Despite the high homology among vertebrate MTs, fish and mammalian MTs exhibit significant differences at the level of primary structure, i.e.. Because this cluster is mainly involved in c
Trang 1A structural and functional comparison
Laura Vergani1, Myriam Grattarola1, Cristina Borghi2, Francesco Dondero3and Aldo Viarengo3,4
1 Department of Biophysical Sciences and Technologies, M & O University of Genova, Italy
2 Department of Biology, University of Genova, Italy
3 Department of Environmental & Life Science, University of Piemonte Orientale, Alessandria, Italy
4 Center on Biology and Chemistry of Trace Metals, University of Genova, Italy
Metallothioneins (MTs) are cytosolic polypeptides
found in almost all organisms, including vertebrates,
invertebrates, plants and bacteria [1] They do not
appear to be essential for life, even though they are
involved in many pathways, such as sequestration of
toxic (Cd, Hg) or essential (Zn, Cu) metals, scavenging
of oxyradicals, inflammation, and infection [2]
MTs exhibit unusual primary sequence, lacking
histi-dines and aromatic residues, and their 3D structure is
unique [3,4] Cysteines represent one-third of the total
amino acids and are distributed in typical motifs con-sisting of CC, CXC or CXYC sequences [5] The beha-viour of MTs is dominated by the nucleophilic thiol group reacting with electrophilic compounds, including many alkylating agents and radical species [6] Verteb-rate MTs have a monomeric dumbbell shape, com-posed of two globular domains connected by a flexible linker consisting of a Lys-Lys segment Each domain contains a ‘mineral core’ enclosed by two large heli-cal turns of the polypeptidic chain The N-terminal
Keywords
absorbance spectroscopy; circular
dichroism; metal release; structure ⁄
function relationship; thermal stability
Correspondence
L Vergani, Department of Biophysical
Sciences and Technologies, M & O.
University of Genova, Corso Europa 30,
16132 Genova, Italy
Fax: +39 010 3538346
Tel: +39 010 3538404
E-mail: Laura.Vergani@unige.it
(Received 6 July 2005, revised 14
September 2005, accepted 26
September 2005)
doi:10.1111/j.1742-4658.2005.04993.x
Metallothioneins (MTs) are noncatalytic peptides involved in storage of essential ions, detoxification of nonessential metals, and scavenging of oxyradicals They exhibit an unusual primary sequence and unique 3D arrangement Whereas vertebrate MTs are characterized by the well-known dumbbell shape, with a b domain that binds three bivalent metal ions and
an a domain that binds four ions, molluscan MT structure is still poorly understood For this reason we compared two MTs from aquatic organ-isms that differ markedly in primary structure: MT 10 from the inverteb-rate Mytilus galloprovincialis and MT A from Oncorhyncus mykiss Both proteins were overexpressed in Escherichia coli as glutathione S-transferase fusion proteins, and the MT moiety was recovered after protease cleavage The MTs were analyzed by gel electrophoresis and tested for their differen-tial reactivity with alkylating and reducing agents Although they show an identical cadmium content and a similar metal-binding ability, spectro-polarimetric analysis disclosed significant differences in the Cd7-MT secon-dary conformation These structural differences reflect the thermal stability and metal transport of the two proteins When metal transfer from Cd7
-MT to 4-(2-pyridylazo)resorcinol was measured, the mussel -MT was more reactive than the fish protein This confirms that the differences in the pri-mary sequence of MT 10 give rise to peculiar secondary conformation, which in turn reflects its reactivity and stability The functional differences between the two MTs are due to specific structural properties and may be related to the different lifestyles of the two organisms
Abbreviations
MT, metallothionein; GST, glutathione S-transferase; PAR, 4-(2-pyridylazo)resorcinol.
Trang 2right-handed b domain binds three bivalent metal ions.
The C-terminal a domain is left-handed and binds four
bivalent ions Zinc is preferentially located in the b
do-main and cadmium in the a dodo-main Therefore, the
b domain would regulate zinc and copper homeostasis,
whereas the a domain may play a central role in heavy
metal detoxification [7] The loosely structured b
do-main is responsible for metal-bridge dimerization,
whereas the a domain is involved in oxidative
dimeri-zation Metal bridge dimerization is reversed by
dilu-tion or addidilu-tion of chelating agents, whereas oxidative
dimers are reduced by reducing compounds [8] As
reported previously [9], oxidative dimerization may
also occur in vivo under conditions of stress, such as
exposure to toxic metals and reactive oxygen species
and in neurological disorders (e.g Alzheimer’s disease)
A different susceptibility to oxidation may be
import-ant for the physiological role of the protein
Despite the high homology among vertebrate MTs,
fish and mammalian MTs exhibit significant differences
at the level of primary structure, i.e displacement of
one cysteine and fewer lysines [10]
Compared with vertebrates, invertebrate MTs show
unusual features in their primary structure The
sequences of only a few MTs from aquatic
inverte-brates (crab, mussel, sea urchin, snail and oyster) have
so far been elucidated [11–17] As in mammals and fish
[18], echinoderm MTs contain two globular domains
binding four and three bivalent ions [19] On the other
hand, in crab (Scylla serrata and Cancer pagurus) the
two domains bind three bivalent metals each [20–22]
In comparison with mammalians, molluscan MTs
usu-ally have higher glycine content ( 15% in mussels),
randomly distributed throughout the sequence Despite
the differences, molluscan MTs appear to be more
clo-sely related to vertebrate MTs than those from other
invertebrate phyla [23,24]
In this study we focused on two MTs from different
aquatic organisms which we had widely investigated in
previous work [25,26]: MT A from Oncorhyncus mykiss
and MT 10 from Mytilus galloprovincialis were selected
as representative of vertebrates and invertebrates, respectively Although MT 10 is longer than MT A, both have a similar number of cysteine residues and identical cadmium content Both recombinant MTs were tested for reactivity to alkylating and reducing agents, to evaluate their susceptibility to oxidative and metal-bridge dimerization Secondary conformation was analyzed in both the metal-free protein and Cd7 -MTs After metal binding, significant differences between the two forms were observed The altered sec-ondary structure influenced the physicochemical prop-erties of the proteins, with MT 10 being more thermostable than MT A When the redox-induced metal transfer from Zn7-MT or Cd7-MT to the specific acceptor 4-(2-pyridylazo)resorcinol (PAR) was meas-ured, MT 10 was much more reactive in terms of cad-mium release This observation is interesting because the redox control of metal bioavailability seems to be
an important physiological function of MTs [27]
Results
Analysis of primary sequence When the primary sequence of mussel MT 10 was compared with that of fish MT A (Fig 1) with Needle-man–Wunsch global alignments [28], a low identity was observed (39%) Because the first extra amino acid number is similar in the two recombinant MTs, we assumed that they affect the two proteins in a similar way Accordingly, experiments using atomic absorp-tion spectroscopy estimated 7 mol cadmium bound per mol recombinant MT in both samples
The b domain of MT A has nine cysteines distri-buted in classic Cys motifs In MT 10, this domain is two residues longer, but it has only eight cysteines with
a similar arrangement of the CXC motifs Major dif-ferences between the two proteins occur at the level of the a domain, which is longer in MT 10 than in MT A (42 vs 29 residues) and has two additional cysteines Moreover, the cysteines are organized differently in
Fig 1 Sequence alignment of fish MT A and molluscan MT 10 The sequences of the two recombinant MTs were aligned with the program Needleman-Wunsch global alignments This program uses the Needleman–Wunsch global alignment algorithm [28] to find the optimum alignment (including gaps) of two sequences when considering their entire length.
Trang 3terms of both the CXC or CXYC sequence and Cys
motif arrangement In summary, MT A has six CXC,
one CXYC and four CXYWC sequences, whereas
mussel MT 10 has nine CXC, one CXYC and five
CXYWC sequences In MT 10, the last a domain Cys
motif is CXXXCC, instead of CXCC, which is typical
of other vertebrates This feature has been reported in
the MT from the Antarctic fish Notothenia coriiceps
[18], but not in mussels In conclusion, this
comparat-ive analysis localizes the major differences between the
fish and molluscan MTs at the level of the a domain
Because this cluster is mainly involved in cadmium
binding and oxidative dimerization, these differences
may reflect functional differences in the two MTs with
regard to Cd release and oxidation
A further difference is the reduced number of lysines
in MT 10 compared with MT A (5 vs 7),
correspond-ing to 6.8% of the amino-acid composition for the
mussel protein and 11.5% for the fish one In spite of
the fewer lysines, MT 10 contains one more CK motif
(5 vs 4), whereas mammalian MTs have seven The
arrangement of the CK motifs also differs between
mussel and fish proteins: the four CK motifs of MT A
are equally distributed between the a and b domain,
whereas in MT 10, four are at the C-terminus and only
one at the N-terminus
As previously reported [29], the hydropathic index
(a parameter that is inversely proportional to
flexibility) is lower in fish MTs than in mammalian
MTs A higher flexibility should facilitate conforma-tional changes in organisms living at low temperatures When the hydropathic index was calculated for the trout MT A and the mussel MT 10 using the protparam tool [30], MT A yielded a negative value ()0.110), similar to that recorded for N coriiceps [29], whereas MT 10 gave a positive value (0.199) consis-tently higher than that for mammalian MTs (0.098) This points to mussel MT having a lower flexibility than either the fish or mammalian counterparts
Oxidative and metal bridge polymerization After chromatographic purification and enzymatic removal of the glutathione S-transferase (GST) tail, proteins were analyzed by SDS⁄ PAGE (15% gel) As expected, MT A showed a lower molecular mass than
MT 10, but also more marked smearing at high molecular mass than MT 10 This effect is due to the presence of polymeric forms typical of native MTs (Fig 2) When both MTs were alkylated with N-ethyl-maleimide, a unique band at a lower molecular mass appeared, representing the monomeric form, and no differences in mass between the two MTs could be observed Moreover, alkylation of the thiol group of
MT A resulted in disappearance of the smearing at high molecular mass A similar effect on the aggre-gates was observed when MT A was reduced with dithiothreitol, which caused the appearance of a single
Fig 2 Electrophoretic comparisons between fish MT A and mussel MT 10 MT A (A) and MT 10 (B) were electrophoresed on SDS ⁄ 15% polyacrylamide gel before (lane 1) and after the addition of an alkylating agent (N-ethylmaleimide) at two different concentrations: 40 and
80 m M (lanes 2 and 3) for 3 h MTs were also treated for the same period with a reducing agent (dithiothreitol) at 40 and 80 m M (lanes 4 and 5) To reduce aggregation, MT samples were handled in anaerobic conditions under nitrogen atmosphere Molecular markers (lane M) from the top: BSA, 66 kDa; chicken egg ovalbumin, 45 kDa; bovine chymotrypsinogen, 25 kDa; lysozyme, 14.3 kDa; ribonuclease A, 11.9 kDa; bovine lung aprotinin 6.5 kDa.
Trang 4band at 12 kDa, corresponding to the dimeric form
of the protein In contrast, no changes occurred in
MT 10 when exposed to the reducing agent, indicating
lower susceptibility of the mussel protein to oxidation
These results suggest that the smearing at high
molecu-lar mass (12–18 kDa) is due to oxidative
polymeriza-tion of the MT molecules, whereas dimer formapolymeriza-tion
( 12 kDa) is probably due to metal bridge effects
The marked differences between the a domain
sequences of the two MTs described above is in line
with the reduced sensitivity to oxidation exhibited by
MT 10, as oxidative polymerization occurs mainly in
the a domain
Characterization of structure and metal binding
UV absorption spectra of both MTs were recorded on
addition of increasing equivalents of cadmium ions to
the metal-free apoprotein, at neutral pH After Cd(II)
titration, a shoulder peak appeared at 254 nm,
reflect-ing the charge-transfer interaction of the cadmium–
thiolate clusters In both curves (Fig 3) absorption at
254 nm increased steadily, until saturation was reached
at seven metal equivalents The slope of the curve was
almost identical for the two MTs, indicating no
signifi-cant differences with respect to cadmium-binding
prop-erties This agrees with the data acquired by atomic
absorption spectroscopy, which estimated 7 mol
cad-mium bound per mol MT
On analysis of the CD spectra of the MTs, both
metal-free thioneins showed a strong negative band at
230 nm (Fig 4), typical of proteins in random coil conformation [31] This confirms that both apo-MTs were unfolded in the absence of metals, and only after binding of the correct number of cadmium equivalents did they assume a stable secondary structure When complexed to the metal, both MTs showed a strong positive ellipticity band above 250 nm, but the peak was red-shifted in the MT 10 spectrum compared with that of MT A The major differences were evident in the region below 250 nm In fact, both the negative band at 245 nm and the positive one at 228 nm, char-acteristic of the fish MT A, were lost in the MT 10 spectrum Considering the spectral peculiarities, we can infer that mussel MT 10 has an atypical secondary conformation, which is probably due to the differences
in primary sequence
Fig 3 Spectrophotometric titration following the binding of Cd(II)
to the apo-MTs The Cd-induced contribution to the absorption
spectrum at 254 nm is plotted against the number of Cd
equiva-lents added, from 0.3 to 8 ratio for both fish MT A (m) and mussel
MT 10 (n) Each curve is representative of at least three
independ-ent sets of measuremindepend-ents.
Fig 4 CD analysis CD spectra were acquired in the near-UV region (from 190 to 290 nm) for fish MT A (A) and molluscan
MT 10 (B) for both Cd7-MT forms and the apoproteins The metal-free protein was obtained by acidification with HCl The measure-ments were performed on three different MT preparations.
Trang 5Thermal stability
We examined whether the differences in primary and
secondary structure affected the thermal stability of
the two MTs UV absorption spectra were acquired
for fish and mussel Cd7-MTs, after exposure to a
ther-mal gradient A254was plotted as a function of
tem-perature As expected, in both cases we observed a
decrease in percentage absorbance with temperature
increase (Fig 5) The absorbance of fish MT A
declined steadily starting at 30C, with a marked
change in slope above 50C, similar to the description
of D’Auria et al [18] The thermal profile of MT 10
showed a similar trend, but the slope change occurred
at a higher temperature (above 60C) Moreover,
MT 10 maintained higher percentage absorbance at all
temperatures than the fish protein (0.7 vs 0.5 at 90C,
respectively) These results suggest that the mussel MT
is much more thermostable at high temperatures than
the fish protein This is in line with the greater rigidity
suggested by the hydropathic index
Kinetics of metal release
Cysteine residues can be oxidized in vitro by mild
cellu-lar oxidants and release metals during the process
[32–34] It has been suggested that oxidoreductive
mechanisms may also modulate in vivo the affinity of
cysteines for metal ions and regulate the bioavailability
of bivalent metals [35]
In the presence of the glutathione redox couple
(GSH⁄ GSSG), we observed zinc release from both
recombinant MTs The kinetics of this process were similar early on, but, at saturation, MT 10 seemed to release slightly more zinc than MT A (Fig 6A) The difference in metal-releasing ability was much more evident when the Cd-complexed MTs were assayed in the presence of the H2O2 redox partner Because MTs have a higher affinity for cadmium than for zinc (typically, Kd¼ 5Æ10)12 m for zinc and Kd¼ 5Æ10)16m for cadmium), a stronger oxidizing agent such as H2O2 was needed to detach cadmium ions [32,36] Cadmium release was much more marked for
MT 10 than for MT A (Fig 6B)
These data point to a pronounced reactivity of the metal–thiolate clusters in the mussel MT 10, which
Fig 5 Thermal stability of fish and mussel Cd 7 -MTs Absorption
UV spectra were acquired for fish MT A (m) and mussel MT 10
(n) as a function of the temperature increase from 20 to 90 C The
absorbance decrease at 254 nm was reported as a fraction of the
standard absorbance (absorbance at room temperature) in order to
compare the denaturation profile of the Cd–thiolate chromophore of
the two MTs Each curve is representative of four independent
sets of measurements.
Fig 6 Kinetics of zinc and cadmium release from recombinant MTs The metal release was followed by the formation of metal– (PAR)2complex at 500 nm Each experimental point represents the difference between the absorbance measured in the presence and absence of the appropriate redox couple: GSH ⁄ GSSG for zinc and
H2O ⁄ H 2 O2 for cadmium We measured (A) the kinetics of zinc release and (B) the kinetics of cadmium release for fish MT A (m) and mussel MT 10 (n) Each curve is representative of at least three independent sets of measurements.
Trang 6releases zinc and cadmium more quickly and effectively
than fish MT A The kinetic observations of a more
pronounced release of cadmium than zinc fits well with
our data indicating that the major structural
differ-ences are at the level of the a domain, which is in fact
responsible for cadmium binding
Discussion
MTs are noncatalytic metalloproteins, the
physiologi-cal function of which is not yet fully understood The
moderate variability of this class of proteins across
phylogenetically distant organisms reflects the highly
conserved function that they exert in living systems
In contrast, the specific environmental requirements
explain the existence of numerous isoforms in the same
organism A comparative analysis of the functional
and structural features of MTs from different
organ-isms may help to clarify their physiological role
Usually, vertebrate MTs contain 61–62 amino-acid
residues, whereas larger chains with 72–74 residues are
found in molluscs and in nematodes, and shorter
chains have been reported in insects and fungi [37]
In this report, we describe the production and
char-acterization of two MTs from evolutionary distant
aquatic organisms, the fish MT A and the mussel
MT 10 Whereas much information is available for fish
MTs [18,38], characterization of mussel MTs has been
a problem until now [39], which we have overcome by
using the recombinant protein
When MT 10 from M galloprovincialis was
com-pared with MT A from O mykiss, the major finding
was a difference in their primary sequence, mainly at
the level of the a domain These differences suggest
that the mussel protein a domain is larger than the
b domain We therefore postulated an asymmetric
dumbbell shape for MT 10 and different behaviour in
terms of cadmium release and oxidative dimerization,
which occur in this region As expected, we found that
oxidative dimerization was less marked in the mussel
MT 10 than in the fish MT A When the kinetics of
metal release were investigated, MT 10 showed more
pronounced reactivity than MT A We wish to
empha-size that this higher mobility was more marked for
cadmium than for zinc Accordingly, the major
struc-tural differences are concentrated only in the domain
binding this toxic metal The different reactivity can be
attributed to a different spatial arrangement of the
mercaptide bonds, altering their accessibility to
oxid-izing agents
Marked differences between the two proteins
appeared also at the level of their secondary
conforma-tion The CD spectrum of MT 10 lacked both the
245 nm negative and 228 nm positive bands that are typical of vertebrate MTs We hypothesized that these striking differences in the CD spectra are due mainly to the lysine residues, which are highly conserved in ver-tebrate MTs, but not in mussel The lower number of lysine residues in MT 10 than in MT A (6.8% vs 11.5%) may also explain the increased ability of the mussel protein to release metals The increased mobility
of cadmium and zinc of MT 10 may be due to a weaker metal–thiolate interaction because of the reduced num-ber of lysines In fact, substitution of three lysines with glutamates in the CK motifs of the a domain modified the metal-binding ability of MT [40]
Finally, the mussel MT 10 showed greater thermal stability than the fish protein, probably because of its longer polypeptide chain Moreover, MT 10 has a pos-itive hydropathic index (0.199), whereas fish MTs are usually characterized by a negative value ()0.110 for trout MT A) As a higher hydropathic index means lower flexibility, this feature may explain the higher thermal stability of MT 10 This is confirmed by 2D NMR spectroscopy data A preliminary analysis of 2D homonuclear (1H) NOESY spectra, acquired for both proteins, indicates a more rigid structure for MT 10 than for MT A, with both the number of NOE peaks and signal spread being greater in the former (Fig 7) All the above data led us to conclude that the mus-sel MT is different, in terms of spatial conformation and functional properties, from vertebrate MTs, even
if the cadmium content is identical The higher metal mobility and rigidity exhibited by MT 10 is probably related to the environment inhabited by mussels, which are subjected to sudden changes in environmental vari-ables (temperature, anoxia, concentration of aquatic pollutants) The modified a domain, which plays a role
in detoxification⁄ sequestering of toxic metals (e.g cad-mium), would allow adaptation to the requirements of these aquatic organisms
Resolution of the 3D structure of MT 10 at the atomic level will allow us to clarify the structural fea-tures supporting the observed different reactivity For both MTs, besides 2D homonuclear (1H) NOESY spec-tra, 2D heteronouclear (113Cd) NMR spectra have also been acquired, and data processing is in progress How-ever, from a comparison of the raw 2D homonuclear (1H) NOESY spectra, the differences between the two proteins have already been confirmed (Fig 7) When spectra are compared in the same chemical-shift win-dow, a greater number of NOE peaks and signal spread
is palpable in the MT 10 sample, providing clear evi-dence of the difference in the level of structural organ-ization The more the spectrum is ‘crowded’ and the wider the chemical-shift range over which the signal is
Trang 7spread, the more the structure can be assumed to be well
defined, therefore these raw data confirm that MT 10
has a better defined and stable structure than MT A
Experimental procedures
Materials
Chemicals and molecular mass markers were supplied by
Sigma Aldrich (Milan, Italy) Reagents for bacterial growth
were purchased from Fluka (Milan, Italy) T4 DNA ligase
and Taq polymerase were from Stratagene (La Jolla, CA,
USA), and restriction enzymes and dNTPs from Promega
Italia (Milan, Italy) Expression vector pGEX 6P-1, E coli
strains BL21 and JM109, precision protease and
glutathi-one–Sepharose 4B matrix were purchased from Amersham
Biosciences (Uppsala, Sweden) Primers for sequencing and
mutagenesis were synthesized by TibMolBiol (Genoa, Italy)
Cloning and amplification of MTs
The coding sequence of the O mykiss MT A gene [41] was
a gift from Professor P E Olsson (Umea University,
Umea, Sweden) and was cloned as previously described
[42] Recombinant molluscan MT 10 from M
galloprovin-cialis (NCBI GeneBank database accession number
AY566248) was prepared starting from the 222-bp coding
sequence, previously cloned by our group By PCR we added a BamHI site upstream from the ATG codon, using the 5¢-end primer (5¢-CTACTACGAATTAGGATCCCCT GCACCTTG-3¢) and the 3¢-end primer (5¢-GTAATACGA CTCACTATAGGGCGAATTGGG-3¢) Amplification was performed as previously described [42] The PCR fragment was eluted from gel using the NUCLEOSPIN-EXTRACT
MN kit (Du¨ren, Germany) and subcloned into the expres-sion vector pGEX-6P-1 Both recombinant MTs were syn-thesized as fusion proteins, with a GST tail at the N-terminus After enzymatic removal of the GST, MT 10 had four additional amino acids (Gly-Pro-Leu-Gly) with respect to the wild-type, with the initial Met substituted with a Ser (Fig 1) The sequence of the recombinant vector and the correct orientation of the cDNA were checked by sequencing it in both directions using the appropriate pGEX primers (Amersham Biosciences)
Bacterial expression and purification Large-scale expression was carried by inoculating 12.5 mL Luria–Bertani medium containing 100 lgÆmL)1 ampicillin and growing the cells at 37C overnight with vigorous sha-king Then 1 L prewarmed 2XYT medium (16 gÆL)1 tryp-tone, 10 gÆL)1 yeast extract, 5 gÆL)1 NaCl, 100 lgÆmL)1 ampicillin) was inoculated with 10 mL of the overnight culture and grown until mid-exponential growth phase To
Fig 7 Comparison of the whole 2D-NOESY spectra of fish MT A and mussel MT 10 The 2D nuclear Overhauser enhancement spectra (2D-NOESY) were acquired on a Bruker Advance 600 MHz spectrometer (Rheimstetten, Germany) using 2 m M solutions of the proteins in 95% H2O, 5% 2 H2O or 2 H2O at pH 7.0 under a nitrogen atmosphere Spectra are shown in the same chemical-shift window.
Trang 8overexpress the recombinant protein, we added isopropyl
b-d-thiogalactopyranoside to a final concentration of
0.5 mm The highest level of nondegraded MT was observed
after 5 h of growth at 30C For preparing Me7-MTs,
0.2 mm CdCl2 was added to the culture medium, or
alter-natively Zn7-MT the same concentration of ZnCl2
Recombinant MTs were purified by affinity chromatography
using glutathione–Sepharose 4B matrix to selectively bind
the GST tag of the fusion protein The expression showed an
average yield higher than 1 mgÆL)1of culture The bacterial
pellet was resuspended in cold NaCl⁄ Pi (140 mm NaCl,
2.7 mm KCl, 10 mm Na2HPO4, 1.8 mm KH2PO4, pH 7.3)
and lysed by mild sonication at 4C After addition of 1%
Triton X-100, the suspension was mixed gently at 4C for
30 min, and the supernatant was mixed for 30 min with
2 mL 50% slurry resin previously equilibrated Recombinant
MT was recovered by enzymatic cleavage using ‘Prescission
Protease’ (120 UÆmL)1resin) to selectively remove the GST
tail Digestion was carried out at 4C for 16 h directly on
the column equilibrated with digestion buffer (50 mm
Tris⁄ HCl, pH 7, 150 mm NaCl, 1 mm dithiothreitol) [42]
SDS/PAGE electrophoresis and metal
quantification
At each step of the purification procedure, the presence of
the recombinant MT was checked by electrophoresis on
12.5% polyacrylamide gel, performed according to the
clas-sical method of Laemmli [43] Because of the small
dimen-sions and the physicochemical features of MTs, the best
resolution was obtained by 16% Tris⁄ Tricine SDS ⁄ PAGE
[44] The cadmium content in the recombinant MTs was
determined with a polarized Spectra AA 558 atomic
absorption spectrophotometer (Varian, Torino, Italy) The
number of molecules of cadmium bound per molecule of
MT was determined using as a standard curve constructed
using a standard solution of cadmium chloride Both fish
and mussel recombinant MTs contain 7 equivalents of
cad-mium per mol protein
Protein quantification
At each step of the purification, total proteins were
quanti-fied by the Bradford assay [45], with BSA as standard At
the end of the purification, MT was quantified by
measur-ing the absorbance of the metal-free protein at 220 nm in
0.1 m HCl using e220¼ 47 300 m)1Æcm)1[46] Although the
absorption coefficient of molluscan apo-MT should be
higher once the amino-acid content is higher (73 vs 61
resi-dues), the protein concentration was calculated using the
absorption coefficient of vertebrates [24] Alternatively MT
was quantified by estimating the -SH groups using Ellmans’
reagent in potassium phosphate buffer (2 m NaCl in 0.2 m
potassium phosphate, pH 8), using the absorption
coeffi-cient e412¼ 13 600 m)1Æcm)1[47]
Absorption and CD spectroscopy Absorption spectra were acquired after resuspending each recombinant MT (0.025 mgÆmL)1) in 5 mm Tris⁄ HCl (pH 7)⁄ 100 mm NaCl UV spectra were recorded in the wavelength range 200–300 nm, using a Jenway 6505 spec-trophotometer (Felsted Dunmow, Essex, UK), both in standard conditions and after exposure to a linear thermal gradient (25–90C) A broad absorption shoulder occurred near 250 nm when thionein binds cadmium To analyse the formation of the metal–thiolate clusters, we subjected fish and molluscan MTs to titration with bivalent metals (zinc and cadmium): 0.025 mgÆmL)1 each protein was resus-pended in 5 mm Tris⁄ HCl (pH 7.5) ⁄ 100 mm NaCl ⁄ 1 mm dithiothreitol and the spectra were recorded in the range 220–300 nm at increasing metal⁄ protein ratios [38]
CD spectra were recorded on a Jasco J-710 spectropola-rimeter (Jasco, Tokyo, Japan) calibrated with a standard solution of (+)-10-camphosulfonic acid All spectra were recorded in a 0.05-cm path-length quartz cell, using the following parameters: time constant 4 s, scanning speed
20 nmÆmin)1, band width 2 nm, sensitivity 10 millidegrees, step resolution 0.5 nm [48] Photomultiplier high voltage did not exceed 600 V in the spectral region under analysis, and the absorbance never exceeded 1.0 Each spectrum was
an average of five scans over 290–190 nm Protein concen-tration was kept below 0.1 mgÆmL)1 in 5 mm Tris⁄ HCl (pH 7)⁄ 100 mm NaCl All the acquired spectra were correc-ted for the baseline and normalized to the amino-acid con-centration, in order to calculate the mean residual molar ellipticity (degreesÆcm)2Ædecimol)1) All experiments were performed in strictly anaerobic conditions, by purging high-grade nitrogen in the sample chamber To characterize the secondary structure of the two proteins, the acquired CD spectra were analyzed by dedicated software [49–51]
Kinetics of metal release Zinc and cadmium release were estimated spectrophotomet-rically by following the formation of the metal–PAR com-plex at 500 nm For zinc kinetics, the MT samples (1.3 lm protein) were resuspended in 0.2 m Tris⁄ HCl, pH 7.4, and incubated with 100 lm PAR in the absence or presence of 1.5 mm GSH⁄ 3 mm GSSG [32] Cadmium mobility was tested by measuring its transfer from Cd7-MT to PAR induced by the presence of the H2O2⁄ H2O redox couple [33] Samples of 4.6 lm MT were added to the reaction buf-fer (100 lm PAR, 50 mm Tris⁄ HCl, pH 7.4) in the absence
or presence of 1 mm H2O2
Acknowledgements
We would like to extend our gratitude to Professor Gabriella Gallo for her scientific collaboration and
Trang 9Dr Mara Carloni for her experimental contributions.
We thank Dr Giuseppe Digilio (Bioindustry Park del
Canavese spa, Ivrea, Italy) and Professor Mauro Botta
for NMR spectra This research was supported by a
grant from the National Research Council (within
the program ‘Biomolecules for Human Health’) and
from the University of Genova Project for the year
2002 This work was also supported by the 5th UE
Framework Program project Biological Effects of
Environmental Pollution in marine coastal ecosystems
(BEEP) (contract No EVK3-2000-00543)
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