The Science of the Total Environment, 105 (1991) 61-71 Elsevier Science Publishers B.V., Amsterdam
61
The reactivity of EDTA, copper ion, and copper citrate with metallothioneins isolated from the digestive gland of cadmium-contaminated lobster
(Homarus americanus) C.L. Chou "~, R.D. Guy b and J.F. Uthe '~ ~Marine Chemistry Division, Physical and Chemical Science Branch, Scotia-Fundy Region, Department of Fisheries and Oceans, P.O. Box 550, Halifax, N.S. B3J 2S7, Canada bDepartment of Chemistry, Dalhousie UniversiO,, Hal([ax, N.S. B3H 4J1, Canada (Received May 22nd, 1990; accepted August 7th, 1990)
ABSTRACT
The reactivity of EDTA, Cu2+, and copper citrate with two metallothionein preparations (MT-I and MT-2) isolated from the digestive gland of Cd-contaminated American lobster (Homarus americanus) was studied. Under pseudo-first-order conditions~ metallothioneins reacted with EDTA for removal of Cd2+ in a multiphasic manner. Cadmium(+ II) removal by Cu2+ was complex and non-stoichiometric, suggesting different binding sites. Rabbit liver metallothionein reacted similarly. Cadmium removal from lobster metallothionein by copper citrate was slow and triphasic in nature. EDTA re~roved Cu:t from lobster metallothionein very slowly. INTRODUCTION Recently, many studies have investigated the reactivity of metal ions with metallothionein preparations (Ot~os and Armitage, 1980; Laib et al., 1985; Vasak et al., 1985). Distribution, relative accessibility, and reactivity of metal ions in metallothionein are of particular interest since metallothioneins may function in the homeostasis of essential metal ions, e.g. Zn 2+ and Cu 2+, and detoxification of others, e.g. Cd 2+ and Hg 2+ (O'Brady, 1982). The metal ion composition of metallothionein can be altered by exposing animals to certain metal ions, in vivo, or by exchange during extraction and isolation (Winge et al., 1975; Otvos and Armitage, 1980). Metallothioneins isolated from organisms previously e~xposed to metallothionein-inducing metal ions contain predominantly the administered metal. For example, metallothionein isolated from the liver of Cd-treated rats contained five atoms of Cd and two of Zn per molecule (Winge et al., 1975). Often, a reconstituted
0048-9697/91/$03.50
© 1991 -- Elsevier Science Publishers B.V.
62
C.L. CHOU ET AL.
metallothionein containing only a single type of metal ion has been studied to avoid problems introduced by heterogeneity. Various metalloproteins have been studied by 113Cd nuclear magnetic resonance (Otvos and Armitage, 1980). Results have yielded important informe.tion on the structure and dynamics ofligand exchange at the metal binding sites. There are few chemical studies of the metal-mercaptide unit of metallothionein. Kinetic studies of the reaction of excess EDTA (pseudo-first-order conditions) with metallothionein containing only Zn 2÷ , or only Cd 2÷ or a mixture of both ions, were first reported by Li et al. (1980). They reported that the ligand substitution reaction of 7n 2+ was multiphasic and that Zn 2÷ was transferred to EDTA faster than Cd 2÷ in a first-order, a dissociative process. In the present work, we studied the reactivity of EDTA, Cu ~÷, and copper citrate with metallothionein preparations isolated from the digestive gland of Cd-contaminated lobsters (Uthe et al., 1980). MATERIALS AND METHODS
Two metallothionein preparations (MT-I and MT-2) were isolated from American lobster (Homarus americanus) digestive gland (Chou et al., 1991). MT-1, exhibiting two major bands by isoelectric focussing, was less pure than MT-2, which yielded a single band. MT-2 demonstrated the spectral properties, amino acid composition (high cysteine, lack of aromatic amino acids), apparent molecular weight, and high Cd content characteristic of metallothioneins isolated from other species. MT-I also showed metallothionein characteristics, although it contains a more complex suite of metal ions and a lower level of cysteine. MT-I may simply be an impure metallothionein or comprise different forms of metallothiopein, e.g. metallothionein containing different metal ions. Rabbit liver metallothionein (Sigma), containing at least two forms of metailothionein, was studied without further purification.
Kinetic proce&o'es The reactions of MT-I and MT-2 with a large excess of ethylenediamine tetraacetate (EDTA) were monitored under pseudo-first-order conditions for EDTA-complexed and protein-bound Cd by use o! a short molecular-sieve column (Fractogel HW-40, 1.5cm i.d. x 15cm long) in the gel-permeation atomic absorption spectrophotometric-chromatographic (GP-AAS) system (Guy et al., 1985). Reactions were studied at room temperature (pH 7.0) in 0.2 M NaNO3 since CI- interferes in the chromatography of Cd 2÷ (Chou et al., 1991). The elution buffer was 0.1 M sodium formate/0.01 M sodium citrate (pH 7.0). Citrate is needed to prevent the non-specific sorption of Cd 2÷ by the column gel during GP-AAS (Chou et al., 1991). The reaction rate was
REACTIVITY
OF EDTA.
Cu’+ . AND
COPPER
CITR4TE
WITH
METALLOTHIONEINS
63
measured by determining either the loss of protein-bound Cd or the formation of EDTA-bound Cd. The logarithms of these values were plotted against time. The reaction time period was up to the point of injection onto the column. Cadmium displacement was also studied using excess Cu’+ or copper citrate under the same conditions. Copper citrate solution contained 10 pg Cu ml-’ in 0.81 N sodium citrate/O.2 M NaNO,. Excess citrate was used to ensure the full complexation of the Cu*+ ion. Citrate (0.01 N) alone did not remove metal ions from lobster digestive gland metallothioneins. Copper removal from MT-1 by excess EDTA was also studied. Cadmium ( + II) removal from rabbit metallothionein by excess EDTA was monitored with a Hewlett-Packard Model HP-84528 Photodiode Array Spectrophotometer. Spectra (200320 nm) were recorded every 4 min over a 2-h period using a 5-s integration time. RESULTS AND DISCUSSION
The reactions of lobster MT-l and MT-2 with various concentrations of EDTA, under pseudo-first-order conditions, are triphasic for removal of Cd (see, fo.r a-a-pie, bA 111 Fig. I), as first reported by Li et al. (1980). The first phase occurred too fast to be monitored by the GP-AAS system. The second and third phases were successfully monitored. The relationships between the pseudo-first-order rate constants and the EDTA concentration for lobster MT-l and MT-2 are shown in Figs 2 and 3, respectively. The rate law equations for steps 2 and 3 are dependent on EDTA concentrations. For example, with 2.6mM EDTA, the k, values for h T-l and MT-2 were
A. 1.0 mM EDTA 8: 2.~ mM EDTA C, LO mM EDTA
0
100 Readion
Time
(min
)
Fig. I. Plots of kinetics of the removal of Cd from lobster digestive gland MT-2 (containing Cd) by EDTL’~.The reaction was monitored by GP-AAS chrcmatographic method.
0.0721 mM
64
C.L. CHOU ET AL.
6
)5 |'
/
/
/
~
1
N
;3
J
Y
2.85 x 10 -4880.1 m
+ 0.128 M R
2
1
m
s3c
1
X
: 0.956 iii
Cone. of EDTA (In raM) 16
|
14
S
12
ii,n,
,.
th
-1
t
N
/
Y : 4'24 x 10 ilsec'1 + 0.055 M" sec" 1 X
t.
R
2
= 0.997
,,
,
Conc. of EDTA (In mM) Fig. 2. E DTA dependence of the reaction between lobster digestive gland MT- ! (containing 0.0157 mM Cd) and EDTA for removal of Cd. The pseudo-first-order rate constants (step 2 = k,, step 3 = k.~) obtained at each concentration of EDTA are plotted against EDTA concentrations (R" = coefficient of determination).
6,18 x 1 0 - 4 s - I and 3.17 × 10-4s -~, whereas k3 values ~,,.~re 1.85 x 10 -4 s -~ and 2.13 x 10 -4 s -~ , respectively. The dependence of ks and k3 on EDTA concentration is complex. Unequivocal interpretation is difficult since we do not know if we are dealing with multiple, different Cd-binding sites within a single protein or multiple metallothioneins. At 1.0mM EDTA, ks for MT-1 was about twice that for MT-2 while ka was only slightly larger. The different rate constants for MT- 1 and MT-2 and the similarity of their reaction kinetics suggest that, in the case of lobster digestive gland, we are dealing with at least two metallothioneins. These rate constants are compared with those reported in the literature for other metallothioneins in Table 1. Lobster MT-1 and MT-2 were less reactive in EDTA-Cd displacement than crab (Cancerpagurus) digestive gland metallothionein and more reactive than horse kidney metallothionein and rabbit
REACTIVITY OF EDTA. Cu 2 +. AND COPPER CITRATE WiTH METALLOTHIONEINS
!4
. . .
65
5
'7,
3
I 1
8 •
Y : 1 . 7 4 x 10 .4 8ec ÷ 0.0SS M "l~e~..1 X 2 R = 0.992
1
Cone. of EDTA (in mM) 5
:i
' 3
R
°'°" M"'°°"
= 0.999
I
0 • Ic
1
0
0
I
2 3 4 Conc. of EDTA (in raM)
5
6
Fig. 3. EDTA dependence of the reaction between lobster digestive gland MT-~ (containing 0.0721 mM Cd) and EDTA for removal of Cd. The pseudo-first-order rate constants (step 2 = k,, step 3 = /,'3) obtained at each concentration of EDTA are plotted against EDTA concentrations (R" = coefficient of determination).
liver metallothionein. The reaction of lobster metallothioneins with l mM EDTA was very similar to that of horse kidney Zn-metallothionein reported by Li et al. (1980). On the contrary, the transfer of Cd from horse kidney Cd-metallothionein to EDTA was characterized by a single stage with a pseudo-first-order rate constant of 3.7 x l0 -6 s -~, which is much slower than those of horse kidney Zn-metallothionein and lobster metallothionein. In the case of plaice (Pleuronectes platessa) metallothionein (Higham et al., 1986), k2 was shown to be slow, with 86% of the Cd removed during the first fast step and only 5% removed in the following slow reaction. Rabbit liver Cdmetallothionein reacted slower with EDTA than lobster metallothionein (Table 1).
Reaction with EDTA for Cu e+ removal The reaction between excess EDTA and MT-I (MTo2 doe~ not contain appreciable Cu) for Cu 2+ removal was much slower than that for Cd 2+
66
C.L. CHOU ET AL.
TABLE !
Rate constants (s -~ ) for reactions of metallothioneins with E D T A for removal of Cd Type of M T
kl
k_,
Horse kidney a
Fast
3.70
Lobster MT-!
Fast
4.15 x 10 -4
9.30 x 10 -5
!.0
MT-I
Fast
6.18 x 10 -4
1.85 x 10 -4
2.6
Lobster MT-2
Fast
2.35 x 10 -4
8.86 x 10 -5
1.0
MT-2
Fast
3.17 x 10 -4
2.13 x 10 -~
2.6
Crab prep. ! h
Fast
!.80 x 10-:
2.20
prep. 2 b
Fast
!.80 x 10 -3
4.20 x 10 -4
Fast
Very slow
Fast
!.38 x 10 -4
Plaicec
k3
x
[EDTA] (mM) !.0
10 - 6
X 10 -4
Rabbit liver MT
2.05 x 10 -6
2.6
"Li et al. (I 980). bHigham et al. (1986). CAlmost all of the 86% Cd was removed rapidly during the first fast step and only 5% o f the total Cd in plaice MT was removed in the following slow reaction (Higham et al., 1986).
removal, requiring many days to go to completion (Fig. 4). The reaction was monophasic with a pseudo-first-order rate constant of 2.72 x 10-6s -~, similar to that observed with horse kidney metaliothionein (l.i et al., 1980). Reaction with Cu 2+ for Cd removal
At least 95% of the Cd 2+ in MT-I was immediately displaced by Cu 2+ under equimolar concentrations with the remaining Cd displacement after 2 h (Table 2). MT-2 did not react so rapidly. The addition of 0.0629mM Cu 2+ (equivalent to 87.2% of the bound Cd) immediately displaced only 46% of the Cd. The rapidity with which Cu 2+ replaced Cd in both MT-1 and MT-2 implies a direct substitution of one ion for the other. However, there may be differences among the binding sites within and between the two protein preparations. Figure 5 presents the GP-AAS chromatogram for Cd and Cu in the presence of rabbit liver metalliothionein treated with Cu 2+. Curve A shows that Cd 2+ is bound to the protein. Curve B shows that Cd is removed almost completdy and instantaneously upon addition of excess Cu 2+ (0.1090.024 mM Cd). ~-towever, a stoichiometric amount of Cu was not taken up.
67
EI)TA. Cu 2 . . AND COPPER CITRATE WITH METALLOTHIONEINS
REACTIVITY OF
100
£
R 10
•
•
I
20
•
•
I
40
"
•
I
•
60
Reaction T i m e
•
2 l
80
= 0.989 •
•
l
100
•
•
I
120
•
•
40
(in h o u r s )
Fig. 4. Plot of kinetics of the removal of Cu from lobster digestive gland MT-! by EDTA. The reaction was monitored by GP-AAS chromatographic method (rate constant k = 2 . 7 2 x 10 "s-'; 2.16 × 10-~mM Cu of MT-! reacting with 7.5raM EDTA).
TABLE 2 Reactions of lobster digestive gland MT- 1 and MT-2 with Cu" ' ion for removal of Cd 0.0157mM Cd of MT-I + 0.0157mM Cu -'+ ion Time (min) 2 13 24 44 71 92 121
Cd-MT-i remaining (%) 5.47 4.95 07
-~ 7 a 3.21 2.56
...o
J
0.0721 m M Cd of MT-2 + 0.0629mM Cu '+ ion Time (min)
Cd-MT-2 remaining (%)
5 15 27 40 56 62 76 88 103
54.19 53.35 52.18 5.5.13 53.37 51.84 51.37 52.28 53.29
68
C.L. CHOU
18
"/
~
I
z
10
Cd*= "."".
~
I
~
I
n,-
AFTER 4 HOURS
I
IJJ U3 0 t'~ (/3 i,i
w COPPER AND MT - - MT ONLY •..... CD AND MT WITH COPPEF
c~-~: •-
1¢
ET AL.
ii1~
6
\
Cu"
'""..
:
-
t
I
I
0
2
4
6
-2
8
TIME(MINUTE)
Fig. 5. GP-AAS chromatograms for the reaction of Cd-metallothionein from rabbit liver (containing 0.024mM Cd + 0.0056raM Zn) with 0.109mM Cu 2+ . (A) Cd-metallothionein, (B) Cd 2+ displaced by Cu '+ , (C) bound Cu + residual Cu 2÷ .
s.o-'~
Y = 7.6209 x l O ' 4 s e c - 1 - 26.26 M " 1 sec" 1 X
7.0-' 6,0"
R2
= 0.971
0~,,s.oi 4.03.0-
o
= zo-
rn 1.0 0.0 0,006
•
'
:° 8 Y=,.,.,0:'..o:, •
+ 9.466
in W
' o,.. 6
I
0.016 Conc. o f Cu Citrate (In raM)
R2
M " • see'" 1 X
j
I
= 0.949
X ,e,*
=4 M P 0
~
2
nO
Conc. of Cu Citrate (in mM)
Fig. 6. Copper citrate dependence of the reaction between lobster digestive gland MT-I (containing 0.0157mM Cd) and copper citrate for removal of Cd. The first-order rate constants (step 2 = k , , step 3 = k~) obtained at each concentration of copper citrate are plotted against copper citrate concentration (R 2 -- coemcient of determination).
.~+
REACTIVITYOF EDTA.Cu" . ANDCOPPERCITRATEWITHMETALLOTHIONEINS
69
4 m
Y ; 3"60 M ' l s e c ' l X
0 Q
J
3 Z
m
.
0 X
.,
2
m w C 0
o
1
Q
0 ,----,--iP--- I 0.02 0.00
•
i
•
0.04
i
-
0.06
i
•
0.08
O.
Conc. of Cu Citrate (in mM)
'7, u
Y = - 1.5173 x 10
-4
sac
-1
+ 3.0487 M "1 sac" 1 X o
m
~
61
The reactivity of EDTA, copper ion, and copper citrate with metallothioneins isolated from the digestive gland of cadmium-contaminated lobster
(Homarus americanus) C.L. Chou "~, R.D. Guy b and J.F. Uthe '~ ~Marine Chemistry Division, Physical and Chemical Science Branch, Scotia-Fundy Region, Department of Fisheries and Oceans, P.O. Box 550, Halifax, N.S. B3J 2S7, Canada bDepartment of Chemistry, Dalhousie UniversiO,, Hal([ax, N.S. B3H 4J1, Canada (Received May 22nd, 1990; accepted August 7th, 1990)
ABSTRACT
The reactivity of EDTA, Cu2+, and copper citrate with two metallothionein preparations (MT-I and MT-2) isolated from the digestive gland of Cd-contaminated American lobster (Homarus americanus) was studied. Under pseudo-first-order conditions~ metallothioneins reacted with EDTA for removal of Cd2+ in a multiphasic manner. Cadmium(+ II) removal by Cu2+ was complex and non-stoichiometric, suggesting different binding sites. Rabbit liver metallothionein reacted similarly. Cadmium removal from lobster metallothionein by copper citrate was slow and triphasic in nature. EDTA re~roved Cu:t from lobster metallothionein very slowly. INTRODUCTION Recently, many studies have investigated the reactivity of metal ions with metallothionein preparations (Ot~os and Armitage, 1980; Laib et al., 1985; Vasak et al., 1985). Distribution, relative accessibility, and reactivity of metal ions in metallothionein are of particular interest since metallothioneins may function in the homeostasis of essential metal ions, e.g. Zn 2+ and Cu 2+, and detoxification of others, e.g. Cd 2+ and Hg 2+ (O'Brady, 1982). The metal ion composition of metallothionein can be altered by exposing animals to certain metal ions, in vivo, or by exchange during extraction and isolation (Winge et al., 1975; Otvos and Armitage, 1980). Metallothioneins isolated from organisms previously e~xposed to metallothionein-inducing metal ions contain predominantly the administered metal. For example, metallothionein isolated from the liver of Cd-treated rats contained five atoms of Cd and two of Zn per molecule (Winge et al., 1975). Often, a reconstituted
0048-9697/91/$03.50
© 1991 -- Elsevier Science Publishers B.V.
62
C.L. CHOU ET AL.
metallothionein containing only a single type of metal ion has been studied to avoid problems introduced by heterogeneity. Various metalloproteins have been studied by 113Cd nuclear magnetic resonance (Otvos and Armitage, 1980). Results have yielded important informe.tion on the structure and dynamics ofligand exchange at the metal binding sites. There are few chemical studies of the metal-mercaptide unit of metallothionein. Kinetic studies of the reaction of excess EDTA (pseudo-first-order conditions) with metallothionein containing only Zn 2÷ , or only Cd 2÷ or a mixture of both ions, were first reported by Li et al. (1980). They reported that the ligand substitution reaction of 7n 2+ was multiphasic and that Zn 2÷ was transferred to EDTA faster than Cd 2÷ in a first-order, a dissociative process. In the present work, we studied the reactivity of EDTA, Cu ~÷, and copper citrate with metallothionein preparations isolated from the digestive gland of Cd-contaminated lobsters (Uthe et al., 1980). MATERIALS AND METHODS
Two metallothionein preparations (MT-I and MT-2) were isolated from American lobster (Homarus americanus) digestive gland (Chou et al., 1991). MT-1, exhibiting two major bands by isoelectric focussing, was less pure than MT-2, which yielded a single band. MT-2 demonstrated the spectral properties, amino acid composition (high cysteine, lack of aromatic amino acids), apparent molecular weight, and high Cd content characteristic of metallothioneins isolated from other species. MT-I also showed metallothionein characteristics, although it contains a more complex suite of metal ions and a lower level of cysteine. MT-I may simply be an impure metallothionein or comprise different forms of metallothiopein, e.g. metallothionein containing different metal ions. Rabbit liver metallothionein (Sigma), containing at least two forms of metailothionein, was studied without further purification.
Kinetic proce&o'es The reactions of MT-I and MT-2 with a large excess of ethylenediamine tetraacetate (EDTA) were monitored under pseudo-first-order conditions for EDTA-complexed and protein-bound Cd by use o! a short molecular-sieve column (Fractogel HW-40, 1.5cm i.d. x 15cm long) in the gel-permeation atomic absorption spectrophotometric-chromatographic (GP-AAS) system (Guy et al., 1985). Reactions were studied at room temperature (pH 7.0) in 0.2 M NaNO3 since CI- interferes in the chromatography of Cd 2÷ (Chou et al., 1991). The elution buffer was 0.1 M sodium formate/0.01 M sodium citrate (pH 7.0). Citrate is needed to prevent the non-specific sorption of Cd 2÷ by the column gel during GP-AAS (Chou et al., 1991). The reaction rate was
REACTIVITY
OF EDTA.
Cu’+ . AND
COPPER
CITR4TE
WITH
METALLOTHIONEINS
63
measured by determining either the loss of protein-bound Cd or the formation of EDTA-bound Cd. The logarithms of these values were plotted against time. The reaction time period was up to the point of injection onto the column. Cadmium displacement was also studied using excess Cu’+ or copper citrate under the same conditions. Copper citrate solution contained 10 pg Cu ml-’ in 0.81 N sodium citrate/O.2 M NaNO,. Excess citrate was used to ensure the full complexation of the Cu*+ ion. Citrate (0.01 N) alone did not remove metal ions from lobster digestive gland metallothioneins. Copper removal from MT-1 by excess EDTA was also studied. Cadmium ( + II) removal from rabbit metallothionein by excess EDTA was monitored with a Hewlett-Packard Model HP-84528 Photodiode Array Spectrophotometer. Spectra (200320 nm) were recorded every 4 min over a 2-h period using a 5-s integration time. RESULTS AND DISCUSSION
The reactions of lobster MT-l and MT-2 with various concentrations of EDTA, under pseudo-first-order conditions, are triphasic for removal of Cd (see, fo.r a-a-pie, bA 111 Fig. I), as first reported by Li et al. (1980). The first phase occurred too fast to be monitored by the GP-AAS system. The second and third phases were successfully monitored. The relationships between the pseudo-first-order rate constants and the EDTA concentration for lobster MT-l and MT-2 are shown in Figs 2 and 3, respectively. The rate law equations for steps 2 and 3 are dependent on EDTA concentrations. For example, with 2.6mM EDTA, the k, values for h T-l and MT-2 were
A. 1.0 mM EDTA 8: 2.~ mM EDTA C, LO mM EDTA
0
100 Readion
Time
(min
)
Fig. I. Plots of kinetics of the removal of Cd from lobster digestive gland MT-2 (containing Cd) by EDTL’~.The reaction was monitored by GP-AAS chrcmatographic method.
0.0721 mM
64
C.L. CHOU ET AL.
6
)5 |'
/
/
/
~
1
N
;3
J
Y
2.85 x 10 -4880.1 m
+ 0.128 M R
2
1
m
s3c
1
X
: 0.956 iii
Cone. of EDTA (In raM) 16
|
14
S
12
ii,n,
,.
th
-1
t
N
/
Y : 4'24 x 10 ilsec'1 + 0.055 M" sec" 1 X
t.
R
2
= 0.997
,,
,
Conc. of EDTA (In mM) Fig. 2. E DTA dependence of the reaction between lobster digestive gland MT- ! (containing 0.0157 mM Cd) and EDTA for removal of Cd. The pseudo-first-order rate constants (step 2 = k,, step 3 = k.~) obtained at each concentration of EDTA are plotted against EDTA concentrations (R" = coefficient of determination).
6,18 x 1 0 - 4 s - I and 3.17 × 10-4s -~, whereas k3 values ~,,.~re 1.85 x 10 -4 s -~ and 2.13 x 10 -4 s -~ , respectively. The dependence of ks and k3 on EDTA concentration is complex. Unequivocal interpretation is difficult since we do not know if we are dealing with multiple, different Cd-binding sites within a single protein or multiple metallothioneins. At 1.0mM EDTA, ks for MT-1 was about twice that for MT-2 while ka was only slightly larger. The different rate constants for MT- 1 and MT-2 and the similarity of their reaction kinetics suggest that, in the case of lobster digestive gland, we are dealing with at least two metallothioneins. These rate constants are compared with those reported in the literature for other metallothioneins in Table 1. Lobster MT-1 and MT-2 were less reactive in EDTA-Cd displacement than crab (Cancerpagurus) digestive gland metallothionein and more reactive than horse kidney metallothionein and rabbit
REACTIVITY OF EDTA. Cu 2 +. AND COPPER CITRATE WiTH METALLOTHIONEINS
!4
. . .
65
5
'7,
3
I 1
8 •
Y : 1 . 7 4 x 10 .4 8ec ÷ 0.0SS M "l~e~..1 X 2 R = 0.992
1
Cone. of EDTA (in mM) 5
:i
' 3
R
°'°" M"'°°"
= 0.999
I
0 • Ic
1
0
0
I
2 3 4 Conc. of EDTA (in raM)
5
6
Fig. 3. EDTA dependence of the reaction between lobster digestive gland MT-~ (containing 0.0721 mM Cd) and EDTA for removal of Cd. The pseudo-first-order rate constants (step 2 = k,, step 3 = /,'3) obtained at each concentration of EDTA are plotted against EDTA concentrations (R" = coefficient of determination).
liver metallothionein. The reaction of lobster metallothioneins with l mM EDTA was very similar to that of horse kidney Zn-metallothionein reported by Li et al. (1980). On the contrary, the transfer of Cd from horse kidney Cd-metallothionein to EDTA was characterized by a single stage with a pseudo-first-order rate constant of 3.7 x l0 -6 s -~, which is much slower than those of horse kidney Zn-metallothionein and lobster metallothionein. In the case of plaice (Pleuronectes platessa) metallothionein (Higham et al., 1986), k2 was shown to be slow, with 86% of the Cd removed during the first fast step and only 5% removed in the following slow reaction. Rabbit liver Cdmetallothionein reacted slower with EDTA than lobster metallothionein (Table 1).
Reaction with EDTA for Cu e+ removal The reaction between excess EDTA and MT-I (MTo2 doe~ not contain appreciable Cu) for Cu 2+ removal was much slower than that for Cd 2+
66
C.L. CHOU ET AL.
TABLE !
Rate constants (s -~ ) for reactions of metallothioneins with E D T A for removal of Cd Type of M T
kl
k_,
Horse kidney a
Fast
3.70
Lobster MT-!
Fast
4.15 x 10 -4
9.30 x 10 -5
!.0
MT-I
Fast
6.18 x 10 -4
1.85 x 10 -4
2.6
Lobster MT-2
Fast
2.35 x 10 -4
8.86 x 10 -5
1.0
MT-2
Fast
3.17 x 10 -4
2.13 x 10 -~
2.6
Crab prep. ! h
Fast
!.80 x 10-:
2.20
prep. 2 b
Fast
!.80 x 10 -3
4.20 x 10 -4
Fast
Very slow
Fast
!.38 x 10 -4
Plaicec
k3
x
[EDTA] (mM) !.0
10 - 6
X 10 -4
Rabbit liver MT
2.05 x 10 -6
2.6
"Li et al. (I 980). bHigham et al. (1986). CAlmost all of the 86% Cd was removed rapidly during the first fast step and only 5% o f the total Cd in plaice MT was removed in the following slow reaction (Higham et al., 1986).
removal, requiring many days to go to completion (Fig. 4). The reaction was monophasic with a pseudo-first-order rate constant of 2.72 x 10-6s -~, similar to that observed with horse kidney metaliothionein (l.i et al., 1980). Reaction with Cu 2+ for Cd removal
At least 95% of the Cd 2+ in MT-I was immediately displaced by Cu 2+ under equimolar concentrations with the remaining Cd displacement after 2 h (Table 2). MT-2 did not react so rapidly. The addition of 0.0629mM Cu 2+ (equivalent to 87.2% of the bound Cd) immediately displaced only 46% of the Cd. The rapidity with which Cu 2+ replaced Cd in both MT-1 and MT-2 implies a direct substitution of one ion for the other. However, there may be differences among the binding sites within and between the two protein preparations. Figure 5 presents the GP-AAS chromatogram for Cd and Cu in the presence of rabbit liver metalliothionein treated with Cu 2+. Curve A shows that Cd 2+ is bound to the protein. Curve B shows that Cd is removed almost completdy and instantaneously upon addition of excess Cu 2+ (0.1090.024 mM Cd). ~-towever, a stoichiometric amount of Cu was not taken up.
67
EI)TA. Cu 2 . . AND COPPER CITRATE WITH METALLOTHIONEINS
REACTIVITY OF
100
£
R 10
•
•
I
20
•
•
I
40
"
•
I
•
60
Reaction T i m e
•
2 l
80
= 0.989 •
•
l
100
•
•
I
120
•
•
40
(in h o u r s )
Fig. 4. Plot of kinetics of the removal of Cu from lobster digestive gland MT-! by EDTA. The reaction was monitored by GP-AAS chromatographic method (rate constant k = 2 . 7 2 x 10 "s-'; 2.16 × 10-~mM Cu of MT-! reacting with 7.5raM EDTA).
TABLE 2 Reactions of lobster digestive gland MT- 1 and MT-2 with Cu" ' ion for removal of Cd 0.0157mM Cd of MT-I + 0.0157mM Cu -'+ ion Time (min) 2 13 24 44 71 92 121
Cd-MT-i remaining (%) 5.47 4.95 07
-~ 7 a 3.21 2.56
...o
J
0.0721 m M Cd of MT-2 + 0.0629mM Cu '+ ion Time (min)
Cd-MT-2 remaining (%)
5 15 27 40 56 62 76 88 103
54.19 53.35 52.18 5.5.13 53.37 51.84 51.37 52.28 53.29
68
C.L. CHOU
18
"/
~
I
z
10
Cd*= "."".
~
I
~
I
n,-
AFTER 4 HOURS
I
IJJ U3 0 t'~ (/3 i,i
w COPPER AND MT - - MT ONLY •..... CD AND MT WITH COPPEF
c~-~: •-
1¢
ET AL.
ii1~
6
\
Cu"
'""..
:
-
t
I
I
0
2
4
6
-2
8
TIME(MINUTE)
Fig. 5. GP-AAS chromatograms for the reaction of Cd-metallothionein from rabbit liver (containing 0.024mM Cd + 0.0056raM Zn) with 0.109mM Cu 2+ . (A) Cd-metallothionein, (B) Cd 2+ displaced by Cu '+ , (C) bound Cu + residual Cu 2÷ .
s.o-'~
Y = 7.6209 x l O ' 4 s e c - 1 - 26.26 M " 1 sec" 1 X
7.0-' 6,0"
R2
= 0.971
0~,,s.oi 4.03.0-
o
= zo-
rn 1.0 0.0 0,006
•
'
:° 8 Y=,.,.,0:'..o:, •
+ 9.466
in W
' o,.. 6
I
0.016 Conc. o f Cu Citrate (In raM)
R2
M " • see'" 1 X
j
I
= 0.949
X ,e,*
=4 M P 0
~
2
nO
Conc. of Cu Citrate (in mM)
Fig. 6. Copper citrate dependence of the reaction between lobster digestive gland MT-I (containing 0.0157mM Cd) and copper citrate for removal of Cd. The first-order rate constants (step 2 = k , , step 3 = k~) obtained at each concentration of copper citrate are plotted against copper citrate concentration (R 2 -- coemcient of determination).
.~+
REACTIVITYOF EDTA.Cu" . ANDCOPPERCITRATEWITHMETALLOTHIONEINS
69
4 m
Y ; 3"60 M ' l s e c ' l X
0 Q
J
3 Z
m
.
0 X
.,
2
m w C 0
o
1
Q
0 ,----,--iP--- I 0.02 0.00
•
i
•
0.04
i
-
0.06
i
•
0.08
O.
Conc. of Cu Citrate (in mM)
'7, u
Y = - 1.5173 x 10
-4
sac
-1
+ 3.0487 M "1 sac" 1 X o
m
~
