate buffer. A ° represents the corrected absorbance at 61o n m of the fully oxidized protein at the beginning of the t i t r a t i o n and A s t a n d s for the corrected a b s o r b a n c e at 61o n m during the titration. Reduction (closed symbols) was obtained by addition of small a m o u n t s of io mM anaerobic ascorbate solution and oxidation (open symbols) was obtained b y addition of air. The concentration in /~M of ceruloplasmin, o c t a c y a n o t u n g s t a t e , ferrihexacyanide, diphenylaminosulfonic acid and f e r r i a m m o n i u m sulfate were in e x p e r i m e n t s described with the s y m b o l s (2) O, 8o, 80, o, 2oo, 8o; V V, 92, 2oo, 2oo, 2oo, o; B, 38, 6o, co, o, o. I n the last case the ceruloplasmin was p r e p a r e d f r o m fresh blood. The sigmoidal line was c o m p u t e d a s s u m i n g the presence of two one-electron acceptors with the same extinction coefficient, b u t with the o x i d a t i o n - r e d u c t i o n potentials 49o and 58o mV, respectively.

REDOX POTENTIAL OF TYPE I C u IN CERULOPLASMIN

327

respect to the oxidation-reduction potential of their Type I copper atoms. Furthermore, the oxidation-reduction behavior was independent whether ascorbate or ferroammonium sulfate was the reductant. The experimental points for the reductive and oxidative titrations follow the same symmetrical sigmoidal line with an apparent midpoint potential of 535 mV. The line in Fig. 3 is computed under the assumptions that the two Type I Cu 2+ both have the same extinction coefficient at 61o nm, but a difference in oxidation-reduction potential of 9 ° inV. The data of Figs 2 and 3 were combined to produce a Nernst plot for the 34o-nm absorption band, shown in Fig. 4, as described in the legend to Fig. 4. j

I

+1

,<

I

% ~ o < :

-1

I 50O Oxidation--reduction

I

J

600 potential

[mV]

Fig. 4. N e r n s t plot of the 61o- and 34o-nm c h r o m o p h o r e s in ceruloplasmin. To o b t a i n the dashed line for 34 ° n m in this figure the o x i d a t i o n - r e d u c t i o n potential was read f r o m the c o m p u t e d line in Fig. 3 a n d the 34o-nm a b s o r b a n c e f r o m Fig. 2, b o t h at the s a m e a b s o r b a n c e at 61o nm. F o r comparison, the 6 I o - n m curve in Fig. 3 is r e p r o d u c e d in this figure. Direct m e a s u r e m e n t s of the a b s o r b a n c e at 34 ° n m u n d e r the conditions of Fig. 3 were e r r o n o u s because of the a b s o r b a n c e of the m e d i a t o r s in the s a m e region.

DISCUSSION

The different preparations of ceruloplasmin do show the same properties with respect to the E P R spectrum, the oxidation-reduction potential of the two Type i Cu z+ and the total electron capacity. However, there is a difference in the rate with which the protein becomes reduced; the time needed to accept one electron per copper ranges from 15 min to I h for preparations used for this work and to IO h for protein in phosphate buffer 8. The potentiometric titrations give an apparent midpoint potential for the absorbance band at 61o nm of 535 mV, which falls in the range suggested b y Fee and MalmstrSm ~.

328

J. DEINUM, T. VANNGXRD

The symmetrical shape of the Nernst plot (Fig. 3) indicates the same extinction coefficient at 61o nm for the two Type I Cu 2+ in ceruloplasmin. The calculated line in Fig. 3 is based on the assumption that the two oneelectron acceptors are unequivalent, having different oxidation-reduction potentials, 58o and 49 ° mV, respectively. This is not a unique interpretation, as one can obtain virtually the same curves for two interacting, but identical one-electron acceptors having a negative cooperativity with a free energy change corresponding to 6o mV. However, a protein consisting of only one polypeptide chain Gcannot have two identical sites. Also, a difference as much as 9 ° mV for two blue Cu 2+ is not unreasonable, when we compare these values with those found by Reinhammar 20 for the Type I Cu 2+ in other blue proteins, stellacyanin (184 mV), Rhus laccase (394 mV) and Polyporus laccase (785 mV). Other reports are consistent with two non-equivalent ions with the same extinction coefficient. In high concentration both azide and cyanate affect the absorption of ceruloplasnfin at 61o mn so that only 5o°/0 of the original absorbance at 61o nm is left e4. In the case of azide this occurs without reduction of the paramagnetic copper (Deinum, J., unpublished). The disappearance of tile absorbance at 61o nm upon anaerobic reduction with norepinephrine e5 or NADH and phenazine methosulfate is biphasic, with each phase corresponding to 5o% of the absorbance of tile oxidized protein. Upon reoxidation of the reduced protein the first 5o% of the original absorbance of the oxidized protein at 61o nm is rapidly restored and the next 50% recovers very slowly 26. Tile last effect is even more pronounced in presence of fluoride, when even after several days only 5o% of the absorbance at 6Io nm can be restored (Deinum, J., unpublished). For the ceruloplasmin preparations used one can distinguish in the E P R spectrum one Type 2 Cu e+ and two Type I Cu e+. It was found by E P R that this Type 2 Cu e~ is affected by azide. In the commercial preparation used by Andr6asson and V/inngSrd 4 the E P R spectrum could be separated into two Type i Cu 2+ and two different Type 2 Cu"~ of which only one Type 2 Cu e+ was affected by azide. The Type 2 Cu e+ which is not changed by azide might have been picked up during tile purification procedure. The procedure earlier used in this laboratory s involved precipitation with (NH4)2SO~, which often contains copper. We did not use special chelating agents to free the protein from non-tightly bound copper. Thus, the socalled "chelexable" copper eT, not present in our preparation, may well be identical with the second Type 2. The value of the absorbance at 6Io nm per total copper is L6 mM 1.cm- 1 as compared to about I. 5 for tile protein used in earlier work 8 (V~tnngfird, T., unpublished), which would be consistent with the higher Type 2 Cu 2+ content reperted previously*. On the basis of the more recent molecular weight determinations 6, tile total number of copper atoms per molecule is likely to be less than eight. It is, however, not sure whether there are seven or six copper atoms in the molecule. The E P R and absorbance data presented here (three EPR-detectable Cu e+ accounting for 43% of the total copper) are totally consistent with the presence of seven Cu atoms per molecule, as was earlier suggested from measurements on magnetic susceptibility es. However, the accuracy of the determinations do not allow a definite choice between the two possibilities, and thus the question whether there are three or four nonparamagnetic Cu e+ in ceruloplasmin is still not settled. The two non-paramagnetic Cu in the laccases have been associated with the

REDOX POTENTIAL OF TYPE I CU IN CERULOPLASMIN

32 9

two-electron acceptor absorbing at 33o nm 2°. No such simple analysis is possible for the corresponding band in ceruloplasmin. This is seen from the Nernst plot in Fig. 4 where the slope indicates an n value in the range o.5-1 rather than 2. In fact, it must be questioned whether this band is at all an indication of the redox state of nonparamagnetic electron acceptors, as under certain conditions 0.5 electron equivalent suffice to abolish both this band and all E P R signal 26. However, Fig. 4 shows, that a direct correlation with the redox state of Type I Cu 2+ is impossible. By analogy, we favour the idea that the 33o-nm electronic transition occurs within the same nonparamagnetic entity in all blue oxidases. Then, in ceruloplasmin this band could be made to disappear not only through the reduction of this entity, but also through other modifications of the protein. One such modification might be the reduction of the Type 2 Cu 2+, a suggestion supported by studies involving F - binding to this ion, which have shown that at least in the laccases there exists a close connection between the Type 2 Cu ~+ and the non-paramagnetic electron acceptor (Andr6asson, et al.29). A gratifying conclusion from the present work is that a number of important properties are independent of whether the protein has undergone hydrolytic attack or not. This suggests that a large portion of the data collected earlier on ceruloplasmin is also valid for the single-polypeptide-chain molecule. On the other hand, the data point to the complicated and asymmetric nature of ceruloplasmin with its three EPR-detectable Cu ions all being different. The nature of the other electron acceptors is essentially unknown, but it is a particularly interesting feature of this protein, that the 33o-nm band apparently can be made to disappear without reduction of these acceptors. Studies on this phenomenon might possibly lead to a better understanding of the chemical structure of this chromophore. ACKNOWLEDGEMENTS

The Cohn fractions were gifts from Mr H. Bj6rling, AB Kabi, Stockholm and the Dutch Red Cross, Amsterdam. At the latter department Miss van der Giesen kindly performed the immunological tests of ceruloplasmin. Drs Sandberg and Lindholm at Blodcentralen, Sahlgrenska Sjukhuset, G6teborg provided invaluable help in the collection of the serum. Fruitful discussions with Drs B. F. van Gelder and B. G. MalmstrSm are gratefully acknowledged. This work was supported by grants from the Swedish Institute (J.D.) and the Swedish Natural Science Research Council.

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Alkjaersig, N., Fletcher, A. P. and Sherry, S. (1959) J. Biol. Chem. 234, 832-837 Deutch, H. F. (1962) Arch. Biochem. Biophys. 99, 225-229 Stokes, R. P. (1967) Clin. Chim. Acta 15, 517-523 Curzon, G. (1967) Biochem. J. lO3, 289-298 13rumby, P. E. and Massey, V. (1967) in Methods in Enzymology (Estabrook, Jl. W. amt Pullman, M. R., eds), Vol. X, pp. 473-474, Academic Press, New York Pecht, I., Levitzki, A. and Anbar, M. (1967) J. Chem. Soc. 89, 1587-1591 Van Gelder, B. F. and Slater, E. C. (1962) Biochim. Biophys. Acta 58, 593-595 Sandel, E. 13. (1959) Colorimetric Metal Analysis, p. 537, Interscience Publ. Inc., New York Heintz, E. A. (1963) in Inorganic Synthesis (Kleinberg, J., ed.), Vol. 7, P. 142, McGraw Hill, New York Reinhammar, B. R. M. (1972) Biochim. Biophys. Acta 275 , 245-259 13adsgaard, H. and Treadwell, W. D. (1955) Helv. Chim. Acta 38, 1669-1679 Clark, W. M. (196o) Oxidation-Reduction Potentials of Organic Systems, The Williams and Wilkins Company, 13altimore, Md. Fee, J. A. and Malmstr6m, B. G. (1968) Biochim. Biophys. Acta 153 , 299 302 Marriott, J. and Perkins, D. J. (1968) Biochim. Biophys. Acta 154, 5Ol-5O6 Walaas, E., Walaas, O. and Lovstad, R. (1966) in The Biochemistry of Copper (Peisach, J., Aisen, P. and Blumberg, V~T. E., eds), p. 537, Academic Press, New York Carrico, R. J., Malmstr6m, B. G. and Viinng~rd, T. (1971) Eur. J. Biochem. 22, 127-133 McKee, D. J. and Frieden, E. (1971) Biochemistry io, 3880-3883 Aisen, P., Koenig, S. H. and Lilienthal, H. R. (1967) J. Mol. Biol. 28, 225-231 Andr6asson, L.-E., Malmstr6m, 13. G., Str6mberg, C. and VXnng~rd, T. (1973) Eur. J. Biochem., in the press