Quantitative electron spin resonance studies on native and denatured ceruloplasmin and laccase

J. Mol. Biol. (1962) 5, 301-310

Quantitative Electron Spin Resonance Studies on Native and Denatured Ceruloplasmin and Laccase LARS BROMAN, Bo

G.

MALMSTROM

I nstitute of Biochemistry, University of Uippsala, Uppsola, Sweden ROLAND AASA AND TORE VANNGMtD

Institute of Physics, University of Uppsala, Uppsala, Sweden (Received 24 May 1962) The effect of urea denaturation on the visible absorption, elec t r on spin resonance spectrum and oxidase a ctivity of ceruloplasmin has be en investigated. Some m easurements of the effect on optical rotation and ultraviolet absorption have also b een p erformed. The denaturation destroys the specific bonding of Cu 2 +, ch aracter ist ic for native ceruloplasmin as evidenced by electron spin resonance spectra. The loss of color a n d oxidase activit y follows t he same time-course and concen t ration d ep endence as t he change in bonding, wh ile com p a ra b le cha nges in opticalrotation and ultra viol et difference spect ra require more drastic denaturation. These findings indicate that the str on g vi sible a bso rption is r elat ed to t he spe cific bonding of Cu 2 +, and that this bonding is n ecessary for ca t alytic activit y . With bo th cerulop las m in and laccase, the Cu 2 + si gn al of the native protein only corresponds to a bout 50% of the total copper content, as determined both by chemical anal ysis and by m easurement s of t he electro n sp in re sonance signal intensities after relea se and oxidation of prot ein-bound copper wi th p erohlorie acid. The po ssibilit y tha t t he sp ecific b onding m a y in volve an in teraction b etween Cu 2 + and Cu 1 + is discussed.

1. Introduction Previous electron spin resonance (ESR) studies on copper proteins and mod el complexes (Malmstrom & Vanngard, 1960) have shown that two proteins with oxidase activity, ceruloplasmin and laccase, contain Cu2+ bonded in a unique manner. These proteins also have unusually strong blue colors (extinction coefficients of the order of loa liter mole-1 em-I; see, for example, Dawson, 1960; Williams, 1962). It has been suggested (Williams, 1961, 1962) that most intensely blue copper complexes contain Cu1+. In hemocyanin, for example, the intense color is probably du e to a Cu1+ . 02 charge transfer complex (Orgel, 1958 ; Nakamura & Mason, 1960). Thus, the que stion arises whether the strong visible absorpt ion of ceruloplasmin and lac case is actually du e to the Cu2+ pr esent in these proteins , as demonstrated by the ESR t echnique. It would appear important to answer this qu estion for at least two reasons. Firstly, analysis of the bonding involves the assumpt ion that the color is due to the 002+ ion (see, for example, Malm strom & Vanngard, 1960). Secondly, measur ements of the visible absorpt ion would offer a convenient method of following t he reduction of the metal, and this approa ch ha s already been used for the determinetion of the redox potential ofla ccase copper (Nakamura , 1958a). 301

302

BROMAN, MALMSTROM, AASA AND VANNGARD

The finding that the unique ESR spectrum is destroyed on denaturation of the protein (Malmstrom & Vanngard, 1960) offers a means to investigate this problem. We will here present results of experiments in which the effects of urea denaturation on visible absorption, ESR spectrum and oxidase activity of ceruloplasmin are correlated. In addition, the amount of Cu2+ present in native ceruloplasmin and laccase, as estimated from measurements of the ESR signal intensities, has been compared with the total amount of copper found by chemical analysis and by measurements of the ESR signal intensities after the protein-bound copper had been dissociated and oxidized with perchloric acid.

2. Materials and Methods (a) Proteins

Preparations of human ceruloplasmin of 60 to 90% purity, prepared from Cohn fractions IV-l and III by an acetone extraction method, developed at the Research Department of AB Kabi, were obtained from AB Kabi, Stockholm, Sweden. We are indebted to Mr. H. Bjorling for generous gifts of this material. A further purification was achieved by chromatography on hydroxylapatite by a procedure described previously (Broman, 1958, 1961). With gradient elution, an inactive peak as well as two or three peaks of ceruloplasmin were obtained; the ceruloplasmin peaks represent different forms of the enzyme, designated ceruloplasmin I, IIa and Hb, respectively (cf. Broman, 1961). In most experiments, material from the first peak, constituting about 80% of the total ceruloplasmin, was used, but some measurements on material from the other peaks have also been performed (see Results). In some experiments, only the inactive material was removed, and a mixture of all three forms of ceruloplasmin was employed. The ratio of the absorbancies at 610 miL and 280 miL was> 0·04 across the portions of the peaks employed, indicating a high degree of purity of the material (cf. Curzon & Vallet, 1960). The solutions from the chromatographic experiments were concentrated according to Hofsten & Falkbring (1960) or, with small volumes, by ultrafiltration through collodion membranes (Membranen-Gesellschaft, Gottdngen, West Germany) according to Mies (1953), to give a final protein concentration of about 8 x 10-4 M, as determined by copper analysis on the basis of a copper content of 0·34% and a molecular weight of 151,000 (see Laurell, 1960). The concentrated solution was dialysed for about 24 hr at 4°C against about 10 times its volume of sodium phosphate buffer, pH 6,8, sometimes including 0·01 M-NaCI, which was found to stabilize the protein; in some cases EDTA was also included in the buffer in order to remove extraneous copper (Broman, 1958). The concentrations of buffer and EDTA varied in the separate experiments, and details are given under Results. Fungal laccase was prepared by a modification (Mosbach, in preparation; we are indebted to Mr. Mosbach for his assistance in the purification work) of the procedure described previously (Malmstrom, Fahraeus & Mosbaoh, 1958); the modification consists mainly in the introduction of a chromatographic step on hydroxylapatite to remove a pigment with absorption maximum at 405 miL (cf. Malmstrom, Mosbach & Viinngard, 1959). For the ESR experiments, the purified protein was concentrated in the same way as described for ceruloplasmin. (b) Reagents Deionized water was -used in making up all solutions. Buffer solutions were purified by dithizone extraction as described earlier (Malmstrom, 1953). Reagent-grade urea was recrystallized in water before use. Reagent-grade copper salts and perchloric acid were used without further purification. (c) Copper analyses

Acid extraction, usually employed for the determination of metals in enzymes in this laboratory (Malmstrom, 1956), was found to give too low values of copper content when the dithizone method (Sandell, 1959) was used, so that with this method wet digestion was necessary. However, most copper determinations were carried out by the determination

ELECTRON SPIN RESONANCE OF COPPER PROTEINS

303

of Cu 1 + with 2,2'-biquinoline (British Drug House, Ltd.) after reduction of the Cu 2 +, essentially as described by Felsenfeld (1960). The sample, containing 1 to 10 f'g of copper (in less than 2 ml.), was pipetted into a graduated test tube, and 1 mi. of an aqueous solution of hydroquinone (2 mg/ml.) was added. After 5 min, 2 mi. of biquinoline reagent in glacial acetic acid (0,5 mg/ml.) was added, and the sample was diluted to 5 ml. with glacial acetic acid-water (2 : 1). After 10 min, the absorption at 540 mf' was measured in a Zeiss PMQ II spectrophotometer with 2 em cells. A series of standards, diluted from a stock solution obtained by dissolving copper metal in HN0 3 , was always analysed in parallel. Standard curves determined with Cu 2 + or with Cu 1 + salts were identical within limits of error. The use of cysteine hydrochloride or ascorbic acid, instead of hydroquinone, as the reducing agent gave the same results. With laccase, ascorbic acid was used, since with hydroquinone an interfering color sometimes developed. The results with the biquinoline method agreed with those obtained by the dithizone method after wet digestion. (d) Activity measurements, visible spectra and optical rotation The activity was measured spectrophotometrically at 25°C with N,N-dimethyl-pphenylenediamine as substrate (cf. Broman, 1958), and calculated as the change in the absorbancy per minute at 323 mf' in a 1 em cell; in general, the absorbancy increase was linear with time during the period of measurement. A Beckman DU spectrophotometer, provided with thermospacers and coupled to a potentiometric recorder, was employed, and 25 or 50 f'1. of enzyme solution was added to 3 ml. of substrate mixture with a plastic plunger. The composition of the substrate mixture was the same as described previously (Broman, 1958). In the experiments with urea denaturation, no urea was included in the substrate since no regain of activity on dilution could be observed. Visible spectra and ultraviolet difference spectra were recorded at 22°C in a Zeiss RRQ20A recording spectrophotometer. This photometer, with the drum connected to a slow constant-speed motor, was also used for continuous recording of the absorption at 610 mf' in denaturation experiments. The optical rotation at 326 mf' was measured in a recording Rudolph spectropolarimeter with a 5 em cell. (e) ESR measurements A Varian V-4500 3 cm spectrometer with 100 kc/s field modulation was used. All spectra were recorded at liquid nitrogen temperature with the tip of the Dewar flask inside the cavity. The microwave frequency was close to 9170 Mc/s. The sample quartz tubes were calibrated at the same temperature with standard solutions of Cu 2 + complexes. The constancy of the cavity Q-value with different samples of similar dielectric properties was checked by placing a small amount of a,a-diphenyl.,8-picrylhydrazyl (DPPH) on the outer Dewar wall. No saturation effects were observed. The integrated ESR absorption intensity was calculated from the first moment of the absorption derivative. Cu 2 + complexes with ligands such as histidine and EDTA (see Malmstrom & Vanngard, 1960) were used as standards. The transition probability depends on the g-value (Bleaney, 1960), and appropriate correction factors were, therefore, applied (Aasa & Vanngard. to be published). For the substances used in this study, these factors differ at the most by about 7%. The measurements of the intensities of the absorption characteristic for native ceruloplasmin (A in Fig. 1) in experiments with partial denaturation (see Results) are rather difficult. One cannot use the total integrated absorption, as the spectrum from the most fully denatured protein (B in Fig. 1) overlaps spectrum A. However, the g-values and hyperfine constants of the two signals differ from each other, as seen in Fig. 1. A characteristic part of the hyperfine structure of spectrum A, between the vertical lines in Fig. 1, appears in a region of the magnetic field where the derivative of signal B is approximately a linear function of the magnetic field. The widths of the hyperfine lines of signal A do not depend on the degree of denaturation, and, therefore, the distance a in Fig. 1 gives a measure of the intensity of signal A. The actual measurements were carried out with greater amplification, and the relevant part of the hyperfine structure in a native and a partially denatured sample is illustrated in Fig. 2.

304

BRO l\fAN , MALM S TROM , . A A S A AND VANN GARD

A

Increasing field B

400 gauss

FIG. 1. ESR spectra of frozen solutions of native (A) and urea denatured (B) ceruloplasmin (concn. 5 x 10- 0 sr) in 0·02 5 M-ph osph ate buffer, pH 6· 8; the solution of den atured protein contains 7·3 M-u re a. The distance a was u sed as a measure of the inten sity of signal A in the d enaturation experiments.

.

Increasing field

FIG. 2. Part of the hyperfine st ruct ure of native (I) and partially denatured (II) ceruloplasmin, recorded with greater amplification than in Fig. 1. The protein (concn. 5 x 10-0 M) was d issolved in 0·0 25 M-phosphate buffer, p H 6·8 ; the denaturation had been carried out for 40 min at 26°C in 3·6 M-urea.

ELECTRON SPIN RESONANCE OF COPPER PROTE INS

305

3. Results The effect of urea denaturation on t he visible ab sorption, oxidase activity and ESR absorption of ceru loplasmin is illustrated in Figs. 3 and 4. Since, at low urea concentrations, the denaturation is relatively slow, the change with time in the parameters concerned could be measured at a constant urea concentration, as shown for 3·6 xr-urea in Fig. 3. The protein used in this experiment had been dialysed

I\O { I

x xx\ 90

100

~

80

\..

~ ~

70 'c OJ

z-

f;

:0

~

~,

60

~o

x~

50

0

0

o

40

o

D

W

~

40

~

~

m

M

W

Time (min)

FIG. 3. The effect of 3·6 M·urea at 26 °C on the absorbency at 610 mp. (0), the distance a (Fig. 1) X 10- 5 M) in 0·025 M-phosphate buffer, pH 6,8, containing 0-001 M·EDTA. The experimental values of all three parameters are expressed in arbitrary units and p lotted on a logarithmic scale. ( 6. ) and the activity ( X ) of ceruloplasmin (concn. 5

against 0·025 sr-phosphate buffer, pH 6,8, containing 0·001 M-EDTA. The absorption at 610 m/-, was followed continuously at 26°C, and at given intervals samples were removed for activity and E SR measure ments. The denaturation reaction was quenched by fre ezing these samples in liquid nitrogen. Two 50-/-'1. por ti ons were used for the activit y measurements, and these were assayed wit hin 1 minute afte r t hawing; the freezing and t hawing did not appreciably affect t he activity. The samples for the ESR measurements were pipet t ed directly into t he ESR sample t ubes and kep t in liquid nitrogen until analysed in the ESR spectrometer. The effect of urea concentration on t he same three parameters is shown in Fig. 4. The protein used in this experiment had been dialysed against 0·1 III-phosphate buffer, pH 6,8, containing O·OIM-NaC1. The ceruloplasmin solutions with varying urea concentrations were allowed to stand for 24 hours at 22°C before the measurements, since no appreciable further change in visible absorption occurred with longer times. The spectrum of each solution was recorded in the wavelength region 500 to 800 uu» with the same concentration of protein in 7·3 M-urea as blank, after which equal portions were pipetted into the ESR sample tubes and frozen in liquid nitrogen. The activities of all solutions were determined with 25 and 50 /-'1. sam ples within approximately 1 hour.

306

BROMAN, MALMSTROM, AASA AND V ANNGARD

No signal with a hyperfine structure splitting intermediate between the splittings of signals A and B has been detected in the experiments with urea denaturation. The effect of urea denaturation on the optical rotation and ultraviolet absorption was also measured. The time course of the change in optical rotation (ex) at two different urea concentrations is compared with the change in visible absorption in Table 1. The protein concentration and other conditions were comparable with those 110,....-----,.---,.----,----,-----,.-----,

o

0.5

1.5

1.0

2.0

2.5

Urea cone. (M)

FIG. 4. The effect of urea concentration after 24 hr at 22°C on the absorbancy at 610 mp. (0), the distance a (Fig. 1) (6) and the activity (X) of ceruloplasmin (concn. 5 x 10-6 M) in 0·10 Mphosphate buffer, pH 6,8, containing 0·01 M-NaCI. The experimental values of all three parameters are expressed in arbitrary units. TABLE

1

The effect of urea at 2rC on the optical rotation (ex) and absorbancy at 610 mo: (AG10 ) of 6·6 X 10-5 u-ceruloplaemim in 0·025 M-phosphate buffer, pH 6·8

Ureaconcn. (M)

Time (min)

-lX

(degrees)

Ano (1 em celI)

Change in % of maximum change (7,3 M-urea) lX

A. I 0

3·1 3·1 3·1 3·1 3·1 3·1 3·1

0 2 29 49 72 152 182

0·260 0·260 0·274 0·280 0·284 0·292 0·294

0·68 0·65 0·50 0·45 0·42 0·36 0·35

0 9 13 16 21 23

4 26 34 38 47 49

3·64 3·64 3·64 3·64 3·64

0 2 7 12 185

0·260 0·280 0·286 0·292 0·340

0·680 0·538 0·470 0·422 0·190

13 17 21 53

21 31 38 72

ELECTRON SPIN RESONANCE OF COPPER PROTEINS

307

in the experiment of Fig. 3, the main difference being the higher temperature and the exclusion of NaCl. Without urea an optical rotation of - 0·260° was found, and this changed to -0'410° in 7·3 M-urea. The difference spectrum in the region 250 to 310 uu« of ceruloplasmin against the urea-treated protein was recorded at a number of urea concentrations. The protein and urea concentrations used as well as other conditions were the same as those in the experiment of Fig. 4, but also some higher urea concentrations were included. Due to the high protein concentration, a 0·2 em light-path was used; with this a slit width of less than 0·4 mm was obtained in the whole wavelength region when the spectrophotometer was operated at high sensitivity. The difference spectra showed two main peaks at 287 and 292 mp" respectively, the former being the largest. Below a urea concentration of 2 M no detectable change occurred, but the absorbancy difference at 287 mp, at some higher urea concentrations is given in Table 2.

TAIlLE

2

The effect of urea concentration after 24 hr at 22°C on the absorbancy at 287 nu: of ceruloplasmin (concn. 5 x 10-s M) in 0·10 u-phosphate buffer, pH 6·8, containing 0·01 M-NaCl

Urea concn. (M)

2·5 3'0 5·0 7·3

Absorbancy difference, native V8. urea-treated protein (0,2 em Iight-path)

<0·002 0·002 0·074 0·123

t Read from the curve in t

Change in % of maximum change (7,3 M-urea) Absorbancy difference

< 1·6 1·6 60 100

Parameter", in Fig. 4

36t 62 t >93~

100

Fig. 4.

This value was obtained with 4·36 M·urea.

Estimations, from the ESR intensities, of the Cu2+ content of native and perchloric acid-treated ceruloplasmin and laccase are compared with results from chemical analysis in Table 3. The protein samples had been dialysed against 0·10 Mphosphate buffer, pH 6'8, containing 0·005 M-EDTA, but EDTA was not included in the case oflaccase; before the experiment, the EDTA was removed by dialysis against the same buffer without EDTA. The perchloric acid-treated samples were obtained by mixing the samples with equal volumes of 70 to 72% perchloric acid and warming on a water bath to about 35°0. This gives a clear, slightly yellowish-green solution, which becomes cloudy reversibly on cooling to room temperature. In the signals from the native proteins we often found superimposed weak signals similar to those obtained from the urea-denatured proteins. Also proteins reduced with a large excess of substrate showed the same type of signal. Its intensity corresponded at the most to 10% of the total copper content, and it has not been subtracted from the values given in Table 3 (see Discussion).

308

BROMAN, MALMSTROM, AASA AND VANNGARD TABLE

3

Concentrations tm-molari of copper in native and perchloric acid-treated ceruloplasmin and laccase, corresponding to the observed ESR signals and obtained by chemical analysis Laccase

Ceruloplasmin Experimental parameter

ESR signal of native protein ESR signal of perchloric acid-treated protein] Total Cu (chemical analysis) Ratios native/acid-treated native/total

t

Kabi

I

lIa

lIb

A

B

0·99

2·96

0·84

0·77

0·90

0·46

1·74 1·92

5·39 6·60

3·86

1·37 1·68

2·10 2·10

0·87 1·08

0·57 0·52

0·55 0·45

0·48

0·56 0·46

0·43 0·43

0·53 0·43

Values corrected for dilution of sample.

4. Discussion The unusual bonding of Cu2+ in ceruloplasmin and laccase is evidenced by their low hyperfine structure constants (Malmstrom & Vanngard, 1960). However, on drastic denaturation the hyperfine structure splitting is increased, as seen from spectrum B in Fig. 1. While in progressive denaturation no intermediates between signals A and B are detected, the facts that a straight line is not obtained in the logarithmic diagram of Fig. 3 and that a threshold concentration of urea is necessary to get appreciable change in the parameters of Fig. 4 indicate that the part of the denaturation process affecting the parameters studied in these experiments involves at least one intermediate. It would thus appear that, when a certain amount of conformational change in the protein molecule has been induced, there is an abrupt transition in the bonding of Cu2+ from the state giving signal A to that giving signal B. The results given in Figs. 3 and 4 show that the same degree of denaturation as causes this transition also leads to a loss of color and of catalytic activity. Since protein denaturation is a complex process, different properties affected by it would not be expected to show the same concentration dependence or time course. For example, urea often inactivates enzymes at concentrations which cause little change in optical rotation (see, for example, Hill, Schwartz & Smith, 1959). With ceruloplasmin, the data of Tables 1 and 2 show that urea can markedly affect visible absorption and Cu2 + bonding at conditions which cause significantly smaller changes in optical rotation and little or no change in absorption at 287 txu». Thus, the fact that the three parameters in Figs. 3 and 4 fall on the same curves, within limits of error, makes it appear very likely that the strong visible absorption of ceruloplasmin is related to the uniquely bonded Cu2 + ion, and, furthermore, that the unique bonding is necessary for catalytic activity. Similar experiments have not been performed with laccase, mainly due to shortage of purified enzyme, but the great similarity in resonance and bonding parameters of the two proteins, both in the native and denatured state (see Malmstrom & Vanngard, 1960), makes it probable that the state of copper is the same in the two cases.

ELE CTRON SP I N R E SO NAN CE OF C O P P E R PROTEIN S

309

Whil e t hus both color and activity appear related to t he Cu2+ ion resp onsible for the E SR signal, the experiments summa rized in Tabl e 3 show t hat this signal only accounts for about 50 % of t he to t al copper content, as determ ined both by chemical analysis and by E SR mea surements. The deviati ons from a ra t io of 0·5 mu st be considered within the limits of error . The un certainty is particularly great for t he a cid-treated samples since t hese did not give ent irely repr odu cible values and the t yp e of signal obtained differed in separa te experiments, indicating t hat complete removal and oxida t ion of the met al were not achi eved . The use of acetic a cid for the lib eration of t he met al ga ve even lower values. Anot her uncertainty arises from t he residual signa l in the fully redu ced enzyme (see R esults), but correction for th is would decrease t he ratios by at t he most 11 %. Furtherm ore, it is likely that this signa l originat es from denatured pr otein molecules, and that the denaturation does not chan ge the intensity very mu ch. In such a case, the residual signa l will not affect the ratios given in Table 3. The data of Table 3 show t ha t all copper atoms are not equivalent in ceruloplasmin and laccase. It is, therefore, interesting that, in the case of cerul oplasmin, Scheinberg & Morell (1957) have shown that only half the copp er is readily exchangeable, and Curz on (1958) has found that only half the copp er becomes dialysable on chyme tryptic digestion. Whil e line broadening may somet imes prevent the det ection of E SR signals fr om param agnetic ions, the Cu2+ ion almost always gives rather narrow signals so that ESR measurements should give a good estimate of th e Cu2+ conce ntration . Thus, the data of Table 3 indicat e tha t both ceruloplasmin and la ccase contain abo ut 50 % of the t otal copp er as Cul+. This ha s been confirm ed by magnetic susceptibility measurements (carried out in collaboration with Dr. A. Ehrenberg ; a detailed report will be published jointly with him). This finding is in cont ras t to results of Nak amura (1958b), which indicate t hat all copper is in the form of Cu2+ in the la cquer-tree enzyme. The presence of appr oximately equimolar concentrations of Cu2+ and Cti 1+ may suggest that the bonding of Cu2+ in cerul oplasmin and laccase involves an interacti on between these tw o ions. H owever, the ESR spe ctra (Fig. 1) exclude the presence of a Cu~ + pair, i.e. one unpaired electron equally shared between tw o copper nu clei, since such a structure would be expected to give a more complicate d resonance st ruc t ure (cf. Bleaney & Bowers, 1952). It is hoped that further studies on modified proteins and on model complexes will permit a more detailed description of the bonding sit uat ion in these two proteins. This investigation was supported by grants from the Swedish N atural Science Research Cou ncil an d the Division of Gen er al Medical Scienc es, U .S. P ublic H ealth Service (R G· 6542·C2). A preliminary ac count of some of th e data was given at t he Int ernational Bi ophysics Congress, St ockholm, 1961. W e are indeb ted to Miss B odil Hid ing for assistanc e with t he cop p er anal ys es and t o Dr. A. R osenber g for m easuremen t s of op ti cal rotation in a R udolph spect rop ola r imeter. REFERENCES Bleaney , B. (1960). P roc, Phys. Soc . A, 75, 621. Bl eaney , B. & Bowers, K. D. (1952). P roc. R oy . S oc. A, 214 , 451. Broman, L. (1958) . Nature , 182, 1655. Broman, L. (1961). W ilson's Di sease, ed. b y J. M. W alsh e & J. N. Cu m ings, p. 69. Oxford: Blackwell Scientific P ublica t ions.

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Curzon, G. (1958). Nature, 181, 115. Curzon, G. & Vallet, L. (1960). Biochem, J. 74, 279. Dawson, C. R. (1960). Ann. N.Y. Acad. Sci. 88, 353. Felsenfeld, G. (1960). Arch. Biochem. Biophys. 87, 247. Hill, R. L., Schwartz, H. C. & Smith, E. L. (1959). J. Biol. Ohern, 234, 572. v. Hofsten, B. & Falkbring, S. O. (1960). Anal. Biochem, I, 436. Laurell, C. B. (1960). The Plasma Proteins, vol. 1, ed. byF. W. Putnam, p. 349. New York: Academic Press. Malmstrom, B. G. (1953). Arch. Biochem. Biophys. 46, 345. Malmstrom, B. G. (1956). Methods oj Biochemical Analysis, vol. 3, ed. by D. Glick, p. 327. New York: Interscience Publishers Inc. Malmstrom, B. G., Fahraeus, G. & Mosbach, R. (1958). Biochim. biophys. Acta, 28, 652. Malmstrom, B. G., Mosbach, R. & Vanngard, T. (1959). Nature, 183, 321. Malmstrom, B. G. & Vanngard, T. (1960). J. Mol. Biol. 2, US. Mies, H. J. (1953). Klin. Wochnschr. 31, 159. Nakamura, T. (1958a). Biochim. biophys. Acta, 30, 44. Nakamura, T. (1958b). Biochim. biophys. Acta, 30, 640; Nakamura, T. & Mason, H. S. (1960). Biochem. Biophys. Res. Comm. 3, 297. Orgel, L. E. (1958). Metals and Enzyme Activity, p. S. Cambridge: The University Press. Sandell, E. B. (1959). Colorimetric Determination oj Traces oj Metals, 3rd ed., p. 454. New York; Interscience Publishers Inc. Scheinberg, I. H. & Morell, A. G. (1957). J. Olin. Invest. 36, U93. Williams, R. J. P. (1961). Fed. Proc, 20 (suppl. 10), 69. Williams, R. J. P. (1962). Symposium IV oj the Vth International Congress oj Biochemistry, ed. by P. Desnuelle and A. E. Braunstein. London: Pergamon Press, in the press.