Both superoxide and H2O2 increase the rate of Keywords antioxidation; fusion protein; hemoglobin; superoxide dismutase Correspondence L.. We show that the engineered SOD–Hb fusion protei
Trang 1protein showing enhanced antioxidative properties
Marie Grey1, Sakda Yainoy1,2, Virapong Prachayasittikul2and Leif Bu¨low1
1 Department of Pure and Applied Biochemistry, Centre for Chemistry and Chemical Engineering, Lund University, Sweden
2 Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand
Introduction
The toxicity of Hb outside of its natural protective red
blood cell environment has been linked partly to the
redox activity of the Hb molecule Consequently, Hb
can react with, and generate, reactive oxygen species
such as the superoxide anion Hb, both inside and
out-side of the red blood cell, readily undergoes
autoxida-tion, but the degree of oxidation is normally restricted
to approximately 3% of the total Hb by the metHb
reductase system However, if damage occurs to the red
blood cells, Hb may be released and, as the normal
pro-tection systems, involving also superoxide dismutase
(SOD) and catalase, are no longer associated with the
Hb, Hb is exposed to oxidative damage Autoxidation
leads to nonfunctional metHb (HbFe3+) and
superox-ide ions (Eqns 1–4) Furthermore, reoxygenation of a
tissue previously deprived of oxygen will produce
super-oxide, a phenomenon known as reperfusion injury [1,2]
HbFe2þO2þ H2O2! HbFe4þ@O þ H2Oþ O2 ð1Þ
HbFe2þO2! HbFe3þþ O
HbFe3þþ H2O2!HbFe4þ@ O þ H2O ð3Þ
HbFe4þ@ O þ H2O2! HbFe3þþ O2 þ H2O!
Superoxide can be reduced either enzymatically by SOD, or spontaneously by dismutation to H2O2 Both superoxide and H2O2 increase the rate of
Keywords
antioxidation; fusion protein; hemoglobin;
superoxide dismutase
Correspondence
L Bu¨low, Department of Pure and Applied
Biochemistry, Centre for Chemistry and
Chemical Engineering, Lund University,
P.O Box 124, SE-211 00, Lund, Sweden
Fax: +46 46 222 4611
Tel: +46 46 222 9594
E-mail: Leif.Bulow@tbiokem.lth.se
(Received 13 May 2009, revised 27 July
2009, accepted 24 August 2009)
doi:10.1111/j.1742-4658.2009.07323.x
Much of the toxicity of Hb has been linked to its redox activity; Hb may generate reactive oxygen species, such as the superoxide anion Superoxide
is intrinsically toxic, and superoxide dismutase (SOD) provides important cellular protection However, if the Hb molecule is located outside the red blood cell, the normal protection systems involving SOD and catalase are
no longer closely associated with it, exposing Hb and its cellular surround-ings to oxidative damage In order to produce less toxic Hb molecules, we have explored gene fusion to obtain homogeneous SOD–Hb conjugates The chimeric protein was generated by coexpressing the human Hb a-chain⁄ manganese SOD gene together with the b-chain gene in Escherichia coli We show that the engineered SOD–Hb fusion protein retains the oxygen-binding capacity and, moreover, decreases cytotoxic ferrylHb (HbFe4+) formation when challenged with superoxide radicals The SOD–
Hb fusion protein also exhibits a 44% lower autoxidation rate and higher thermal stability than Hb alone
Abbreviations
DSC, differential scanning calorimetry; ROS, reactive oxygen species; SOD, superoxide dismutase.
Trang 2metHb formation [3] and eventually lead to heme
degradation The catalase present intracellularly may
be sufficient to remove H2O2 by conversion to water
[4]; however, there is not sufficient SOD to eliminate
the superoxide Chang [2], Alagic [4] and Tarasov [5]
have characterized Hb chemically conjugated to SOD
alone or SOD combined with catalase, i.e Hb–SOD
and polyHb–SOD–catalase, respectively The latter
Hb conjugate has been shown to have antioxidative
properties and offer protection against reperfusion
injury [6]
In order to create proximity between SOD and Hb,
we consequently explored gene fusion Such closeness
generates a favorable microenvironment in which the
formed superoxide ions can be directly taken care of
The SOD–Hb protein was prepared by linking the
human Hb a-chain gene with the human manganese
SOD gene Manganese SOD was chosen because it
shows lower product inhibition by H2O2 and has a
much longer half-life in serum, 5–6 h as compared to
6–10 min for copper and zinc SOD [7,8] This
approach enabled us to produce the SOD–Hb in
Escherichia coliand to obtain a homogeneous product
We characterized this novel enzyme by analyzing the
stability, activity and antioxidative properties of the
fused SOD–Hb, and we then compared it with human
Hb, also produced in E coli
Results
Protein expression and purification
It is notoriously difficult to produce human Hb in
microorganisms, because the a-chains and b-chains
must be produced in equal amounts to give functional
Hb molecules The a-chains are especially prone to
precipitation and degradation if expression is
unbal-anced [9] By linking the SOD gene to the a-chain
gene, we could stabilize fusion protein expression The
proteins were purified using affinity chromatography;
details can be found in Doc S1 After optimization of
the expression protocol, SOD–Hb was expressed well,
with a yield of 8 mgÆL)1, which is four times higher
than the yields obtained with other Hb fusion proteins
[10] The expression level was lower for SOD–Hb than
for wild-type Hb, but may be further optimized by
introducing compensatory mutations to increase
expression and improve heme transport into the cell
[11] Denaturation SDS⁄ PAGE analysis (Fig 1)
showed bands of the expected sizes: 16 kDa for
b-chain Hb, and 38 kDa for SOD–a-chain Hb In
addition, SOD–Hb showed the characteristic red color
of Hb
Molecular mass determination Molecular masses were determined by gel filtration on
a Superdex 200 prep grade column, which gave four components Two of these, corresponding to almost 50% of the peak area (Fig 2), were complexes larger than 160 kDa The remaining peaks corresponded to SOD–Hb in the monomer and dimer forms at equal concentrations (where the Hb b-chain is included; Fig 2)
Activity measurements The SOD activity assay showed a SOD activity of 1.3· 105UÆlmol)1 for SOD–Hb, which means that the fusion protein retained more than half (52%)
of the native SOD activity This corresponds closely
to the amount of high molecular mass SOD–Hb As the native SOD is functional as a tetramer, it is possi-ble that the dimer and monomer fractions of SOD–Hb exhibit much lower activity, or may even be nonfunc-tional [7,12]
In the CO and O2 binding assays, the protein sam-ples were reduced with sodium dithionite and then gently bubbled with CO or O2 (Fig 3) SOD–Hb showed the typical absorption spectrum characteristics
of human Hb, with peaks located at wavelengths of
417, 536 and 566 nm, which are almost identical to those of HbCO (417, 537 and 567 nm) An O2 spec-trum was also recorded, and SOD–Hb again showed the typical peaks at 414, 538 and 573 nm, similar to HbO2 (413, 539 and 574 nm) In particular, the O2 peaks of the fusion protein in the visible region appear to be slightly broader than those of Hb, and there also appears to be a minor peak at 630 nm on the CO spectrum These could be explained by very low amounts of ferric protein in both the SOD–Hb spectra [13]
Fig 1 The results of SDS ⁄ PAGE on crude extract and purified sample Lane 1: molecular mass marker Lane 2: crude extract of SOD–Hb Lane 3: purified SOD–Hb.
Trang 3Autoxidation studies were performed at room tem-perature (20–22C) for 48 h, using an Hb concentration
of 8 lm, and followed using visible spectrophotometry The rate constant for Hb (0.18 h)1) was almost twice as high as that for SOD–Hb (0.10 h)1)
Stability Thermal stability was investigated using differential scanning calorimetry (DSC) The asymmetric shape of the DSC curve (Fig 4) suggests a complex denatur-ation path, which is irreversible under the present con-ditions Thermal denaturation of Hb begins with the dissociation of the tetramer into monomers [14], and ends with a certain degree of aggregation The
Fig 2 Schematic representation of SOD–Hb
showing possible conformations resulting
in (from top to bottom): the high molecular
mass complex, an SOD–Hb dimer, and finally
an SOD–Hb monomer.
A
B
Fig 3 Reduced SOD–Hb and Hb were bubbled with O 2 (A) or CO
(B) SOD–Hb shows the absorption spectrum characteristics typical
of human Hb for both O2and CO Critical regions of the protein
spectra have been enlarged The scale on the y-axis is offset for
clarity.
Fig 4 DSC was performed on 60 l M Hb (dashed line) or 30 l M SOD–Hb (solid line) Cp, heat capacity.
Trang 4broader, less well-defined peak of SOD–Hb indicates
contributions from more than one thermal process,
and thus a more intricate mixture of complexes (e.g
higher-order ‘aggregates’), in agreement with the
results of the gel filtration experiments showing several
species In addition, both Hb and SOD are normally
tetramers, which could account for the numerous
ther-mal processes as they dissociate into dimers and
mono-mers In addition to dissociation into dimers, the
complexity of the DSC curve is increased by the
ther-mal inactivation of the SOD enzymatic activity,
result-ing in smaller conformational changes [15], which
probably contribute to the broadness of the peak
Apparent Tmvalues were calculated using the Microcal
software origin, and found to be 48.7C (Hb) and
55.1C (SOD–Hb), corresponding to the Tm value of
53.2C reported by Olsen [16] (HbACO) SOD–Hb
thus exhibits greater heat stability than Hb, although
lower stability than SOD alone (Tm of 68–94C)
[12,17]
Heme reactivity
Heme loss is dependent on the geometry of the protein
and, possibly, also water shielding Oxidation of the
heme to the met form (Fe3+) increases the probability
of heme loss as the fifth coordination bond to the
proximal histidine is weakened [18] The fast phase has
been associated with heme loss from the b-chains,
whereas the slow phase is attributed to the a-chains
[19] SOD–Hb showed a heme loss rate of
0.19 ± 0.05 min)1 for the fast phase, whereas the loss
rate of Hb was almost three times lower
(0.069 ± 0.008 min)1) Although the b-chains do not
take part directly in the fusion, the effect is more
profound here, suggesting that the b-chains are less
protected during fusion than Hb As expected, for the
slow phase both Hb and SOD–Hb a-chains
exhi-bited lower heme loss rates (0.011 ± 0.001 and
0.017 ± 0.003 min)1, respectively), indicative of higher
heme affinity
SOD–Hb and Hb were also incubated together with
a xanthine⁄ xanthine oxidase superoxide-generating
sys-tem After 30 min of incubation, sodium sulfide, which
reacts with ferrylHb to form sulfHb, was added As
shown in Fig 5, SOD–Hb was significantly more
effec-tive in reducing the amount of ferrylHb formed
(P = 0.0018) than Hb
Heme degradation of Hb and SOD–Hb by H2O2
was measured by monitoring the fluorescence of the
degradation products (Fig 6) For all H2O2
concentra-tions used (1, 2 and 4 mm), the amount of heme
degra-dation for SOD–Hb was much lower than for Hb
alone For both proteins, increasing the H2O2 concen-tration increased the denaturation, as indicated by flu-orescence measurements Blank measurements without
H2O2 gave no contribution to the fluorescence signal (data not shown)
Discussion
The proximity between two proteins is often impor-tant For instance, the physical closeness between two or several enzymes catalyzing sequential reac-tions is a feature of many metabolic pathways The frequently observed improved kinetic behavior of such associated proteins has been explained by the formation of a favorable ‘microenvironment’ in
Fig 5 FerrylHb formation was measured with 10 l M Hb or SOD–
Hb *Significantly different from Hb (P = 0.0018, Student’s t-test).
Fig 6 The formation of fluorescent heme degradation products was measured at a fixed concentration of oxyHb (15 l M ) and vary-ing concentrations of H2O2: e, Hb with 4 m M H2O2; D, Hb with
2 m M H2O2; h, Hb with 1 m M H2O2;¤, SOD–Hb with 4 mM
H 2 O 2 ; , SOD–Hb with 2 m M H 2 O 2 ; , SOD–Hb with 1 m M H 2 O 2
Trang 5which the local concentration of intermediates is
higher Similarly, a second protein in an aggregate
can provide protection against a toxic compound or
intermediate
There are several ways of creating proximity
between proteins A frequently used technique involves
chemical cross-linking of the relevant proteins This
normally generates protein conjugates with random
linking and random orientation of the active centers
Proximity between two or more proteins may also be
achieved by performing in-frame gene fusion of the
corresponding structural genes The chimeric gene then
encodes a polypeptide chain carrying two or more
active centers This strategy resembles the proposed
model for the evolution of naturally occurring
multi-functional proteins [20] Through comparison of DNA
sequences from different species, it has been
demon-strated that such proteins probably evolved through
gene translocation followed by gene fusion Extensive
studies of such naturally occurring protein aggregates
have enabled the three-dimensional structures to be
determined for some of them, notably phosphoribosyl
anthranilate isomerase⁄ indole glycerol phosphate
syn-thase and tryptophan synsyn-thase Besides generating
homogeneous conjugates, this method also allows for
in vivo testing of the fusion proteins, as opposed to
chemical approaches
Superoxide is intrinsically toxic, and SOD provides
important cellular protection Superoxide radicals
and⁄ or impaired SOD functionality have been
impli-cated in diabetes, cancer and neurodegenerative
dis-eases, in addition to cellular damage such as lipid
peroxidation, protein oxidation, and DNA damage
[21,22] Hb may generate reactive oxygen species,
which can be particularly deleterious in clinical
appli-cations, e.g in blood substitutes When a gene fusion
approach is used, the link between the SOD and Hb
is always present between the same amino acid
resi-dues, which precludes heterogeneity Conversely, the
chemically linked Hb–SOD products described by
Alagic [4] and Tarasov [5] have cross-links introduced
at different residues, as well as reactive groups, which
must be blocked in a separate step In these chemical
linking approaches, usually both intra-tetramer and
inter-tetramer bonds are introduced, with the
involve-ment of multiple side chains [23] In addition, it has
been shown that shorter cross-links may provide
better stabilized Hb [24] The SOD–Hb was designed
with a very short linker of only one alanine, in order
to promote proximity, as well as to reduce
proteo-lytic degradation during production However, our
SOD–Hb shows the presence of different aggregation
forms, with large complexes as well as dimers and
monomers The gel filtration step described could, however, be used to remove these fractions As it is likely that the monomer and dimer forms are inac-tive, this simple step could increase the homogeneity
of the product as well as removing the less active fractions
SOD–Hb retains about half of the specific activity
of native SOD This lower activity may be due to the steric difficulties encountered in forming the necessary SOD tetramers [7,12] The tetrameric conformation is important for the formation of an active site suitable for Mn ligation [12] An alternative strategy could be
to coexpress native SOD with our fusion protein, which may facilitate tetramer formation Similarly, we can coexpress native Hb a-chains together with SOD–
Hb to generate a free N-terminal end of this polypep-tide chain It is therefore essential to consider subunit interactions when engineering fusion proteins, particu-larly when producing larger Hb conjugates As indi-cated earlier, catalase is a key component when generating functional Hb-based blood substitutes We have prepared an SOD–catalase fusion protein that can be coexpressed with SOD–Hb in E coli By exploring the natural SOD subunit interactions, we can then form a SOD–catalase–Hb protein The poly-Hb–SOD–catalase developed by Chang [2] also showed lower activity, 85–90% of that of native SOD, depend-ing on the conjugation ratio, implydepend-ing that this prepa-ration also suffers from steric hindrance However, the chemically linked Hb–SOD [4] showed identical activ-ity for both conjugated and free SOD, probably owing
to locking of the SOD in the tetrameric form during conjugation
The genetic fusion of Hb and SOD does not seem to perturb the globin structure noticeably, as both the O2 and CO spectra are essentially identical to those of
Hb All peaks in the Soret region correspond well to that of Hb, indicative of full functionality The autoxi-dation rate constants were determined to be 0.18 h)1 for Hb and 0.10 h)1 for SOD–Hb at room tempera-ture The rate constant for SOD–Hb is thus approxi-mately half of that for Hb, although higher than the values reported by Vandegriff (0.007–0.0021 h)1) [18] This may partly be explained by the residual catalase activity, as the Hb in their study was derived from outdated donated blood
An in vitro test was performed to evaluate the antioxidative properties of SOD–Hb SOD–Hb and
Hb were incubated together with a superoxide⁄ H2O2 -generating system The formation of ferrylhemoglobin with SOD–Hb after 30 min was significantly lower (P = 0.0018) than with Hb (Fig 5) FerrylHb is cytotoxic and has been implicated in oxidative stress
Trang 6situations in a range of diseases [25,26] The ability to
reduce the formation of this toxic species means that
SOD–Hb has considerable promise for the future
development of a clinical product Interestingly,
extra-cellular Hb polymers with SOD activity, with a
molec-ular mass of over 3800 kDa, occur naturally in the
blood of earthworms [32,33]
Heme degradation of Hb and SOD–Hb by H2O2
was measured by monitoring the fluorescence of the
degradation products The oxyHb first reacts with one
molecule of H2O2, forming oxyferrylHb (HbFe4+=O)
When a second molecule of H2O2 reacts with the
oxy-ferrylHb, metHb and a superoxide radical (Eqns 2–4)
are produced, initiating the degradation process The
damage is irreversible, and leads to a cascade of
reac-tions that ultimately result in iron release and
fluores-cent degradation products The superoxide radical,
which has a lifetime of 0.2 s [27], is perfectly located to
react with the heme group Moreover, the superoxide
exhibits higher reactivity, owing to the heme pocket
environment [28,29] As can be seen in Fig 6, heme
degradation in SOD–Hb was much lower than in Hb,
and both showed an increase in denaturation when the
H2O2 concentration was increased The combination
of SOD and Hb thus protects the heme molecule from
denaturation during oxidative stress in vitro, probably
via a proximity effect
The heme degradation is dependent on the lifetime
of ferrylHb and the susceptibility of the heme to
super-oxide-induced damage The close association of SOD
with Hb can allow removal of the superoxide radicals
quickly, leading to less degradation, as shown in this
study Additional modifications of SOD–Hb, e.g by
introducing mutations affecting ferryl reduction
kinet-ics, may further reduce the Hb toxicity We have
previ-ously shown that either introducing or removing
suitably located tyrosines affects ferryl reduction
kinet-ics in human Hb [26] Introducing the same mutations
in SOD–Hb could result in a SOD–Hb molecule that
is even more suitable for practical use Additionally, to
allow further in vivo studies of our construct, suitable
protein surface protection, such as pegylation or
encapsulation, needs to be developed
In conclusion, the engineered SOD–Hb exhibits a
lower autoxidation rate and higher thermal stability
than Hb alone, and the process creates a homogeneous
link between the Hb and the SOD, which chemical
conjugation does not Additionally, in vitro tests show
that cytotoxic ferrylHb formation is significantly
decreased in the presence of superoxide radicals
Con-sequently, the combination of SOD and Hb protects
the Hb molecule from denaturation during oxidative
stress
Experimental procedures
Construction of human manganese SOD and human SOD–Hb
Please see the Supporting information Primers used for site-directed mutagenesis or cloning can be found in Table S1
Protein expression and purification Protein expression and purification were performed essen-tially as described previously [26] Details can be found in Doc S1
Molecular mass determination Gel filtration chromatography was used for molecular mass determination on an A¨KTA purifier system controlled by unicorn software (GE Healthcare, Uppsala, Sweden) Highly purified protein was loaded on a HiLoad 16⁄ 60 Superdex 200 column (also GE Healthcare), equilibrated with 50 mm phosphate buffer (pH 7.2) containing 0.15 m EDTA, and eluted with the same buffer at a flow rate of 1.0 mLÆmin)1 Standard proteins with molecular masses ranging from 6.5 to 158.0 kDa (GE Healthcare) were used
to produce standard curves
SOD activity assay The assay used to determine SOD activity was a slightly modified version of that described by Ewing [30] In prin-ciple, the assay is based on the ability of SOD to inhibit nitroblue tetrazolium reduction by an aerobic mixture of NADH and prenazine methosulfate, which produces superoxide at nonacidic pH Details can be found in Doc S1
DSC DSC measurements were performed with a Microcal differ-ential scanning calorimeter (Microcal, Northampton, MA, USA) with a cell volume of 0.5072 mL All samples were degassed for 15 min at room temperature prior to scanning Baseline scans were obtained with buffer in both the refer-ence and sample cells, and these were later subtracted from sample scans Protein samples (60 lm Hb or 30 lm SOD– Hb) in 70 mm sodium phosphate (pH 7.2) were scanned in the temperature range 20–90C at a rate of 60 CÆh)1
Heme loss rates The heme exchange rate between metHb and human serum albumin was determined as described by Benesch [19] and
Trang 7Jeong [31], with slight modifications Potassium ferricyanide
was added in excess in order to oxidize the proteins to the
ferric form, and this was followed by removal of
ferricya-nide and ferrocyaferricya-nide with a Sephadex G-25 column (GE
Healthcare) equilibrated with 0.05 m bis-Tris and 0.1 m
NaCl (pH 7.5) Each Hb sample was then mixed with
human serum albumin (5 lm final concentration of each),
and 0.5 m Tris buffer (pH 9.05) was added to a total
vol-ume of 2 mL The absorbance (A) at 578 and 620 nm was
then recorded every minute for 90 min, using a Beckman
Coulter DU-800 spectrophotometer The amounts of
metHb and methemalbumin were calculated using the
following two equations:
metHb
½ ¼ 146:03 A578 134:48 A620
and
methemalbumin
½ ¼ 61:95 A578þ 220:01 A620
The data were then fitted to a double-exponential decay
curve, essentially as described by Vandegriff [18]:
Y¼ A ek fast tþ ek slow t
þ C:
Autoxidation
The autoxidation analysis was performed as described by
Jeong [31], but with a lower Hb concentration of 8 lm CO
was removed by shining light on the sample, which was
kept on ice, while gently oxygenating the solution using a
stream of oxygen gas The reaction was carried out in
0.1 m sodium phosphate (pH 7.0) at room temperature
(20–22C) Spectra from 400 to 700 nm were collected at
specific times, using a Beckman Coulter DU-800
spectro-photometer The baseline was adjusted by setting the
absor-bance at 700 nm to zero The experimental data were then
fitted to a single-exponential equation of the form [18]:
Y¼ DYmax 1 ekt
þ Y0 where Y is the relative metHb concentration (%), DYmaxis
the total relative change in metHb at the end of the
reac-tion, k is the rate constant, t is time, and Y0is the relative
metHb concentration at t = 0
Measurement of ferrylHb formation
FerrylHb formation was measured using xanthine⁄ xanthine
oxidase as the oxidation system, based on a method
described by D’Agnillo [2,3] Ten micromolar Hb or
SOD–Hb in 70 mm sodium phosphate (pH 7.2) was reacted
with 100 lm xanthine and 10 mUÆmL)1 xanthine oxidase,
both from Sigma-Aldrich (Stockholm, Sweden) The Hb
concentration was calculated on the basis of heme, using
the relation: e523= 7.12 mm)1Æcm)1[32] At given times, an
excess of catalase (Roche, Mannheim, Germany) was added
to remove residual H2O2, after which 2 mm sodium sulfide was added; finally, the absorbance at 620 nm (A620 nm) was measured
Measurement of heme degradation by fluorescence
Fluorescence measurements originally described by Nagababu [33] were slightly modified OxyHb (15 lm) was incubated with H2O2(1, 2 or 4 mm) in 50 mm potas-sium phosphate buffer (pH 7.4) in a total volume of
2 mL at 25C Reagent-grade H2O2 (30% v⁄ v) was obtained from Sigma-Aldrich, and standardized using an extinction coefficient of 72.8 m)1Æcm)1 at 230 nm [34] The fluorescence signal was recorded for 30 min at excita-tion and emission wavelengths of 460 and 525 nm, respec-tively, using a fluorimeter (PTI Photon Technology International, London, Canada)
Acknowledgements
This study was supported by European Union Frame-work VI Eurobloodsubstitutes project and the Staff Development Project by the Ministry of Education, Thailand (SY)
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Supporting information
The following supplementary material is available: Doc S1 Experimental procedures
Table S1 Primer sequences
This supplementary material can be found in the online version of this article
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