The Science of the Total Environment, 105 (~991) 41-59 Elsevier Science Publishers B.V., Amsterdam

41

Isolation and characterization of metal-binding proteins (metallothioneins) from lobster digestive gland (Homarus americanus) C.L. Chou"', R.D. G u y b and J.F. Uthe ~ "Marine Chemistry Divtswn, Physical and Chemical Sciences Branch, Scotia-Fundy Region, Department of Fisheries and Oceans, P.O. Box 550, Halifax, N.S. B3J 2S7, Canada hDepartment of Chemistry, Dalhousie University, Halifax, N.S. B3H 4J!, Canada (Received May 22nd, 1990; accepted August 7th, 1990)

ABSTRACT Two metailothionein (low-molecular-weight, metal-binding proteins) preparations, MT-I and MT-2, have been isolated from the digestive gland of American lobster (Homarus americanus) contaminated with Cd. MT-I contains Cd- and Cu-binding proteins, whereas MT-2 is a reasonably pure Cd-binding protein. The properties of MT-1 and MT-2 with respect to amino acid and elemental compositions, heat stabilities, polarographic, high-performance liquid chromatography (HPLC), and isoelectric focussing behaviors are reported. Lobster metallothioneins share a number of similarities with mammalian metallothioneins with respect to the presence of Cd and Cu, apparent molecular weights, amino acid compositions, UV absorption spectra at various pH, and polarographic behavior, but differ substantially in their electrophoretic behavior.

INTRODUCTION

High levels of Cd (500mgkg -~ wet wt) have been found in the digestive gland of the American lobster (Homarus amerieanus) caught in Belledune Harbour, New Brunswick, Canada (Uthe et al., 1980). The harbour is the site of a lead smelter which processes materials containing Cd as an impurity. Metallothioneins (metal-binding proteins) are responsible for the accumulation of toxic trace metals in animals (Kojima et al., 1976; Olafson et al., 1979; Overnell and Coombs, 1979). Metallothionein was first isolated by Margoshes and Vallee (1957), who showed that Cd was bound to protein. Metallothionein is a low-molecular-weight protein with high metal affinity, high cysteine content, heat stability, and lack of aromatic amino acids (Margoshes and Vallee, 1957; Kagi and Vallee, 1961; Weser et al., 1973). The inducible

0048-9697/91/$03.50

© 1991 - - Elsevier Science Publishers B.V.

42

C.L. CHOU ET AL.

nature of this protein suggests that metallothioneins play a role in the detoxification of heavy-metal ions (Piscator, 1964; Shaikh and Lacis, 1971; Squibb and Cousins, 1974). In this work, two lobster digestive gland metallothionein fractions were isolated and characterized. EXPERIMENTAL

Materials and methods

Whole digestive gland was removed from a live lob;ter and homogenized with three volumes of 3% NaCl + 0.1% NaH2PO4 + l0 -4 M 0e-toluenesulfonyl flouride (TSF, Sigma), pH 7.0, in a Potter-Elvehjem glass homogenizer with a PTFE pestle for 3 rain. The homogenate was centrifuged three times at 30000g for 30rain at +4°C and the final supernatant lyophilized. Approximately 1.0 g was dissolved in 15 ml of either 1 M ammonium acetate or 0.0 ! N sodium citrate/0 l N sodium formate (pH 7.8) containing l0 -4 M TSF and applied to a 2.5cm i.d. x 100cm long Fractogel HW-50 (EM Science) gelpermeation column. The isocratic elution was monitored by UV absorption at 254/280 nm (LKB Uvicord II) and atomic absorption :;~ectrophotometry (AAS). Fractions containing Cd and eluting in the medium-molecular-weight range were combined and dialyzed in Spectrum 3 dialysis tubing [cutoff limit, 3500 daltons (Da)] against H,O for 24h then concentrated in an Aminco Stirred Cell Concentrator. The material was applied to a Fractogel DEAE650M ion-exchange (EM Science) column (l.6cmi.d. x 28cm long). Proteins were eluted using a linear gradient slarting with H20 and ending with either 0.5 M sodium formate or 0.4 M Na2SO4 containing 0.1 M NaCl (both at pH 7.6). Two Cd-containing peaks, hereafter reii~rred to as MT-I and MT-2, were isolated and lyophilized. Absorption spectra (220-320nm) of MT-I and MT-2 were determined using a Unicam SP1750 spectrophotometer. lsoelectric focussing electrophoresis analysis of MT-I and MT-2 was carried out using a LKB Model 2217 Ultraphor Etectrofocussing Unit, 5% polyacrylamide cast and 2% ampholytes (LKB) (pH 3.5-10.0), at a constant current of 30mA until a final voltage of 1000 V was reached. The gels were stained with silver stain (Bio-Rad) after initial settling for l h in 30% methanol/10% trichloroacetic acid/3.5% sulphosalycylic acid and washing for at least 2 h in multiple changes of 30% methanol/10% trichloroacetic acid to remove ampholytes. The amino acid compositions of lyophilized MT-I and MT-2 were determined by the Hits (1967) performic acid decomposition method. High-performance liquid chromatographic analysis was carried out

ISOLATION AND ('HARACI'ERIZATION OF MI-TAL-BINDING PROTEINS

43

using an Aquapore RP-300 (Brownlee Labs, Santa Clara, CA), 7.0mmi.d. x 350mm long column and a Waters 510HPLC chromatography system, using a linear gradient of eluting buffers: (A) 0.05 M Tris, pH 7.6; (B) 0.05M Tris containing 60% acetonitrile, gradient 0-45% in 30min. Sampled-DC polarography of 2-mercaptoethanol (Kodak) of MT-1, Mq 2, and rabbit liver metallothionein (Sigma) was carried out with a Princeton Applied Research Model 174A Polarographic Analyzer at a 0.5 s drop time and 5 mV s-~ scan rate using a Ag/AgCI reference electrode. The polarograms were stored in an IBM computer. Differential pulse polarography was carried out with a Princeton Applied Research Model 174A at a potential scan rate of 2 m V s -~, a drop time of 2s, and a modulation of 50mV using a KCIbridged saturated calomel reference electrode. RESULTS AND DISCUSSION

Fractogei H W-50 gel permeation chromatography The 254 nm UV absorption profile (Fig. 1) shows the presence of at least eight major peaks (I-VIII, in order of elution). Peak | eluted at the void volume, as determined by Blue Dextran, whereas Peak V eluted at the inclusion volume, as determined by K ÷ elution. Peaks VI-VIII eluted after the inclusion volume and must have done so because of non-specific interaction with the column packing material. Cadmium eluted in a single peak in the medium-molecular-weight range, corresponding to a molecular weight of ~ 12 000 Da based upon the elution of Cytochrome C (tool. w~ 12400 Da). This fraction also contained significant amounts of Zn and Cu. A high-molecular-weight fraction containing both Zn and Cu, and a low-molecular-weight fraction containing Zn were also present. Copper was evenly distributed between the high- and mediummolecular-weight fractions, while Zn was present in approximately equal, and major amounts, in the medium- and low-molecular-weight fractions with only a small amount in the high-molecular-weight fraction. Silver eluted in the medium-molecular-weight fraction. Metallothioneins are heat stable (Cherian, 1974; Shaikh and Smith, 1975; Winge et al., 1975), an expected property of their low molecular weight and single polypeptide chain nature. Heating lobster digestive gland supernatant at 80°C for 10min was investigated. After centrifugation to remove coagulated material, the supernatant was subjected to gel-permeation chromatography (Fig. 2). All high-molecular-weight, metal-containing material was removed by heat treatment. Cadmium, Zn, Cu, and Ag were still present in the medium-molecular-weight material, al, hough the majority of the Zn

44

C,L. CHOU ET AL 300

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Fig. I. Fractogel H W - 5 0 e l u t i o n profile o f lobster digestive gland extract ( c o l u m n , 2 . 5 c m i . d .

long; eluent, ! M NH4OAc).

x 80¢m

was now present in the low-molecular-weight fraction, i.e. the region of unbcund catiol~ elution.

DEAE-650M ion-exchange chromatography The behavior of Cd 2÷ on Fractogel DEAE-650M was investigated. Five micrograms Cd 2÷ was eluted using various anions such as CI- and SO~- as

45

ISOLATION A N D C H A R A C T E R I Z A T I O N O F M E T A L - B I N D I N G PROTEINS

600 500 r,:E 'o

400 30o

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--7 MMW

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s~o

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_q 60 'E

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40

~. ~o > 0

100

200 300 400 soo Elution Volume (in mL)

600

Fig. 2. Fractogel HW-50 elution profile of lobster digestive gland extract in the presence of 10-4MTSF, following heat treatment at 80°C for 10rain.

0.5 M NaCI or NaCI/Na2SO4 mixtures. Cadmium(+ II) in 0.5 M NaCI was strongly adsorbed onto the gel matrix (Fig. 3, curve A) with increasing amounts being eluted as the proportion of SO~- in the eluting solution increased. The adsorption of Cd 2+ in the presence of Cl- is likely due to the conversion ofCd 2 + tO CdCl,_, (x > 2, n = x - 2), species which bind more strongly to the cationic DEAE groups than does CI-. Addition of SO~- and

~w-

C.L CH()IJ E l AL.

46

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2

"

4

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Retention Time (Minutes)

Fig. 3. Chromatogram of 5llg Cd:' ion in: (A) 0.5 M NaCI; (B) mixture o1"0.5 M NaCI/0.5 M (NFI4):SO4. 1: 2, v/v: (C) mixture of 0.5 M NaCI/0.5 M (NH4).,SO.~, 1"4, v/v, ,.,n a Fractogel DEAE-650M column (I.6cm i.d. × 14on( h, ng).

reduction of the CI- concentration in the eluent inhibit formation of CdCI,_, complexes. The elution of Cd -'+ by 0.5 M concentrations of NO3-, CO~-, HCO3-, PO:~-, S O 4 , formate, acetate, citrate, and tartrate (all as the Na salt) was investigated, as was elution by Tris/HCl (pH 7.0). Quantitative eiution was found in all cases except for Tris/HCl. Ion-exchange chromatography (Fig. 4) of the medium-molecular-weight fraction yielded two Cd-containing protein fractions, MT-1 and MT-2, which eluted at 250 and 500/~scm -~, respectively, with a linear gradient of H,O/ 0.25 M sodium formate (pH 7.6). Am#to acM and metal compositions

Both MT-I and MT-2 contain relatively high proportions of cysteine and are lacking in aromatic amino acids (Table 1). Differences exist in MT-I and MT-2 in that MT-I contains more glycine, alanine, valine, leucine, and arginine than MT-2, but less cysteine and serine. MT-I contains more Cu (60.68 mol%) than Cd (39.32mo1%) and is lacking in Zn (Table 1). In contrast, MT-2 contains a very high proportion of Cd (84.20 mol%), less Cu (! 3.32 tool%) and still less Zn (2.5 mol%) (Table I). The high ratio of Cu to Cd in MT-I and its lower cysteine content suggests either that the ,?action may be impure, e.g. containing a significant amount of Cu-containing metal-

47

ISOLATION AND CHARACTERIZATION OF METAL-BINDING PROTEINS 1.2 1.0.

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Fig. 4. Fractogel DEAE-650M chron'u:mgrams of desalted crude lobster digestive gland metallothionein isolated after gel permeation on Fractogel HW-50 (colunm, 1.6cm i.d. x 30cm long). Chromatography was performed at 0.5 ml min ' and elution with a linear gradient from distilled H,O to 0.25 M sodium forrnate (pH 7.8).

lothionein-like protein, or that MT-I is a Cu- and Cd-containing metallothionein-like protein which represents a different class of metal-binding, cysteine-rich proteins. It is known that certain Cu-thioneins are substantially

48

C.L. CHOUET AL.

TABLE 1

Amino acid composition (mol%) and metal content (moles) of MT-I and MT-2 from lobster digestive gland Amino acid

MT-I

MT-2

Cys Asp

19.9 i 8.97 8.02 7.54 9.13 1 !.39 8.45 2.82 0.50 2.59 2.44 ! 5.74 2.51

36. ! 5 7.90 7.38 10.03 8.16 3.30 5.55 0.82 0.49 2.19 1.3 i ! 5.58 1.14

Thr

Ser Giu Gly Ala

Val Met lie

Leu Lys Arg Cd MT-! MT-2

5.90 8.94

Cu (39.32) ~ (84.20)

9.10 !.41

Zn (60.68) (13.32)

0.26

(2.50)

~Mol% of metal in parentheses.

different from metallothionein. Studies of Cu-thionein from chicken (Rupp and Weser, 1974) showed a lower S/Cu ratio (2:1), as did Cu-thionein from yeast (Prinz and Weser, 1975), than the metallothionein. Premakumar et al. (1975) isolated a similar protein and called it Cu-chelatin. The composition of MT-2, on the other hand, agrees with that of mammalian metallothioneins, e.g. those from rat, rabbit, and horse (Nordberg et al., 1972; Winge et al., 1975: Kojima et al., 1976), i.e. a high cysteine content (25-35%), relatively high serine (7-17.5%) and lysine content (10-15%), and an absence of aromatic amino acids. MT-l contains 6 tool Cd, 9 tool Cu and no Zn for a total of 15 mol metal (Cd + Cu + Zn) per mole of protein, rounded to the nearest whole number. MT-2 contains 9 mol Cd and a total number of moles metal (Cd + Zn + Cu) of 11 (Table l). Compared with metallothioneins from horse, crab, and oyster (Table 2), MT-1 and MT-2 have higher metal contents per mole. Garner et al. (1982) reported that sheep liver metallothionein contained 5-6mol Zn per mole, while Otvos et al. (1982) showed that crab metallothionein binds 6 mol Zn per mole in two clusters, each containing three metal ions. Yeast metal-

49

ISOLATION AND CHARACTERIZATION OF METAL-BINDING PROTEINS

lothionein binds either eight Cu ÷ or four Cd 2+ (Winge et al., 1985). Neilson et al. (1985) reported that 12 atoms Cu bind to metallothionein in two clusters of six atoms each. The ratio of the sum of moles of metal (Cd + Zn + Cu) to the number of cysteinyl residues in MT-2 indicates that an average of three cysteinyl residues are involved in the binding of one metal ion, whereas the average ratio in MT-I is 1:1 (Table 2). The ratio for MT-2 is similar to that reported for horse liver and kidney metallothionein (Kojima et al., 1976). Olafson et al. (1979) reported approximately five cysteinyl residues per divalent metal ion in metallothionein from Scylla serrata, but also suggested the protein was not saturated with metal ions. Overnell (1982) found three cysteinyl residues per metal ion. Metal-binding protein from Crassostrea virginica (Ridlington and Fowler, 1979) contained substantial amounts of dicarboxylic amino acids, low cysteinyl and metal contents, and a cysteinyl/metal ratio of 5:1, thus differing from mammalian metallothionein. Obviously such results could also reflect an impure metallothionein preparation. In the case of MT-I, the low ratio may reflect the high Cu content. In a study of Cu-binding proteins from liver of rats pretreated with Cu 2+, Irons and Smith (1977) reported a cysteinyl/Cu catio of 1.5:1. A similarly low ratio was reported by various workers for fish liver metallothionein (Evans et al., 1975; Winge et al., 1975; Takeda and Shimizu, 1982).

TABLE 2 Moles of metal per molecule of metallothionein from horse liver and kidney, and crab, oyster, and lobster digestive glands Metal

Cd Cu Zn Total metal SH/TM°

Horse liver" 0.33 0.18 5.72 6.23 2.88 (3.1)r

Horse kidney~ 4.61 0.1 ! 2.55 7.27 2.85 (3.2)f

Crab (Scylla serrata)"

Crab (Cancer irroratus) ~

Oyster ( Crassostrea virginica) 't

2.28 0.28 0.21 2.70 5.26

9.4 0.2 0.21 I 1.5 2.85

1.14 0.29 !.9 145 5.24

Lobster MT- I

MT-2

5.9 9.10

8.94 1.42 0.26 10.6 2.85

~5.0 I. 18

"Kojima et al. (1976). bOlafsoe et al. (1979). cO~ernel3 (1982). dRidlington and Fowler (1979). eRatio of total SH/total metals, calculation based on the amino acid analysis. rDetermined by the para-chloromercuribenzoic acid method.

50

C.L CHOU ET AL.

UV absorption spectra MT-I (Fig. 5) possesses a concave curve from 220-320 nm, whereas MT-2 has a shoulder at about 254 nm. The 254•280 ratios were 3.5 and 14.2 for MT-1 and MF-2, respectively, with the higher ratio of MT-2 again reflecting a similarity with other purified metallothioneins (Kagi and Vallee, 1961; Nordberg et al., 1972). The different ratios may reflect the difference in Cd content between the two forms, since Kagi and Vallee (1961) have shown that it is the Cd-mercaptide bond which absorbs at 254 nm. Pulido et al. (1966) suggested that the absorption at 254 nm is due to a charge-transfer transition of Cd-mercaptide. Kagi and Vallee (1961) and Weser et al. (1973) reported that acidification destroys this Cd-mercaptide chromophore by displacing Cd 2+ . The absorption spectra of MT-2 at pH 7.0, 4.0 and 3.0, and MT-1 at pH 7.0, 3.0 and 2.0 are given in Figs 6 and 7, respectively. Increasing acid concentrations result in decreasing absorption at 254 nm, which, in the case of MT-2, essentially ceased by pH 3.0. Readjustment to neutrality restored the absorption at

0 . 2 0 AU

|

310

m

I

290

I

2

|

270

~

L

|

250

WAVELENGTH ( n m )

Fig. 5, Ultraviolet spectra of lobster MT-I and MT-2.

A

230

J

ISOLATION AND CHARA('TF.RIZATION OF MI'TAL-BINI)ING PROTI:INS

5]

MT-2

T 0 . 2 0 AU

°.,// pH 4.0

)H 3.0

300

280

260

WAVE LENGTH

240

2 20

( n m)

Fig. 6. Effect of pH on absorption spectrum of lobster MT-2.

254 nm. MT-I did not fully lose its 254 nm absorbance until the pH reached 2.0. Full absu, bant;e was regained when neutrality was restored.

lsoelectric./bcussing gel eh'ctrophoresis ( IEF) Over the ampholyte pH range 3.5-10.0, MT-I yielded two bands with isoelectric points (pl) of 9.85 and 10.1, while MT-2 showed a single band with a pl of 5.0 by isoelectric focussing electrophoresis (Fig. 8). The difference in isoelectric points between the preparations may be due to differences in metal and amino acid compositions. Pulido et al. (1966) found that the metal content was lower in one of their human kidney metallothionein components than in the other, and stated that this could explain the differences in electrophoretic mobility. Reported pl values of other metallothioneins are < 5.0. Two rabbit metallothioneins were reported by Nordberg et al. (1972) to have pl values of 3.9 and 4.5, while Cherian (1974) reported that the pl values for two rat liver metallothioneins were 4.2 and 4.7. Given the divergence charac-

52

C.L. CHOU ET AL.

f,~i -1

T 0.20

AU

L

;)H 7.0 pH 3.0 )H 2.0

310

2~O 270 WAVELENGTH ( n m )

250

2~O

Fig. 7. Effect of pH on absorption spectrum of lobster M T - I . 12

10

O G

Z

m

¢i. ca 8

G

• 'E

m

B STD Marker • MT-1 B MT-2

a rs

o,

",'0

3'o ,'o s'o 6'o ;o

Isoele©trofocusing

Reading (in mm)

Fig. 8. lsoelectric points of lobster MT-! and MT-2 versus standard markers (pl/~-lactoglobulin, 5.20; bovine carbonic anhydrase B, 5.95: human carbonic anhydrase B, 6.55; horse myoglobin, 6.85; horse myoglobin, 7.35; lentil lactin, 8.15; lentil lactin, 8.45; lentil lactin, 8.66; trypsinogen, 9.30; cytochromes,

I0.25),

terizing MT-I, the presence of two proteins (with similar molecular weights) with basic pl values, it is suggested that MT- 1 is comprised of partially altered metallothioneins or metaiiothioneins different from those found in most at!reals. Lobsters may also contain Cd-binding proteins which function in

ISOLATION AND CHARACTERIZATION OF METAL-BINDING PROTEINS

53

their normal physiology. It is known that Cd occurs naturally at surprisingly high concentrations in this species and that the species is remarkably tolerant to dietary Cd (Chou et al., 1987)

High-performance liquid chromatography It"can be seen (Fig. 9A, B) that MT-I was separated into three peaks by HPLC (method of Klauser et al., 1983), with a major component and two minor components, whereas MT-2 yielded one major and one minor peak. Multiple metallothioneins in metallothionein preparations isolated by "conventional" techniques have been reported (Nomiyama and Nomiyama, 1982; 0 In

(A)

MT-1 50

'O.05AU

i

4o

g so 2o

i

~ o

io

o t 0

~

, 4

'

!

!

8

I

I

12

EIution

Time

! 16

I

~ 20

I

i 24

I

i 28

,

i 32

(Minutes)

Fig. 9. Reversed-phase HPLC elution profile of lobster digesth,e gland: (A) MT-1; (B) MT-2 (see page 54), in 0.05 M Tris buffer, pH 7.6. Chromatography was performed at !.5 ml rain-i on an Aquapore RP-300 column (7.0 mm i.d. × 25 cm long) with a linear gradient formed between a starting buffer solution, 0.05 M Tris, and a limiting buffer solution, 0.0S M Tris, containing 60% acetonitrile, pH 7.6. The eluate was continuously monitored at 220 nm.

54

C.L. CHOU

El" A L .

(B)

MT-2

T O.05AU

50

J. 40

30

C O

_=

0 (/) m C

20

| - -"

I

0

I

4

I

i

8

I

I

12

I

I

I

16

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20

1 ....

J--

24

1[

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28

!*__1'

O

A

I 32

EluUon T i m (Minutes)

Fig. 9(B).

Klauser et al., 1983; Suzuki and Yajima, 1984; Waalkes and Perantoni, 1986). High-performance liquid chromatography yielded higher resolution than isoelectric focussing, although this may reflect the sensitivity of the two detection systems rather than separation efliciencies.

ISOLATION AND CHARACTERIZATION OF METAL-BINDING PROTEINS

55

Polarographic studies The MT-2 polarogram (Fig. 10) contained three major peaks, at -0.43, -0.782 and - l . 1 0 V . Titration of MT-2 with Cu 2+ produced increasing amounts of free Cd 2÷ ( - 0 . 6 0 V) and reduction in the peaks at - 0 . 4 3 aild --1.10V. Following EDTA addition, three peaks, at - 0 . 3 5 , -0.73, and - 1.20 V, were observed. The MT-I polarogram (not shown) yielded the same peaks as MT-2, suggesting that there is much similarity between them with regard to their polarographic behavior. Commercial rabbit metallothionein also gave the same results (Fig. 1l). The peak at - 0 . 4 3 V may be due to sulphydryl. Oxidized glutathione (G-S-S-G), for example, has a reduction peak at - 0 . 3 4 V at pH 4.0, and at

5 0 0 nAmp

i

r

!1'

•

I

I

-0.1

-0.3

-0.5

I

-0.7

i

-0.9

I

-1.1

~

I

-1.3

-1.5

Potential vs SCE (Volts)

Fig. 10. Differential pulse polarograms of lobster MT-2 (A) sample; (B) A + 2 x ; (C) A + 5 x ' of 2.0/~g Cu 2+ ion; (D) (C) + I mmol EDTA in l M (NH4)2SO4 (peak potentials (V): l, II, and Ill, -0.43, -0.78, and - 1.10; I', If', f i r , - 0 . 3 5 , -0.73, and - 1.20V, respectively).

56

C.L. CHOIJ ET AL.

It

T

\

A

A B

-0,1

-(13

-(15

-0.7

-G9

-1.1

-1.3

Potential ve 8CE ( V o l t l ) Fig. I !. DPP polarograms of rabbit metallothionein (A) sample; (B) A + 2 x ; (C) A + 5 x of2.5~g Cu "+ ion; (D) (C) + I mmol EDTA in 1 M (NH4),SO4 (peak potentials - - see Fig. 10).

- 0 . 6 6 V at pH 10.3 (Milner, 1957). Reduced glutathione (G-SH) has an anodic peak at - 0.37 V. Thioglycolate has an anodic peak at - 0.47 V at pH 8.0. Figure 12 presents the differential pulse and sampled-DC polarograms of 2-mercaptoethanol. The sampledoDC polarogram was recorded to confirm the electrochemical reactions occurring at the electrodes. Two peaks, at - 0 . 4 6 and - 0 . 5 8 V, were observed due to reaction at the mercury electrode. This indicates that the peak at - 0 . 4 6 V may involve the oxidation process 2RSH + Hg ~-Hg(SR)2 + 2H + + 2e~,i. the s~rface of the mercury electrode (Rabenstein and Saetre, 1977). The second peak at - 0 . 5 8 V may be due to the presence of a disulphide in the

57

ISOLATION A N D CHARACTERIZATION OF METAL-BINDING PROTEINS

10 8-

--

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DPP SAMPLED D e

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I-Z I,i n,* n,"

-~t', _,.000

, ......."""i .................... ,.................... _0.6oo _0.,o0 _0.~00 0.00o

_0.~00

POTENTIAL(VOLTSVS Ag/AgCI) Fig. 12. DPP and sampled-DC polarograms of 0.01 M 2-mercaptoethanol in 0.01 M acetic acid solution (pH 5.0).

surface of the electrode (Casassas et al., 1985). In contrast, rabbit metallothionein yielded two peaks, at - 0 . 4 5 and - 0 . 8 0 V (Fig. 13). The first peak was anodic, and similar to that of 2-mercaptoethanol, suggesting the oxidation of MT at the mercury electrode. However, the - 0.80 V peak had a corresponding cathodic current, which generally indicates an adsorption process. This peak is not completely understood. CONCLUSIONS

Metallothionein-type proteins occur in Cd-contaminated lobster digestive gland. Their high absorbance, particularly for MT-2, at 254 nm is characteris-

- - DPP ...... SAMPLED DC

0.500n

v

E o =I. 0.200-

Z

• . ..... ,.,.

i,i

............

rY

D

".. ''".,

-0.100

.......... .....

............. ....

-0.400 - 1.000

t -0.800

t -0.600

t -0.400

t -0.200

.,....

0.000

POTENTIAL(VOLTSVS Ag/AgCl) Fig. 13. DPP and sampled-DC polarograms of rabbit MT in 0.01 M acetic acid solution (pH 5.0).

58

C.L. CHOU ET AL.

tic of other metallothioneins from Mammalia and other classes. Unlike other metallothioneins, the major proteins in MT-1 had unusually high pl values, as determined by isoelectric focussing. MT-I, with a relatively low cysteine content, may not be a true metaUothionein in spite of its similarities to this class of protein with respect to molecular weight, metal-ion binding characteristics and amino acid composition. Polarographically, MT-1 and MT-2 behaved similar to rabbit metallothionein. REFERENCES Casassas, E., C. Arino and M. Esteban, 1985. Cathodic stripping voltammetry of 2-mercaptoethanol. Anal. Chim. Acta, 176:113-119. Cherian, M.G., 1974. Isolation and purification of cadmium binding proteins from rat liver. Biochem. Biophys. Res. Commun., 61: 920-926. Chou, C.L., J.F. Uthe, J.D. Castell and J.C. Kean, 1987. The effect of dietary cadmium on growth, survival, and tissue concentrations of cadmium, zinc, copper, and silver in juvenile American lobster (Homarus americanus). Can. J. Fish. Aquat. Sci., 44: 1443-1450. Evans, G.W., M.L. Wolenetz and C.I. Grace, 1975. Copper-binding proteins in neonatal and adult rat liver soluble fraction. Nutr. Rep. Int., 12: 261-269. Garner, C.D., S.S. Hasnain, I. Bremner and J. Bordas, 1982. An EXAFS study of the zinc sites in sheep liver metallothionein. J. lnorg. Biochem., 16: 253-256. Hits, C.H.W., 1967. Determination ofcystine as cysteic acid. In: C.H.W. Hirs (Ed.), Methods in Enzymology, Vol. 11. Academic Press, New York, pp. 59-65. Irons, R.D. and J.C. Smith, 1977. Isolation of a non-thionein copper-binding protein from liver of copper injected rats. Chem.-Biol. Interact., 18: 83--89. Kagi, J.H.R. and B.L. Vallee, 1961. Metallothionein: a cadmium and zinc-containing protein from equine renal cortex. J. Biol. Chem., 236: 2435-2492. Klauser, S., J.H.R. Kagi and K.J. Wilson, 1983. Characterization of isoprotein patterns in tissue extracts and isolated samples of metallothionein~ by reverse-phase high-pressure liquid chromatography. Biochem. J., 209: 71-80. Kojima, Y., C. Berger, B.V, Vallee and J.H.R. Kagi, 1976. Amino acid sequence ofequine renal metailothionein-IB. Proc. Natl. Acad. Sci. U.S.A., 73: 3413-3417. Margoshes, M. and B.L. Vallee, 1057. A cadmium protein from equine kidney cortex. J. Am. Chem. Soc., 79:4813-4814. Milner, G.W.C., 1957. The Principles and Applications of Polarography. Longmans, Toronto, pp. 619-628. Neilson, K.B., C.L. Atkin and D.R. Winge, 1985. Distinct metai-binding configurations in metallothionein. J. Biol. Chem., 260: 5342-5350. Nomiyama, K. and H. Nomiyama, 1982. High-performance liquid chromatographic determination of tissue metallothionein in monkeys chronically exposed to cadmium. J. Chromatogr., 228: 285-291. Nordberg, G.F., M. Nordberg, M. Piscator and O. Vesterberg, 1972. Separation of two forms of rabbit metallothionein by isoelectric focusing. Biochem. J., 126: 491--498. Olafson, R.W., R.G. Sim and K.G. Boto, 1979. Isolation and characterization of the heavy metal-binding protein metaUothionein from marine invertebrates. Biochem. Physiol., 62B: 407-4 !6. Otvos, J.D., R.W. Olafson and I.M. Armitage, 1982 Structure of an invertebrate metalIothionein from St3'lla serrata. J. Biol. Chem,, 257: 2427-2431.

ISOLATION AND CHARACTERIZATION OF MET,~.L-BINDING PROTEINS

59

Overnell, J., 1982. A method for the isolation of metallothionein from the hepatopancreas of the crab Cancer pagurus that minimizes the effect of tissue proteases. Comp. Biochem. Physiol., 73B: 547-553. Overnell, J. and T.L. Coombs, 1979. Purification and properties of plaice metallothionein, a cadmium binding pr'~z~in from the liver of the plaice (Pleuronectes platessa). Biochem. J., 183: 277-283. Piscator, M., 1964. Om kadmium: normala manniskonjurar samt redogorelse for isolering au metallothionein ur lever fran kadmium exponerade kaniner. Nord. Hyd. Tidskr., 45: 76-82. Premakumar, R., D.R. Winge, R.D. Riley and K.V. Rajagopalan, 1975. Copper-chelation: isolation from various eucaryotic sources. Arch. Biochem. Biophys., 170: 278-288. Prinz, R. and U. Weser, 1975. Cuprodoxin. FEBS Lett., 54: 224-229. Pulido, P., J.H.R. Kagi and B.L. Valee, 1966. Isolation and some properties of human metallothionein. Biochemistry, 5:1768-1778. Rabenstein, D.L. and R. Saetre, 1977. Mercury-based electrochemical detector for liquid chr'~matography for the detection of glutathione and other sulfur-containing compounds. Anal. Chem., 49: ! 036-1040. Ridlinglon, J.W. and B.A. Fowler, 1979. Isolation and partial characterization of a cadmiumbinding protein fi'om the American oyster (Crassostrea virghlica). Chem.-Biol. Interact., 25: 127-138. Rupp, H. and U. Wcser, 1974. Conversion of metailothionein into Cu-thionein, the possible low molecular weight form of neonatal hepatic mitochondrocuprein. FEBS Lett., 44: 293-296. Shaikh, Z.A. and O.J. Lucis, 1971. Isolation of cadmium-binding proteins. Experientia. 27: 1024-1025. Shaikh, Z.A. and J.C. Smith, 1975. Cadmium induced synthesis of hepatic and renal metallothionein. Fed. Proc., 36: 266-269. Squibb, K.S. and R.J. Cousins, 1974. Control ofcadmium-bindir, g protein synthesis in rat liver. Environ. Physiol. Biochem., 4: 24-30. Suzuki, K.T. and T. Yajima, 1984. Separation of metallothionein into isoforms by cohlmn switching gel permeation and ion-exchange columns with high-performance liquid chromatography-atomic absorption spectrophotometry. J. Chromatogr., 303:13 i- 136. Takeda, H. and C. Shimizu, 1982. Existence of the metallothionein-like protein in various fish tissues. Bull. Jpn. Soc. Sci. Fish., 48(5): 711-715. Uthe, J.F., C.L. Chou and D.G. Robinson, 1980. Cadmium m Atnerican Iooster (ttomarus americamls) trom the area of Beiledune Harbour, New Brunswick. In: J.!:. Uthe and V. Zitko (Eds), Cadmium Pollution of Belledune Harbour, New Brunswick, Canada. Can. Tech. Rep. Fish. Aquat. Sci., 963, pp. 65-72. Waalkes, M.P. and A. Perantoni, 1986, Isolation of a novel metal-binding protein from rat testes, characterization and distinction from metallothionein. J. Biol. Chem., 261: 1309713103. Weser, U., H. Rupp, F. Donny, F. Linnemann, W. Voeter, W. Voetsch and G. Jung, 1973. Characterization of Cd, Zn-thionein (metallothionein) isolated from rat and chicken liver. Envi ,n. J. Biochem., 39: 127-140. Winge, D.R., R. Premakumar and K.V. Rajagopalan, 1975. Metal-induced formation of metallothionein in rat liver. Arch. Biochem. Biophys., 170: 242-252. Winge, D.R., K.B. Neilson, W.R. Gray and D.H. Hanner, 1985. Yeast metaliothionein, sequence and metal-binding properties. J. Biol. Chem., 260: 14464-14470.