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Food Chemistry 123 (2010) 231–236
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Catalytic mechanisms of metmyoglobin on the oxidation of lipids in
phospholipid liposome model system
B. Min a, K.C. Nam b, D.U. Ahn a,c,*
a
Department of Animal Science, Iowa State University, Ames, IA 50011, USA
Department of Animal Science and Technology, Sunchon National University, 540-742, Republic of Korea
c
Department of Agricultural Biotechnology, Major in Biomodulation, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-921, Republic of Korea
b
a r t i c l e
i n f o
Article history:
Received 19 August 2009
Received in revised form 7 March 2010
Accepted 1 April 2010
Keywords:
Metmyoglobin
Lipid oxidation
Liposome system
Iron chelating agents
a b s t r a c t
The catalytic mechanism of metmyoglobin (metMb) on the development of lipid oxidation in a phospholipid liposome model system was studied. Liposome model system was prepared with metMb solutions
(2.0, 1.0, 0.5, and 0.25 mg metMb/mL) containing none, diethylenetriamine pentaacetic acid (DTPA), desferrioxamine (DFO), or ferric chloride and lipid oxidation was determined at 0, 15, 30, 60, and 90 min of
incubation at 37 °C. Metmyoglobin catalysed lipid oxidation in the liposome system, but the rate of lipid
oxidation decreased as the concentration of metMb increased. The amount of free ionic iron in the liposome solution increased as the concentration of metMb increased, but the rate of metMb degradation
was increased as the concentration of metMb decreased. The released free ionic iron was not involved
in the lipid oxidation of model system because ferric iron has no catalytic effect without reducing agents.
Both DFO and DTPA showed antioxidant effects, but DFO was more efficient than DTPA because of its
multifunctional antioxidant ability as an iron and haematin chelator and an electron donor. The antioxidant activity of DTPA in liposome solution containing 0.25 mg metMb/mL was two times greater than
that with 2 mg metMb/mL due to the increased prooxidant activity of DTPA-chelatable compounds. It
was concluded that ferrylmyoglobin and DTPA-chelatable haematin generated from the interaction of
metMb and LOOH, rather than free ionic iron, were the major catalysts in metMb-induced lipid oxidation
in phospholipid liposome model system.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Myoglobin has been recognised as a major catalyst for lipid oxidation in meat, but its mode of action for catalysing lipid oxidation
is controversial. It has been suggested that the interaction of metmyoglobin (metMb) with hydrogen peroxide (H2O2) or lipid hydroperoxides (LOOH) results in the formation of ferrylmyoglobin,
which can initiate free radical chain reactions (Chan, Faustman,
Yin, & Decker, 1997; Davies, 1990; Egawa, Shimada, & Ishimura,
2000; Kanner & Harel, 1985; Min & Ahn, 2005; Rao, Wilks, Hamberg, & Ortiz de Montellano, 1994). In addition, ferrylmyoglobin
as well as metMb can degrade LOOH to free radicals such as alkoxyl
and peroxyl radicals (Reeder & Wilson, 1998, 2001), which can initiate and/or catalyse a series of propagation and termination step
in the free radical chain reactions of lipid oxidation (Frankel,
1987; Halliwell & Gutteridge, 1990). However, others limited the
role of myoglobin as only a source for free ionic iron or haematin
(Ahn & Kim, 1998; Kanner, Shegalovich, Harel, & Hazan, 1988;
* Corresponding author at: Department of Animal Science, Iowa State University,
Ames, IA 50011, USA. Tel.: +1 515 294 6595; fax: +1 515 294 9143.
E-mail address: duahn@iastate.edu (D.U. Ahn).
0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.04.013
Puppo & Halliwell, 1988). They indicated that free ionic iron and/
or haematin released from myoglobin in the presence of H2O2 or
lipid hydroperoxide, rather than ferrylmyoglobin, were the major
catalysts for lipid oxidation in meat. The ratio of peroxides to metMb is a determining factor for the formation of ferrylmyoglobin or
the release of free ionic iron or haematin (Rhee, Ziprin, & Ordonez,
1987). Haematin is released from myoglobin in the presence of
H2O2, followed by the liberation of free ionic iron from haematin
(Prasad, Engelman, Jones, & Das, 1989). Haematin reacts with
H2O2 or lipid hydroperoxide to form haematin with higher oxidation state (Ferrylhaematin, Fe(IV = O)), which can initiate and propagate lipid oxidation (Kim & Sevanian, 1991). Dix and Marnett
(1985) indicated that LOOH such as linoleic acid hydroperoxide
were more efficient for haematin-catalysed lipid oxidation than
H2O2, and ferrylhaematin and alkoxyl radical (LO) generated from
the interaction of haematin with LOOH were responsible for the
haematin-catalysed lipid oxidation. Haematin can be easily intercalated into membrane due to its hydrophobicity and catalyse lipid
oxidation (Schmitt, Frezzatti, & Schreier, 1993).
The concentration of metMb is a determining factor for its
prooxidative activity in the presence of fatty acid or LOOH (Baron,
Skibsted, & Andersen, 2002; Lapidot, Granit, & Kanner, 2005). In
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B. Min et al. / Food Chemistry 123 (2010) 231–236
addition, myoglobin shows a pseudo-hydroperoxidase activity in
the presence of reducing agents such as ascorbic acid and phenolic
antioxidants to remove lipid hydroperoxides (Gorelik & Kanner,
2001; Harel & Kanner, 1989).
Iron chelators such as diethylenetriamine pentaacetic acid
(DTPA) and desferrioxamine (DFO) have been widely used to elucidate the mechanism of iron compounds on lipid oxidation (Ahn,
Wolfe, & Sim, 1993; Harel, Salan, & Kanner, 1988). DFO has been
known as an excellent chelating agent for ferric ion and DTPA for
ferrous and ferric ions (Kanner & Harel, 1987; Rahhal & Richter,
1989). Both DFO and DTPA have chelating ability to haematin
(Radi, Turrens, & Freeman, 1991). DFO can also act as an electron
donor to ferrylmyoglobin to suppress the prooxidant activity of
ferrylmyoglobin and release free ionic iron from metMb as well
as to free radicals to break down the free radical chain reaction
of lipid oxidation (Rice-Evans, Okunade, & Khan, 1989).
The objectives of this study were to determine the concentration effect of metMb and the effect of ferric ion and chelators such
as DFO and DTPA on the metMb-induced lipid oxidation in the
phospholipid liposome model system.
2. Materials and methods
2.1. Chemicals and reagents
Metmyoglobin (from equine skeletal muscle), linoleic acid, 2thiobarbituric acid (TBA), ferrozine (3-(2-pyridyl)-5,6-bis (4-phenyl sulphonic acid)-1,2,4-triazine), neocuproine (2,9-dimethyl1,10-phenanthroline), ferric chloride, diethylenetriamine pentaacetic acid (DTPA), desferrioxamine (DFO), chelex-100 chelating resin (50–100 dry mesh, sodium form), butylated hydroxytoluene
(BHT), and Tween-20 were purchased from Sigma (St. Louis, MO).
All other chemicals and reagents used were of reagent grade.
Deionised distilled water (DDW) by Nanopure infinity™ ultrapure
water system with ultraviolet (UV) (Barnstead, Dubuque, IA) was
used for the preparation of all reagents and buffers. All DDW and
buffers were treated with the chelex-100 chelating resin to remove
any free metal ion before use.
2.2. Preparation of metmyoglobin solution
An appropriate amount of metMb was dissolved in 50 mM acetate buffer (pH 5.6) at 4 °C. The metMb solution was centrifuged at
3000g at 4 °C for 60 min to remove undissolved impurities. The concentration of metMb and percentages of metMb in the solution
were calculated according to Krzywicki (1982). The metMb concentration of solution was adjusted to 2.0, 1.0, 0.5, and 0.25 mg/mL
with 50 mM acetate buffer (pH 5.6). The average concentration of
metMb and percentages of metMb were 2.02 ± 0.02, 1.02 ± 0.01,
0.5 ± 0.01, and 0.25 ± 0.00 mg/mL and 100.82 ± 0.19%, 100.90 ±
0.24%, 100.84 ± 0.12%, 100.28 ± 0.86%, respectively. DTPA (2 mM;
final concentration), DFO (2 mM; final concentration), and ferric
chloride (5 lg/mL; final concentration) were added to the metMb
solutions. The metMb solution was treated with Chelex-100 chelating resin to remove any free ironic ion present before use.
2.3. Lipid oxidation potential in metmyoglobin–liposome model
system
The metMb–liposome model system was prepared using egg
phospholipids. The fatty acid composition of the phospholipids
(Table 1) was determined by the method of Ahn, Wolfe, and Sim
(1995). An aliquot of phospholipids dissolved in chloroform was
placed in a scintillation vial and evaporated under nitrogen gas
to make thin film on the wall. The metMb solution containing
none, DTPA, DFO, or ferric chloride was added to a phospholipidcoated vial and shaken vigorously for 2 min to make metMb–liposome solution with final concentration of 3 mg phospholipids per
mL. The solution was incubated at 37 °C for 90 min to accelerate lipid oxidation. Lipid oxidation was determined at 0, 15, 30, 60, and
90 min. An aliquot (0.5 mL) of the solution was mixed with 10 lL
BHT solution (6% BHT in ethanol), added with 1 mL TBA/TCA solution (15 mM TBA/15% trichloroacetic acid (TCA; w/v)), and incubated in boiling water bath for 15 min. After cooling, the mixture
was centrifuged at 15,000g for 10 min. The absorbance of the
supernatant was determined at 531 nm against a reagent blank. Lipid oxidation was expressed as mmol malondialdehyde (MDA)
equivalents (eq.) per kg phospholipids, calculated from the molar
extinction coefficient of 1.56 105 M 1 cm 1. In addition, the generation of nonheme iron during the incubation was measured at 0,
15, 30, 60, and 90 min using the ferrozine method of Min and Ahn
(2009) with modification. In brief, sample (0.6 mL) and ascorbic
acid (0.2 mL, 1% in 0.2 M HCl, w/v) were thoroughly mixed with
11.3% TCA solution (w/v, 0.4 mL). After 5 min at room temperature,
the mixture was centrifuged at 3000g for 15 min at 20 °C. The
supernatant (1 mL) was mixed with 0.4 mL of 10% ammonium acetate (w/v) and 0.1 mL of the ferrozine colour reagent. After colour
development at room temperature for 10 min, the absorbance
was determined at 562 nm against a reagent blank. The concentration of nonheme iron released from metMb during reaction was
expressed as lg iron/mL metMb–liposome solution. All measurements were quadruplicated.
2.4. Lipoxygenase-like activity of metmyoglobin
Lipoxygenase-like (LOX-like) activity of metMb (1 mg/mL) was
measured by the method of Gata, Pinto, and Macias (1996) with
some modifications. Linoleic acid (10 mM) in 0.02 M NaOH solution emulsified with Tween-20 was used as a substrate solution,
which was flushed with and kept under nitrogen. The reaction
mixture was composed of 80 lL of the substrate solution, 80 lL
of each metMb solution as an enzyme solution, and 50 mM acetate
buffer (pH 5.6) to a final volume of 1 mL. Lipoxygenase-like activity
was assessed by the increase of absorbance at 234 nm due to the
generation of conjugated dienes from linoleic acid at 27 °C. The results were expressed as units of activity (U) per mL, calculated
from the molar extinction coefficient of hydroperoxyl linoleic acid
(e = 25,000 M 1 cm 1). One unit of lipoxygenase-like activity was
defined as the amount of enzyme catalysing the formation of
Table 1
Fatty acid composition of phospholipids used in model system.
Fatty acid
Content (%)
Myristic acid
Palmitic acid
Palmitoleic acid
Margaric acid
Margaroleic acid
Stearic acid
Oleic acid
trans-Vaccenic acid
Linoleic acid
c-Linolenic acid
Gondoic acid
Arachidonic acid
DTA
DPA
DHA
0.19 ± 0.03
28.70 ± 0.22
1.28 ± 0.18
0.28 ± 0.01
0.12 ± 0.03
16.25 ± 0.14
27.01 ± 0.20
1.59 ± 0.15
15.38 ± 0.14
0.17 ± 0.02
0.21 ± 0.03
6.68 ± 0.09
0.41 ± 0.08
0.14 ± 0.02
1.59 ± 0.04
Means was expressed with the standard deviation. n = 4.
Abbreviations: DTA, all cis-7,10,13,16-docosatetraenoic acid; DPA, all-cis7,10,13,16,19-docosapentaenoic acid, DHA, all cis-4,7,10,13,16,19-docosahexaenoic
acid.
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1 lmol of hydroperoxide per minute. All measurements were
quadruplicated.
2.5. Statistical analysis
All the analyses were performed on the samples with four replications. Data were analysed using the JMP software (version
5.1.1; SAS Institute Inc., Cary, NC). Differences among mean values
were determined by the Student-Newman–Keuls’ multiple range
test (P < 0.05) (Kuehl, 2000).
3. Results and discussion
1.2
20
Mb0.25
Mb0.5
Mb1.0
Mb2.0
PL
15
Nonheme iron content (µg / mL)
TBARS value (mmol MDA eq. / kg PL)
Metmyoglobin, at all concentrations, induced lipid oxidation
and increased the TBARS values linearly in phospholipid liposome
model system during the 90 min-incubation (Fig. 1). However, the
increasing rate of TBARS values significantly decreased with the increase of metMb concentration (P < 0.05): the highest rates at lower metMb concentrations (0.199 and 0.194 mmol MDA eq./kg
phospholipid per min at 0.25 and 0.5 mg/mL, respectively), followed by 0.177 mmol MDA eq./kg phospholipid per min at
1.0 mg/mL and 0.157 mmol MDA eq./kg phospholipid per min at
the highest metMb concentration (2.0 mg/mL) (P < 0.05). Especially, after 60 and 90 min of incubation, the TBARS values at the
highest metMb concentration (2 mg/mL) (11.43 and 15.52 mmol
MDA eq./kg phospholipids, respectively) was significantly lower
than those at the lowest concentration (0.25 mg/mL) (12.50 and
18.43 mmol MDA eq./kg phospholipid) (P < 0.05). The presence of
LOOH was detected right after the preparation of the liposome
model system (data not shown). Trace amount of LOOH during
the preparation of liposome solution have been widely recognised
(Halliwell & Gutteridge, 1990; Kim & Sevanian, 1991). This result
indicates that the concentration of metMb is a critical factor for
determining prooxidant activity of myoglobin in the presence of
LOOH and/or fatty acid: at low concentrations, metMb acts as a
prooxidant (Baron et al., 2002; Lapidot et al., 2005).
The amount of free ionic iron significantly increased during
incubation, and was proportional to the concentration of metMb
(Fig. 2). The concentrations of free ionic iron after 90 min of incubation were 15.93, 11.82, 11.10, and 7.88 lM at 2.0, 1.0, 0.5, and
0.25 mg metMb per mL metMb–liposome solution, respectively,
indicating that 13.94%, 20.69%, 38.85%, and 55.14% of metMb in
2.0, 1.0, 0.5, and 0.25 mg/mL, respectively, were decomposed and
liberated free ionic iron. These results agreed with many previous
reports (Baron & Andersen, 2002; Lapidot et al., 2005), which suggested that the interaction of H2O2 or LOOH with metMb caused
the liberation of free ionic iron as well as haematin. Thus, the LOOH
preexisted or generated during the incubation should be the major
catalysts to release free ionic irons from metMb because H2O2 was
not added in this study.
Prasad et al. (1989) suggested that haematin was released from
myoglobin before free ionic iron release in the presence of H2O2
and the amount of haematin released from metMb during incubation was greater than that of free ionic iron released. Thus, the
amount of haematin and free ionic iron produced during incubation should be proportional to the concentration of metMb in the
liposome system. The release of haematin was confirmed by Chiu
et al. (1996) but it was readily decomposed by LOOH to release free
ionic iron (Kim & Sevanian, 1991). Haematin-catalysed lipid oxidation more efficiently than ionic iron because of its hydrophobicity
that allowed it to permeate into membrane (Schmitt et al., 1993).
Although haematin was more active than other hemeproteins
and ferrous ion (Chiu et al., 1996; Kaschnitz & Hatefi, 1975), the ratio of haematin to lipids was the determining factor for its prooxidant activity (Schmitt et al., 1993). They suggested that haematin
formed either dimer at low ratio or aggregated at high ratio in
aqueous solution: a dimer was less effective than a monomer for
lipid oxidation but could permeate to membrane where it was degraded to monomer, and aggregates were inactive. The haematin
monomer within membrane interacted with LOOH to form alkoxyl
radical and haematin-containing hypervalent iron (Fe(IV) = O) both
of which were regarded as initiators and catalysts for the haematin-catalysed lipid oxidation (Dix & Marnett, 1985; Kim & Sevanian, 1991). Therefore, a high amount of haematin at a high
concentration of metMb in a liposome system (2 mg/mL) should
be partially responsible for the lower lipid oxidation rate, compared to that at lower metMb concentrations (<1 mg/mL) in Fig. 1.
The addition of ferric ion did not affect myoglobin-catalysed lipid oxidation in phospholipid liposome model system (Fig. 3), indicating that either ferrylmyoglobin or haematin generated from
metMb rather than free ionic iron was the major catalyst for metMb-induced lipid oxidation in this system. It has been suggested
that the oxidation state of iron is more important than the amount
of iron for the development of lipid oxidation in model system
10
5
Mb0.25
Mb0.5
Mb1.0
Mb2.0
PL
1
0.8
0.6
0.4
0.2
0
0
0
10
20
30
40
50
60
70
80
90
Reaction time (min)
Fig. 1. Lipid oxidation potential of metMb with various concentrations in
phospholipid liposome model system during incubation at 37 °C for 90 min (TBARS:
mmol malondialdehyde (MDA) equivalents/kg phospholipid (PL)). The concentrations of metMb in 50 mM acetate buffer (pH 5.6) were 2 (Mb2.0), 1 (Mb1.0), 0.5
(Mb0.5), and 0.25 (Mb0.25) mg per mL, respectively. Phospholipid liposome model
system with buffer alone was used as a control (PL). Means with standard deviation
were expressed. n = 4.
0
10
20
30
40
50
60
70
80
90
Reaction time (min)
Fig. 2. Formation of nonheme iron in a phospholipid liposome model system with
various concentrations of metMb during incubation at 37 °C for 90 min (lg
nonheme iron/mL metMb–liposome solution). The concentrations of metMb in
50 mM acetate buffer (pH 5.6) were 2 (Mb2.0), 1 (Mb1.0), 0.5 (Mb0.5), and 0.25
(Mb0.25) mg per mL, respectively. Phospholipid liposome model system with buffer
alone was used as a control (PL). Means with standard deviation were expressed.
n = 4.
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B. Min et al. / Food Chemistry 123 (2010) 231–236
A. 0.25 mg metmyoglobin / mL
Mb
Fe(III)
DTPA
DFO
PL
Mb
Fe(III)
DTPA
DFO
PL
20
TBARS value (mmol MDA eq. / kg PL)
20
TBARS value (mmol MDA eq. / kg PL)
B. 1.0 mg metmyoglobin / mL
15
10
5
0
15
10
5
0
0
10
20
30
40
50
60
70
80
90
Reaction time (min)
0
10
20
30
40
50
60
70
80
90
Reaction time (min)
Fig. 3. Lipid oxidation potential of metMb treated with desferrioxamine (DFO, 2 mM; final concentration), diethylenetriamine pentaacetic acid (DTPA, 2 mM; final
concentration), or ferric chloride (Fe(III), 5 lg/mL; final concentration) in phospholipid liposome model system during incubation at 37 °C for 90 min (TBARS value, mmol
malondialdehyde (MDA) equivalents/kg phospholipid (PL)). The final concentrations of metMb in liposome solution were 0.25 (A) and 1.0 (B) mg per mL, respectively.
Phospholipid liposome model system with metMb and buffer were used as a control (Mb) and blank control (PL), respectively. Means with standard deviation were
expressed. n = 4.
(Ahn & Kim, 1998). However, the released free ionic iron may play
a significant role in the acceleration of lipid oxidation in meat
where the ferric ion-reducing capacity has been detected (Ahn &
Kim, 1998; Kanner, Salan, Harel, & Shegalovich, 1991).
Iron chelators, DTPA and DFO, showed different antioxidant
effects in the liposome model system (Fig. 3). DFO inhibited
myoglobin-catalysed lipid oxidation effectively, but DTPA showed
only partial inhibitions. Both DTPA and DFO are known as strong
iron chelators and inhibit free ionic iron-catalysed lipid oxidation
(Graf, Mahoney, Bryant, & Eaton, 1984). However, DFO showed
stronger antioxidant activity than DTPA. The antioxidant activity
of DTPA was affected by the ratio of DTPA to free ionic iron,
but DFO was not. DFO can act not only as an efficient iron chelator but also an electron donor or hydrogen donor to ferrylmyoglobin, resulting in the suppression of ferrylmyoglobin-catalysed
lipid oxidation (Rice-Evans et al., 1989). Rice-Evans et al. (1989)
suggested that DFO can prevent the release of free ionic iron from
myoglobin by reducing ferrylmyoglobin and breaking free radical
chain reactions. In addition, DFO can interact with haematin via
the iron moiety to prevent their catalytic and membrane-intercalating activity for lipid oxidation (Baysal, Monteiro, Sullivan, &
Stern, 1990). On the other hand, DTPA can inhibit iron-catalysed
lipid oxidation by occupying all six coordination sites of iron.
Also, DTPA can inhibit haematin-catalysed lipid oxidation (Radi
et al., 1991). Free haematin may have one or two unoccupied or
loosely bound coordination sites. It is assumed that DTPA or
DFO may bind to those coordination sites to inactivate the catalytic activity of haematin, but no evidence is available. DTPA
did not inhibit lipid oxidation catalysed by ferrylmyoglobin (Harel
& Kanner, 1988). Consequently, the high inhibitory effect of DFO
was from the synergistic effect of DFO as a chelator for chelatable
compounds, probably haematin, and an electron donor to ferrylmyoglobin and free radicals, whereas the partial effect of DTPA
was attributed to its chelating ability, indicating that DTPA-che-
latable compounds, haematin (Baysal et al., 1990; Radi et al.,
1991), was partially responsible for the metMb-induced lipid oxidation in the liposome model system. Free ionic iron (ferric form)
was already ruled out because it did not show any prooxidant
effect in model system (Ahn & Kim, 1998). The antioxidant activity (95.24%) of DFO at low myoglobin concentration (0.25 mg/mL)
was higher than that (89.43%) at high myoglobin concentration
(P < 0.05). Moreover, the antioxidant activity (36.24%) of DTPA
in liposome model system with low concentration of metMb
(0.25 mg/mL) was twice as high as that (18.08%) with high concentration (>1.0 mg/mL) (Fig. 3A and B) (P < 0.05), indicating that
DTPA-chelatable compound, probably haematin, was contributed
more to the development of lipid oxidation at lower than at higher concentration of metMb.
LOX-like activity is related to the generation of conjugated
diene at initial stage of lipid oxidation. LOX-like activity of
metMb was not changed by ferric ion in the absence of reducing
agents (Fig. 4), indicating that free ionic ion released from
myoglobin was not involved in the initiation of lipid oxidation
in metMb-induced lipid oxidation. The addition of DFO and DTPA
to the liposome model system decreased LOX-like activity of
metMb, but DTPA (34.98%) suppressed it more effectively than
DFO (15.57%).
In this study, the catalytic mechanism of metMb on lipid oxidation was investigated in phospholipid liposome solutions
incubated at 37 °C, which is different from the refrigerated temperature conditions (4 °C) for normal meat storage and distribution. In
general, the reaction rates increase as the reaction temperature increase. Although temperature at or below 37 °C is not likely to
change the nature of metMb, it may affect the reactivity and/or solubility of metMb and other compounds such as lipids and haematin. In addition, meat products contain various anti- and
prooxidative factors. Therefore, further studies on the effect of
low temperature and other factors on metMb-induced lipid oxida-
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B. Min et al. / Food Chemistry 123 (2010) 231–236
Lipoxygenase-like activity (Unit / mL)
12
a
10
a
b
8
c
6
4
2
0
1
Control
DFO
DTPA
Fe(III)
Fig. 4. Lipoxygenase-like activity (Unit/mL) of metMb solution treated with none
(control), metMb (1 mg/mL; final concentration) desferrioxamine (DFO, 2 mM; final
concentration), diethylenetriamine pentaacetic acid (DTPA, 2 mM; final concentration), or ferric chloride (Fe(III), 5 lg/mL; final concentration) in 50 mM acetate
buffer, pH 5.6. Means with different letters (a–c) are significantly different
(P < 0.05). n = 4.
tion are needed to strengthen the proposed catalytic mechanism of
metmyoglobin on lipid oxidation in this study.
4. Conclusion
Lipid oxidation in phospholipid liposome model system was
accelerated in the presence of metMb. Increases in metMb concentration in model system decreased lipid oxidation, due to the ratio
of myoglobin to LOOH or fatty acid and the ratio of haematin to
lipids. The concentration of free ionic iron released from metMb increased during incubation but was not involved in the development of lipid oxidation. The addition of DFO and DTPA inhibited
lipid oxidation. DFO was more effective than DTPA because DFO
can inactivate haematin and reduce ferrylmyoglobin and free radicals whereas DTPA only binds to haematin. The metMb-induced lipid oxidation was caused by both ferrylmyoglobin and haematin
generated from the interaction of metMb with LOOH in phospholipid liposome system, rather than the released free ionic irons.
Acknowledgement
The work has been supported by the National Integrated Food
Safety Initiative/USDA (USDA Grant 2002-5110-01957), Washington DC, and WCU (World Class University) program (R31-10056)
through the National Research Foundation of Korea funded by
the Ministry of Education, Science and Technology.
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