Reaction of Human Ceruloplasmin and Anion Treated Ceruloplasmin with Diethyldithiocarbamate

Martine Herve, Arlette Gamier-Suillerot Laboratoire de Chimie Bioinorganique, U.E.R. de MPdecine et Biologie Humaine, Universite’ Paris

Lucia Tosi Dkpartement de Recherches Physiques, Universitk Pierre et Marie Curie, Paris

Marion Steinbuch De’partement de Fractionnement, Centre National de Transfusion Sanguine, Orsay

ABSTRACT

The reaction of human ceruloplasmin and anion treated ceruloplasmin with diethyldithiocarba-

mate was studied at pH 5.5. The analysis of optical and EPR spectra at 9 GHz showed that ceruloplasmin contains five pammagnetic copper ions, two of which, X and Y, not involved in

enzymatic activity, are chelated by diethyldithiocarbamate; the complex thus formed is easily

removed by high-speed centrifugation. However, the enzyme depleted of these two X and Y

copper ions is able to compete with the Cu(II)-diethyldithiocarbamate complex, as time elapses, recovering both Cu(II) atoms. In addition diethyldithiocarbamate acts as a reducing agent for the

two type-1 copper atoms when added in large excess to the enzyme or the anion treated enzyme.

INTRODUCTION

Ceruloplasmin is a “blue” multicopper mammalian serum metalloenzyme possessing oxidase activity towards Fe(U) and several aromatic amines and phenols [l]. Its activity is inhibited by anions such as azide, thiocyanate, and fluoride.

Address reprint requests to Dr. A. Gamier-Suillerot, Laboratoire de Chimie Bioinorganique, U.E.R. de Medecine et

Biologie Humaine, Universim Paris XIII, 74 rue Marcel Cachin, 93012 Bobigny, France

Journal of Inorganic Biochemistry 25, 121-130 (1985) 0 1985 Elsevier Science Publishing Co., Inc. 121

52 Vanderbilt Ave., New York, NY 10017 0162-0134/85/$.03.30

122 M. Herve et al.

Like the other blue copper oxidases, ceruloplasmin contains three spectroscopically distinct copper ions, i.e., type 1, characterized by an intense electronic absorption at 600 nm and an abnormally small hyperfine coupling constant; type 2, with EPR hyperfine structure similar to that found in Cu(II) tetragonal complexes, and type 3, a non-EPR detectable copper associated with an intense absorption at 330 nm fl]. The number of copper atoms in the molecule of ceruloplasmin is still uncertain; values of six, seven, and eight atoms per molecule have been reported, the discrepancy being due in part to the different values of molecular weight on which the number of coppers is based. On the basis of a molecular weight of 132oo0, six to seven atoms per molecule have been obtained [ 11. From them, two are of type 1, one of type 2, and two of type 3. The latter is always present in pairs and is assumed to be a Cu(II)-Cu(I1) antiferromagnetic pair [I]. The two type I Cu(II) ions, although they absorb at the same wavelength (ecu = 5500 M-i cm-‘, possess different EPR parameters [2] different CD [3] and resonance Raman spectra [4], different redox potential [5], and reoxidation rates [6]. One of them, labeled la, has its Cu-S(Cys) bond disrupted by the addition of some inhibitory anions such as SCN- and N3- [7] and is more sensitive to pH changes [8], while the other, called lb, is not perturbed by inhibitory anions. In addition, on the basis of EPR measurements, it has been demonstrated that the inhibitory anions azide and fluoride interact with type 2 copper [9, lo].

The five Cu(II) ions mentioned above are essential to enzymatic activity. Huber and Frieden [ 1 I] have shown that Chelex treatment leads to the loss of another copper without affecting catalytic and spectral properties. On the other hand, to fit a stoichiometry of six, one “type 4” copper has also been assumed by Ryden and Bjork [12]. These authors have suggested, in addition, that a value of more than six copper atoms per molecule arises from partial occupation of this “chelexable” site.

In the present report we show that, for a molecular weight of 132000, ceruloplasmin contains five paramagnetic copper ions, two of which are chelated by diethyldithiocar- bamate and are not involved in enzymatic activity.

The complex thus formed may be easily removed by high-speed centrifugation. However, the protein depleted of these “chelexable” copper atoms is able to compete with the Cu(II)-diethyldithiocarbamate complex as time elapses, recovering both Cu(I1) atoms.

In addition diethyldithiocarbamate acts as a reducing agent for type la and type lb Cu(I1) when added in large excess to the enzyme.

MATERIALS AND METHODS

Human ceruloplasmin was prepared as indicated previously [ 191. Purity was controlled by the t6i0/e2a0 ratio, which was higher than 0.04. The ceruloplasmin dissolved in 0.5 M sodium acetate was kept in liquid nitrogen until used. Working solutions were obtained by dilution with 0.05 sodium acetate buffer pH 5.5. All solution were prepared with doubly distilled water. The experiments have been carried out at 25°C unless otherwise stated. Sodium acetate, NaN3, NaSCN, NaF, DDC, were commercial product of the best grade available and were used without further purification.

Crystals of p-phenylenediamine dihydrochloride were prepared and purified according to the Ravin method [13]. A 0.5 M solution of the purified substrate was prepared just prior to use for assay of enzymatic activity. DDC-treated ceruloplasmin

Ceruloplasmin; Diethyldithiocarbamate; Type I Copper Reduction 123

solutions were diluted in 0.4 M acetate buffer, pH 5.5 and added to p-phenylene- diamine dihydrochloride solution in order to obtain final ceruloplasmin and p- phenylenediamine dihydrochloride concentrations of 3 x lo-’ M and 10e3 M, respectively.

Absorption spectra were recorded with a Cat-y 219 spectrophotometer, circular dichroism with a Jobin Yvon Mark V dichrograph, electron paramagnetic spectra were obtained, at approximately 9 GHz, with a Varian type E-9 spectrometer.

RESULTS

DDC-Ceruloplasmin Interaction. The spectra are recorded 10 min after addition of DDC to ceruloplasmin . As illustrated in Figure 1, the addition of DDC to ceruloplasmin led to the appearance of one band at 445 nm in the protein absorption spectrum and no further spectral change was noticeable during the following 30 min. The band at 445 MI is characteristic of DDC chelated Cu(JJ) [14, 151 and, as shown in Figure 1 insert, the absorption in this region levels off when the molar ratio [DDC]/ [enzyme] reaches 4, giving an absorption coefficient of about 16000 M- ’ cm- l. Since the molar absorptivity of Cu(DDC)*, in several organic solvents, at this wavelength is approximately 8000 M- ’ cm-’ [ 151, one can estimate that two Cu(II) have been

FIGURE 1. Absorption spectrum of native ceruloplasmin and DDC treated ceruloplasmin. 70 PM

ceruloplasmin in 0.05 Msodium acetate buffer, pH 5.5 [l] was incubated at 2S’C for 10 min with DDC at

[DDC]/[enzyme] = 6 [2]. Insert: the molar absorption coefficient at 445 nm is plotted as a function of the

molar ratio [DDC]/[enzyme]; the time of incubation is 10 min.

2x10- cm-l

124 M. Herve et al.

chelated by DDC, forming two moles of the complex. Up to ten moles were added to one mole of ceruloplasmin without any change in absorption (see Fig. 1 insert). At higher molar ratios precipitation occurs.

The CD spectrum of DDC treated ceruloplasmin, on the other hand, does not show any appreciable modification. This fact, and the lack of optical activity of the transition at 445 nm, strongly suggests that both DDC chelated Cu(I1) ions are completely removed from the protein. Moreover, we did not observe any modification in the ultraviolet CD spectrum, indicating that the addition of DDC does not induce a noticeable change in the secondary structure of the enzyme. Actually, after 10 min incubation the CU(DDC)~ complex is easily separated from the protein by high-speed centrifugation at 30,000 g for 20 min. The absorption of the supematant does not exhibit the band at 445 nm and is similar to that of native ceruloplasmin. Moreover, subsequent addition of DDC does not give rise to reappearance of any absorption at 445 mn. Obviously there are two Cu(I1) ions, hereafter called X and Y, which are easily removed from the protein by treatment with DDC and subsequent centrifuga- tion.

Quite different results are obtained when, instead of using centrifugation 10 min after addition of DDC, the DDC treated ceruloplasmin is left to incubate for a longer period of time. Under such conditions one observes a very slow decrease in intensity of the band at 445 nm, which, after 24 hr of incubation, completely disappears. Concomitantly a slight yellow precipitate is formed and the spectrum of native ceruloplasmin is finally recovered. By subsequent addition of DDC to the supernatant the 445-nm band reappears. As illustrated in Figure 2 this experiment can be repeated several times with the same ceruloplasmin sample, indicating that after longer periods of incubation Cu(I1) is finally removed from the CU(DDC)~ complex reentering presumably the same X and Y Cu(I1) binding sites.

In addition the DDC reaction with ceruloplasmin causes progressive changes in the EPR spectrum that level off at [DDC]/[enzyme] > 4. Thus, in order to clarify the nature of the DDC-ceruloplasmin interaction, the EPR of the following samples have been recorded.

Sample 1: native ceruloplasmin; sample 2: DDC treated ceruloplasmin at [DDC]I [enzyme] = 6 after 10 min incubation; sample 3: DDC treated ceruloplasmin at [DDC]/[enzyme] = 6 after 10 min incubation and subsequent centrifugation; sample 4: DDC treated ceruloplasmin at [DDC]/[enzyme] = 6 after 24 hr incubation.

FIGURE 2. DDC treated ceruloplasmin. Variation of the molar absorption coefficient at 445 nm as a

function of time. 70 PM ceruloplasmin in 0.05 M sodium acetate buffer, pH 5.5 was incubated with DDC at [DDC]/[enzyme] = 6. At time indicated by arrows the solution was centrifuged to eliminate the yellow

precipitate formed, and DDC was subsequently added to the supematant.

Ceruloplasmin; Dietbylditbiocarbamate; Type I Copper Reduction 125

2800 3000 3200 Magnetic Field -Gauss-

FIGURE 3. EPR spectra of native ceruplasmin and DDC treated ceruloplasmin. 120 PM ceruloplasmin in

0.05 M sodium acetate buffer, pH 5.5, was incubated at 25°C for 10 min and then centrifuged at 30,000 g

for 20 min. Line 1, enzyme alone; line 2, enzyme plus DDC at [DDC]/[enzyme] = 6 before centrifugation;

line 3 after centrifugation. EPR spectra were recorded at 9.27 GHz frequency, 20 mW power, 10 G modulation amplitude, and - 180°C sample temperature.

As illustrated in Figure 3 after addition of DDC to ceruloplasmin and 10 min of incubation (sample 2), the EPR signal of the low-field region decreases, whereas an increase is observed in the high-field region. On the other hand, the low-field EPR signal of the DDC treated ceruloplasmin after 10 min incubation remains constant when the sample is centrifuged (sample 3), although it is weaker in the high-field region. However, the EPR spectrum of ceruloplasmin is completely recovered after 24 hr incubation (sample 4).

To quantify the DDC-ceruloplasmin reaction the areas under the absorption EPR of samples l-4 have been measured and compared with that of CuEDTA as unity [ 161. The values thus obtained are reported in Table 1. As can be noted, area EPR data of native ceruloplasmin can be accounted for by five cupric centers, while that of sample 3 by only three, confirming that both X and Y Cu@) have been removed by centrifugation. Besides, when the X- and Y-depleted ceruloplasmin is left to incubate for 24 hr in the presence of the CU(DDC)~ complex the same area is obtained that for the X- and Y-containing ceruloplasmin EPR spectrum (compare samples 1 and 4 in Table 1).

Prior to quantifying the EPR spectrum of sample 2, the double integrated EPR spectrum of CU(DDC)~ solution of known concentration was measured using the same method as above. The area for each CU(DDC)~ is about 0.6 that of CuEDTA. As shown in Table 1 the value thus obtained for sample 2 can be accounted for by the

126 M. Herve et al.

TABLE 1. EPR data for ceruloplasmin and F- treated ceruloplasmin in the absence and in the

presence of DDC.

Sample N”

Type of copper EPR detected ~.__~~ .~ - -~~..-~..~~.-_..~ ___..

Ia Ib II X Y Cu(DDC)J

CU(DDC)~

(1) Native Cpb

(2) Cp-kDDC

[DDC]/[Cp] = 6

At= 10 min’ (3) (2) Centrifugedd

(4) Cp+ DDC

[DDC]/[Cp] = 6

At=24 hr’ (5) (3) + 2 Cu(DDC)z

At = 24 hr’

(6) Cp+DDC

[DDC]/[Cp] = 10 At= 1 minC

(7) Cp-F-

(8) Cp-F- + DDC

[DDC]/[Cp] = 10 At=1 min’

(9) (8) centrifuged d

(10) (9)

At=2 days’

0.6 1(x0.6)

5 1 1 1 1 1 4.2 1 1 1 2(x0.6)

3 1 1 1

5 1 1 1 1 1

5 1 1 1 1 1

3.9 0.8 0.8 1 2(x 0.6)

5 1 1 1 1 1

2.2 1 2( x 0.6)

1 1

2.6 0.8 0.8 1

___.-I_. -_____.

L N is the number of paramagnetic centers defined as the ratio between the area under the absorption EPR spectrum of the sample and that of CuEDTA taken as reference. b Cp stands for ceruloplasmin. c At is the time of incubation. d The sample is centrifuged at 30,000 g for 20 min.

three Cu(I1) ions of ceruloplasmin (la, lb, and 2) plus the two X and Y CU(DDC)~ complexes.

The enzymatic activities of the four samples have been determined and appear to be essentially the same, showing that native ceruloplasmin contains two extra nonenzy- matic Cu(II) ions, which are easily removed by reaction with DDC and subsequent centrifugation.

In addition, when X- and Y-depleted ceruloplasmin (sample 3) is treated with a solution of Cu(DDC)z, the EPR (Table 1, sample 5) and the absorption spectra of the native enzyme are recovered.

Hence, the above results may be summarized by the following equilibria:

Cp + nDDC + “Cp” + 2Cu(DDC)* + Cp + nDDC IO min 24 hr

where Cp stands for native ceruloplasmin and “Cp” for X- and Y-depleted ceruloplasmin (n > 4).

DDC-Ceruloplizsmin Interaction. The spectra are recorded within the 10 min following the addition of DDC to ceruloplasmin at fDDCJ/fenzyme/ > 4. In this case the band at 445 nm is immediately observed, and, as mentioned above, it does not

Cenhplasmin; Diethyldithiocarbamatemate; Type I Copper Reduction 127

E c 0

5 5x103 -

Y m

0

minutes FIGURE 4. DDC treated ceruloplasmin. Variation of the molar absorption coeffkient at 610 NII as a function of time. 70 pM ceruloplasmin in 0.05 A4 sodium acetate buffer, pH 5.5, was incubated with DDC at [DDC]/[enyzme] = 10.

evolve during the following 40 min. Concomitantly with the appearance of the 445- run band a decrease in absorption at 610 nm is noted, which is more pronounced at higher ratios of [DDC]/[enzyme]. As shown in Fig. 4, at [DDC]/[enzyme] = 10 the molar extinction coefficient at 610 nm decreases from 11000 to about 8000 M- l cm-’ within 2 min; however, the initial value is recovered after 10 min of DDC addition.

As in the preceding set of experiments the EPR signal evolves concomitantly with the 610~nm band. Thus, the area under the EPR spectrum of a solution containing DDC-treated ceruloplasmin at [DDC]/[enzyme] = 10 one minute after DDC addition (sample 6) is slightly lower that of sample 2 (see Table 1). These results may be interpreted as arising from partial reduction of the type 1 and eventually the type 2 Cu(II) centers.

DDC-Anion Treated Ceruloplasmin Interaction. It is well documented that the addition of inhibitors such as Ns- or SCN- gives rise to the appearance of charge transfer transition bands in the electronic spectrum of ceruloplasmin. Concomitantly a decrease of 50% in the absorption at 610 nm is observed [7].

In contrast, the spectrum of F--treated ceruloplasmin is very similar to that of the native enzyme [9].

Vanngard and co-workers, on the other hand, studying the EPR spectrum of ceruloplasmin upon addition of N3- and F- , concluded that both anions interact with type 2 copper. The same conclusion for F- has been obtained in EPR studies by Gray and co-workers [17].

Thus, in order to clarify the nature of the DDC-ceruloplasmin interaction at [DDC]/ [enzyme] = 10 we have studied the effect of DDC upon inhibitory anion treated ceruloplasmin .

DDC-NJ- Treated Ceruloplasmin Interaction. This set of experiments was, in fact, performed either with Ns- or SCN-. Since the results are strictly analogous, only the data concerning Nr - will be reported here.

N3- was added to a solution of native ceruloplasmin in amounts high enough to make the absorption band at 380 nm (characteristic of the charge transfer Ns- +

128 M. Herve et al.

nm

800 600 400

2x104 3x104 c rn

-1

FIGURE 5. Reaction of DDC with N3--treated ceruloplasmin. 70 HM ceruloplasmin in 0.05 M sodium

acetate buffer, pH 5.5 was treated with a lo3 excess of N3- and then incubated with DDC at [DDC]/ [enzyme] = 6. Curve a, enzyme alone; curve b, enzyme plus N3-; curve c, N,- treated enzyme incubated with DDC.

CL@)) level off, i.e., [N3-]l[enzyme] = 1000, c3&, = 8000 M-’ cm-‘. Concomi- tantly the 610-nm band of ceruloplasmin decreases to 50% of its original value owing to disruption of type la Cu(LI)-S(Cys) bond [7] (see Fig. 5, curve b). Addition of DDC to this sample at [DDC]/[enzyme] > 4 led to the immediate appearance of the 445 nm absorption band characteristic of the Cu(DDQ complex, which is now superimposed on the N3 - -+ Cu(I1) charge transfer band (see Fig. 5, curve c). The difference of absorptivities at 445 nm between the two spectra (c and b) is approximately 16000, which corresponds, as above, to the chelation of X and Y Cu(I1).

Moreover, the addition of Nj- either to DDC treated ceruloplasmin or to X- and Y- depleted ceruloplasmin led to the appearance of the 380-nm band in their spectra with the same amplitudes as in curves c and b (Fig. 5), respectively.

The foregoing results clearly indicate that N3- and DDC sites of complexation are quite different. N3- coordinates to type 2 Cu(II), which is not chelated by DDC. On the other hand, the X and Y Cu(II), which are easily chelated by DDC, are unable to bind N3 -. Similar conclusions hold for SCN - .

DDC-F- Treated Ceruloplasmin Interaction. A second set of experiments was performed by addition of DDC to F--treated ceruloplasmin. As is well known, both the absorption and CD spectra of ceruloplasmin are not appreciably perturbed by treatment with F- ([F-]/[enzyme] = 103), whereas the EPR spectrum is strongly

Ceruloplasmin; Diethyldithiocarbamate; Type I Copper Reduction 129

I L

2750 3000

Magnetic Field -Gauss-

I

3250

FIGURE 6. EPR spectra of F- treated ceruloplasmin in the absence and in the presence of DDC. 120 $4 ceruloplasmin in 0.05 M sodium acetate buffer, pH 5.5 was treated with a 10’ excess of F- , incubated with DDC at iDDC]/[enzyme] = 10 for 1 min and then centrifuged at 30,000 g for 20 min. Line 7, F- treated enzyme; line 8, F- treated enzyme plus DDC before centrifugation; and line 9 after centrihgation.

modified, leading to the splitting of the lowest field EPR transition (see Fig. 6, line 7). The doubly integrated EPR signal is the same as that of native ceruloplasmin (see Table 1, sample 7). When this sample is treated with DDC at [DDC]/[ceruloplasmin] > 4, one observes the same phenomenon as in the case of ceruloplasmin and N3-- treated ceruloplasmin, i.e., the appearance of an absorption band at 445 nm corresponding to two DDC chelated copper. If this mixture is left to incubate, the band at 445 nm slowly disappears, but much more slowly than in the previous case: it takes about 15 days instead of one to recover the original spectrum of F- treated ceruloplasmin.

Furthermore, other changes are detected within the first minute after addition of DDC . The intensity at 6 10 ntn decreases depending on molar ratio and the absorptivity becomes negligible at [DDC]/[enzyme] = 10. Concomitantly the EPR signal decreases (Fig. 6, line 8) and the measured area indicates the reduction of two enzyme Cu(II) centers (Table 1, sample 8), which are different from the X and Y Cu(II), since the 445 nm band remains unchanged.

Moreover, if centrifugation is performed some minutes after the addition of DDC to the F--treated ceruloplasmin (at [DDC]/[enzyme] = lo), the band at 445 nm disappears and the area under the EPR spectrum indicates the presence of only one paramagnetic copper. This center seems obviously to be F--bound type 2 Cu(II) (see Fig. 6, line 9). As time elapses the band at 610 nm slowly reappears (80% in two days) as well as the EPR spectrum (Table 1, sample 10).

130

DISCUSSION

M. Hetve et al.

The foregoing results clearly show that the ceruloplasmin contains five paramagnetic copper centers per molecular weight of 132000, two of which, labeled X and Y, are not essential for enzymatic activity and easily chelexable by DDC. In addition X- and Y-depleted ceruloplasmin may be obtained by high-speed centrifugation, the CU(DDC)~ complex formed being removed from the solution. However, if the X- and Y-depleted enzyme is left to stand for a longer period of time in the presence of Cu(DDC)*, the X- and Y-binding sites compete successfully with the chelate and the metal reenters the protein. These results indicate than an equilibrium between the X- and Y-depleted enzyme and Cu(DDC)z is established, which may arise from local conformational changes (not detected by CD measurements) that are reversible.

Another interesting result that emerges from our experiments is that DDC can act as a reducing agent for type 1 when added in large excess to ceruloplasmin. As we have shown, a partial decrease in absorption at 610 nm and in the EPR signal is observed in the first minute after addition of DDC in large excess to the enzyme. In the presence of air subsequent reoxidation takes place gradually, taking approximately 10 min for practically complete recovery. However, if the reduction of type 2 Cu(I1) is blocked

by F- , reduction of type 1 copper centers is complete and reoxidation takes place more slowly (approximately 80% of the type 1 copper is reoxidized in two days). Experiments with lactase (that contains one type 1, one type 2, and two type 3 coppers) indicate that type 1 and type 2 sites contribute one electron each to the reduction of the two electrons accepting type 3 unit [ 181. Since ceruloplasmin contains two type 1 copper centers, the mechanism of electron transfer should be somewhat different. Our results suggest that the type 2 copper may function as a shuttle between type la, type lb, and type 3 coppers.

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Received April 30, 1985; accepted May 7, I985