Reconstitution of Helix pomatia hemocyanin with cyanocuprate(I)

Recoanstitutionof HeZixpomatia Hemocyanin with Cyanocuprate(I) Raphael Witters, Dominique Groeseneken, Paul Jacobs,

and Rent5Lontie Laboratorium

voor Biochemie. Kathoiieke Universiteit te Leuven. Belgium

The possibility to reconstitutehemocyaninwith copper(I1)ions can be explainedby the reduction of these ions by residualcyanide and by the formation of a stable copper(I) complex that introduces copper(I) into the active site of hemocyanin. The reconstitution of hemocyanin with cyanocuprate(1)in air is disturbed by a secondary reaction whereby the copper-oxygen band is destroyed irreversiblyas a function of time. The formation of hydroxyl radicalsat the activesite of hemocyaninseems partiallyresponsible for its destruction.

INTRODUCTION Hemocyanin reversibly binds one dioxygen molecule per two copper atoms; these copper(I) atoms can be removed from the protein by high concentrations of cyanide thereby forming apohemocyanin. At concentrations of cyanide below 150 @¶, however, only dioxygen is expelled from oxyhemocyanin while the copper remains [l] _ Reconstitution of hemocyanin can only readily be performed with copper(I) complexes. Up to 83% of the copper could be removed from Octopus vu&ris hemocyanin by cyanide and a 50% reconstitution was achieved with chlorocuprate(I) in the absence of oxygen [2] _ A nearly quantitative reconstitution (95%) of Helir pomdb hemocyanin was obtained with a copper(I) acetonitrile complex [3]. Traces of cyanide, left in the apohemocyanin preparation after dialysis, could possiily have acted as a carrier in the reconstitution of 0. vulgar& hemocyanin with solid C&O and Cu,S [4] and even as a reducing agent in the reconstitution of Panulirus infemrptus hemocyanin with copper sulfate [5]. In order to prove this hypothesis, reconstitutions were performed with copper sulfate in the presence of increasing amounts of cyanide. In addition to the actual recorrstitution a secondary reaction was noticed when operating in air. Addition of cyanide to a solution containing copper(In ions causes the precipitation- of Cu(CN),, which rapidly decomposes into cyanogen and C&N, soluble in exAddress reprintrqUeSS Belgium.

to Prof. R. Lontie, I..abOfatOriumvoor Biochemie,Dekenstraat6, B-3000 L.euven,

Journalof Inowic Biochemistry16,237-243 (1982) 0 EIsAvierSciencePublishingCo.. Inc 1982 52 Vanderbilt Ave., New York, NY 10017

237 0162-0134/82/030237-07$2.75

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cess cyanide with the formation of cyanocuprate(I) complexes. These ought to reconstitute hemocyanin. A reconstitution of H. pomatia hemocyanin of could be achieved with cyanocuprate(I) in the absence of oxygen; in air a reaction was observed [6] _ This reaction was studied here by the action cuprate on oxyhemocyanin.

to be able up to 94Yo secondary of cyano-

MATERIALS AND METHODS Hemocyvlin of H. pomatziz was isolated from the hemolymph by precipitation with ammonium sulfate at pH 5.3. After centrifugation the precipitate was dissolved in 0.1 M sodium acetate buffer, pi-I 5.7, and the solution dialyzed exhaustively against this buffer in order to remove ammonium sulfate. Apohemocyanin was prepared by the dropwise addition of a 1 M potassium cyanide solution, adjusted to pH 8.5 with acetic acid, to a solution of hemocyanin inO. M sodium acetate buffer, pH 5.7, 10 mM calcium acetate, until it became yellowish. This solution was dialyzed for 1 day at 4°C against 50 mM potassium cyanide, 10 mM calcium acetate, adjusted to pH 8.5 with acetic acid. Cyanide was removed by dialysis at 4°C against 0.1 M sodium acetate buffer, pH 5.7. Potassium tetracyanocuprate(I) was prepared by mixing stoichiometric amounts of cuprous chloride, suspended in a minimal amount of water, and potassium cyanide dissolved in water. The scrvent was evaporated at 100°C and the product recrystallized from ethanol 88% at -20°C. The crystals were dried at room temperature. Copper was determined photometrically at 454 nm with 0.1% 29dimethyl-IJOphenanthroline in glacial acetic acid in the presence of 0.1% hydroxylammonium chloride. Low copper concentrations were determined with an atomic absorption spectrophotometer (Perkin Elmer 372, Norwalk, CT, USA)_ epr measurements were carried out with an E-109 spectrometer (Varian, Palo Alto, CA, USA) at --?7O”C. The second integral was computed with a HewIett Packard calculator HP 9825A, utilizing software provided by Varian. The ammo acid composition was determined with a modified Beckman Model 12oC amino acid analyzer equipped with a one-coiumn system and a photometer BT 6620 (Biotronik, Frankfurt/Main, F.R.G.). Spectrophotometric measurements were carried out on a Perkin Elmer 554 spectrophotometer or on a Cary 16 spectrophotometer ~‘aarian, Monrovia, CA). In anaerobic experiments oxygen was expelled by nitrogen A28 (L’Air Liquide Beige, Likge, Belgium). Reagents, used as scavengers for hydroxyl radicals, were of analytical reagent grade: dirnethyl sulfoxide (Merck, Darmstadt, FXG.); 5,5dimethyl-1-pyrroline N-oxide

(Aldrich, Milwaukee, WI); N-acetyltyrosinamide (Aldrich); znd mannitol (Hoffmann-La Roche, Basle, Switzerland).

RESULTS AND DISCUSSION influence of the Cyanide Concentration

on the Reconstitution

with Copper(H)

When copper(H) ions were added to H. pomatia apohemocyanin in an amount twice that needed to reconstitute the active site, a very slow increase of the copper oxygen

Reconstitution

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239

FIGURE 1.Specific absorp lion coefficient a of the coppervxygen band of H. pornaria hemocyanin as a function of time on treating apohemocyanin with copper ions (twice the amount of copper in hemocyanin) in 0.1 M sodium acetate buffer, pH 5.7, after removal of cyanide, which is used in the preparation of apohemocyanin; (a) by dialysis, (b) by dialysis followed by treatment with Amberlite IRA-400, or (c) with 1 mM zinc acetate.

baud at 346 nm was observed (Fig. la). The disappearance of this band -when the solution was brought under nitrogen suggested a real reconstitution of the active site. After 14 days, a maximal value of about 47% was reached for the specific absorption coefficient at 346 nm and a copper content of 55% of the normal value in hemocyanin. Cyanide, used in excess in the preparation of apohemocyanin, but probably incompletely eliminated by dialysis, was suspected to play a part in this reaction. In order to eliminate more cyanide, the solution of apohemocyanin was treated with an anion exchanger (Amberlite IRA-400); thereby the reconstitution was decreased but not abolished (Fig. lb). After treating the apohemocyanin solution with 1 r&I zinc acetate, followed by a treatment with 10 mM ethylenediaminetetraacetic acid (EDTA), and removing both by dialysis, the reconstitution with copper@) ions was completely blocked (Fig. lc). Addition of small amounts of cyanide to the reaction mixture favored the reconstitution shown in Figure 2. At 150 &_Mcyanide, however, a secondary reaction was observed; the copper band reached a maximum after about 2 days. The decrease differed from the formation of methemocyanin [7] as there was no regeneration with redxing agents. On Figure 3 are plotted the final values of the copper band and of the copper content after 10 days reconstitution of apohemocyanin in air at pH 5.7 with copper(D) ions in the presence of increasing amounts of ad.ded cyanide. After that time the reagents were removed by dialysis and the soIutions treated with EDTA, which also was removed by dialysis. About 70% of the original amount of copper could be reintroduced in H. pomariiz hemocyanin, but only part of this could bind oxygen reversibly_ This oxygen binding capacity, characterized by the absorption band at 346 nm, decreased in the region between 40 and 300 fl cyanide, most likely due to the abovementioned SecGndary reaction.

240

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I

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FIGURE 2. Reconstitution of H. pomtia hemocyanin (3 g/liter) in 0.1 M sodium acetate buffer, pH 5.7, in the presence of CL3 mM copper sulfate and of increasing concentrations of potassium cyanide: (a), 0. (b) 20. (c) 60, and (d) 150 JM_

Reconstitution with Cyanocuprate(1) under Nitrogen If traces of cyanide are unable to remove copper from hemocyanin plus cyanide form a complex abIe to reconstitute hemocyanin,

and if copper(II)

it must be possible to

use cya3ocuprate(I) directly_ The complex CI$XU)~~is soluble in water and disnciates into Cu(CN)a 2-, Cu(CN)z-, and CN--. At pH 5.7, where the reconstitution

FIGURE 3. F&l vaIue of the copper-oxygep band at 346 nm (0) and of the copper content (e) after 10 days of reconstitution of H_ pomatia apohemc-yanin (3 g/hter) in air in O-1 hf sodium acetate buffer, pH 5-7. at room temperature, in the presence of 0.3 n&I copper sulfate and of increasing concentrations oi po’zssium cyanide.

0

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l

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100

200

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241

with Cyanocuprate(1)

TABLE 1. Specific Absorption Coefficient at 346 nm and Percentage of Copper of Fresh and Aged Hemocyanin Preparations (9.5 mg/ml; 34 @S 0~)~ H. pomam Hemocyanin

Native Hemocyanin =346nm

%Cd

Fresh

0.272

0.235

id.regener. Aged id.regener.

0.334 0.204 0.319

id. id. id.

Apohemocyti %Cu

Reconstituted =346nm

Regenerated =346nm

Wh.l

0.030

0.231

0.236

0.291

0.025 0.039 0.031

0.220 0.230 0.227

0.234 0.235 0253

0.294 0.285 C-281

o Regeneration was performed in the presence of 0.34 mhl hydroxylammonium reconstitution in the presence of 69 PM cyanocuprate(i) under nitrogen.

chloride and

reactions were carried out, CUBseems the main species [8]. The reconstitution with this complex was very effective using small amounts of the reagent in the absence of oxygen [6] _ When copper was removed either from fresh hemocyanin or from an aged preparation, characterized by a lower specific absorption coefficient at 346 mn, a reconstitution of up to 74% could be obtained whether or not the initial starting material was regenerated with hydroxylamine (Table 1). As can be concluded from these data, the copper content was completely restored but part of the copper groups were in the oxidized form and unable to bind oxygen. Regeneration of this fraction with hydroxylamine resulted in an almost complete reconstitution (94%) of the copper band. The efficiency of these reconstitutions was also tested by taking circular dichroic spectra and

FIGURE 4. Specific absorption coefftcient at 346 nm of H. pomatita oxyhemacyanin (1 g/liter) at room temperature in the presence of 0.36 mM cyanocuprate(I) in 0.1 M acetate buffer, pH 5.7,under 1 atmosphere of nitrogen (o), air (e), or oxygen (0). Experimental points were measured after saturation of samples of the solutions with air.

0.25

15

h

20

R_ Witters et al.

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oxygenation curves, whereby no significant differences were found compared original hemocyanin preparations.

to the

Study of the Secondary Reaction Observed with Cyanocuprate@) in Air When a reconstitution with cyanocuprate(I) at pH 5.7 was performed in air the fast increase of the copper band at 346 run was followed by a slow decrease_ The copper band did not reappear when the reagent was removed by dialysis, nor when hydroxylamine was added in order to regenerate hemocyanin [6 ] _The same effect was observed in the reactirn of oxyhemocyanin with cyanocuprate(I) in air (Fig. 4) To acceIerate the reaction, an amount of cyanocuprate(1) ten times the amount of copper present in hemocyanin was added to hemocyanin solutions of about 1 mg/rnl. There was no marked difference between experiments in air and under 1 atm of oxygen; under these conditions the percentage of oxyhemocyanin in the solution amounted to 97 and 100, respectively. Under nitrogen, there was no secondary reaction at all (Fig. 4) This suggests an essential role of oxygen in the secondary reaction and the formation of an intermediate product that irreversibly destroyed the oxygen binding capacity of the copper group, as it could not be regenerated with hydroxylamine nor with hydrogen peroxide. When cyanocupratt$I) reacts with dioxygen in solution a superoxide anion might be formed that easily dismutates to dioxygen and hydrogen peroxide_ Analogous to the Fenton reaction, hydroxyl radicals could be formed in the reduction of hydrogen peroxide with copper(I)_ These hydroxyl radicals could be responsrble for some essential damage of the active site in ii. pomatia hemocyanin. To verify this hypothesis several additions were made. There was no influence on the course of the reaction of copper penicillamine (4.6 nmol/ml) nor of catalase (7 &ml). Probably catalase was itself inhibited by the presence of cyanide ions in the solution_ Dimethyl sulfoxide (DMSO), S,S-dirnethyl-l-pyrreline N-oxide (DMPO) (40 n&l), mamritol (S%), and _rV-acetyltyrosinamide (5 mM) were used as scavengers for hydroxyl radicals_ Only DMSO inhiiited the reaction (Fig. 4). This inhiiition, however, was not directly proportional to the concentration of DMSO in the range from 5 to 15%; higher concentrations (20%) showed no further increase of the inhibitory effect. According to McCord and FriZovich some unidentified impurity in DMSO is responsrble for the scavenging effect [9] ; therefore, DMSO was treated according to these authors with a small amount of HaOar distilled under vacuum, and used in another experiment: no inhibitory effect was noticed then. In conclusion, only the addition of DMSO resulted in an inhibition of the secondary reaction_ This suggests the formation of hydroxy1 radicals in situ at the active site of hemocyanin in the reaction of cyanocuprate(I) with oxygen bound as peroxide to the copper group. The active substance in DMSO, probably dimethyl sulfide, might reach the active site and react with the hydroxyl radicals. The other products are too large to penetrate into the protein, they can only scavenge hydroxyl radicals formed in solution. In order to characterize the fmal product, amino acid analyses, epr, absorption, and circuhu dichroic measurements were carried out on a concentrated hemocyarrin solution (I l-5 mglml) after 24 hr reaction in the presence of 0.4 r&l cyanocuprate(I) in O-1 M acetate buffer, pH 5_7_ Computed from the absorption at 346 run, 29% of oxyhemocyanin was left. No difference in amino acid composition, including tryptophan,

Reconstitution of Hemocyanh

with Cyanocuprate(1)

243

was found compared to oxyhemocyanin, although by circular dichroic measurements considerable differences were observed in the region between 250 and 300 mn, in opposition to the great similarity of the second derivative of the absorption spectra in that region. The copper content did not change, but 34% of the copper present in hemocyanin became epr detectable as mononuclear copper(

CONCLUSIONS The reconstitution of Helix pomati hemocyanin with an amount of copper(H) twice that originally present was greatly enhanced by the addition of small amounts of cyanide. Under nitrogen a reconstitution up to 94% was obtained at pH 5.7 with cyanocuprate in a ratio of two to one of the original amount of copper. The secondary reaction, observed with cyanocuprate(I) in air, whereby the active site is destroyed irreversr%ly, seems to be due to a direct reaction of cyanocuprate(1) with the bound peroxide at the active site. A formation of hydroxyl radicals in solution in the presence of oxygen cannot account for the specificity of the reaction_ Moreover, there was only a slight inhrbition by mannitol and a very slight inhibition by N-acetyltyrosinamide. The inhibition by dimethyl sulfoxide was abolished by a treatment with HaOa according to McCord and Fridovich [9]. We suspect the elimination of traces of dimethyl sulfide. This inhibition was not proportional with concentration, which could indicate a saturation of the binding site. The authors wish to thank IWE M Debroye and Mr. P. Hendrir for their experimental contribution. i%ey are indebred to the lnstituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en L-andbouw for graduate felIowships (to D-G_ and P-J_) and the Fends voor ColIectief Fundamenteel Onderzoek [Contract No. 20016.76). They are gratefil to Roj? H. A_ 0. Hill, Oxford, England. for suggesting the possibility of a reaction of cyanocuprate(I) at the active site of oxyhemocyanin and to Dr. E. Len&elder. Munich, F.R.G., for the sample of copper penicillamine.

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Dekker, New York. 1974. Vol. 3, Chap. 6.

7. R. witters and R. Lontie, FESS Letr. 60,400 (1975). 8. J. H. Baxendale and D. T. Westc0tt.L Chem Sot. 2347 (1959). 9. 1. M. MeCord and I. Fridovich./. Biol. Chem. 244,6056 (1969). Received November

18, 2 980; accepred June 22.198I