Download Protein Cleavage Due to Pro-oxidative Activity in Some Spices

Protein Cleavage Due to Pro-oxidative Activity in Some Spices
Sittiwat Lertsiri
Department of Biotechnology
Faculty of Science, Mahidol University
Phayathai, Bangkok 10400
Thailand
Kanchana Dumri
Department of Chemistry
Faculty of Science, Chiang Mai University
Chiang Mai 50200
Thailand
Keywords: hydroxyl radical, lemongrass, nutmeg, oxidative stress, safflower
Abstract
To investigate the pro-oxidative activity of nutmeg, safflower, and
lemongrass, methanolic extracts of each were incubated with bovine serum albumin
(BSA) in the absence or presence of Fe3+/EDTA/H2O2, Fe2+/ascorbate, or Cu2+/H2O2
at 37°C. The protein cleavage by SDS-polyacrylamide gel electrophoresis (SDSPAGE), protein and free amino groups contents were determined. As a result, BSA
degradation was observed on SDS-PAGE. This corresponded with decrease in
protein concentration and free amino group elevation in the reaction mixture. Such
degradation was inhibited in the presence of mannitol, the hydroxyl radical
scavenger, indicating that the pro-oxidative activity in these spices caused hydroxyl
radical formation.
INTRODUCTION
Oxidative stress describes a state of damage by reactive oxygen species (ROS),
which affects specific molecules or the entire organism. ROS, such as singlet oxygen,
superoxide anion, peroxide radical and hydroxyl radical, are generated both intra- and
extra-cellularly in all aerobic organisms. They are harmful by their action on vital cellular
components including lipids, proteins and nucleic acid. Recent studies showed that
various vegetables, fruits, tea, herb, and spices exerted an antioxidant action. The
antioxidant capacity of these plant foods is due to the presence of anti-oxidative vitamins
(tocopherols, ascorbate), polyphenols, flavonoids, terpenes and various phytochemicals.
Some antioxidants such as ascorbic acid, gallic acid and carotenoids, are found to show
pro-oxidative activity, depending on the reduction-oxidation potential, concentration and
the biological environment. Several flavonoids can also undergo auto-oxidation and
generate ROS, such as hydrogen peroxide (Palozza, 1998; Bagnati et al., 1999). This
study was conducted to determine the pro-oxidative activity of spices, focusing on the
damage of protein due to oxidative stress. The results may lead to better understanding
the pro-oxidative activity in the spices.
MATERIALS AND METHODS
Powdered Spice Samples
Nutmeg (Myristica fragrans H.), safflower (Carthamus tinctorius L.), and
lemongrass (Cymbopogon citrates L.), were purchased from a local market in Chiang
Mai, Thailand. Ten grams of each powdered spices were twice extracted with 75 mL
methanol, and filtered through Whatman filter paper No.1, prior to evaporation.
Effect of Safflower, Lemongrass, and Nutmeg Extracts on Metal-induced
Fragmentation of BSA
Effect of spice extracts on metal-induced fragmentation of bovine serum albumin
(BSA) was investigated by treating albumin 2 mg/mL under three different conditions: 50
µM FeCl3/3 mM H2O2/100 µM EDTA, 50 µM FeSO4/50 µM ascorbic acid, and 100 µM
CuSO4/3 mM H2O2. The systems were in 20 mM phosphate buffer (pH 7.4), incubated at
37°C in the presence or absence of spice extracts as a control in the experiment. Portions
were withdrawn from the reaction mixture every 4 h until 24 h and adding butylated
Proc. WOCMAP III, Vol.6: Traditional Medicine & Nutraceuticals
Eds. U.R. Palaniswamy, L.E. Craker and Z.E. Gardner
Acta Hort. 680, ISHS 2005
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hydroxytoluene methanolic solution to terminate the oxidation. Then, BSA was
precipitated from the reaction mixture by adding 20% w/v TCA (final concentration), and
centrifuged at 10,000 rpm for 20 min. Fragmentation of albumin was monitored on
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Kocha et al.,
1997; Winterbourn, 1981). Protein content and free amino groups were measured by
Bradford method (Bradford, 1976) and by 2,4,6-trinitrobenzenesulfonic acid (TNBS;
Habeeb, 1966), respectively.
Effect of Mannitol as Hydroxyl Radical Scavenger on Metal-induced BSA
Fragmentation
BSA was treated with three different mixtures: 50 µM FeCl3 (0.25 mL)/3 mM
H2O2 (0.5 mL)/100 µM EDTA (0.25 mL), 50 µM FeSO4 (0.5 mL)/50 µM ascorbic acid
(0.5 mL), and 100 µM CuSO4 (0.5 mL)/3 mM H2O2 (0.5 mL) in the presence of mannitol
10 mM. The reaction mixture was analyzed as mentioned above.
RESULTS AND DISCUSSIONS
Effect of Safflower, Lemongrass, and Nutmeg Extracts on Metal-induced
Fragmentation of BSA
The fragmentation of BSA in three different systems were observed on SDSPAGE (Fig. 1, 2 and 3). BSA was fragmented markedly by oxidation reaction in the
Cu2+/H2O2 system since the oxidizing power of the Fe2+/ascorbate and Fe3+/ EDTA/H2O2
on the fragmentation of BSA were much weaker than the Cu2+/H2O2 system (Kocha et al.,
1997). The pattern of protein bands detected on SDS-PAGE suggested that
Fe3+/EDTA/H2O2, Fe2+/ascorbate and Cu2+/H2O2 exerted different oxidation actions in
fragmentation of BSA. Moreover, after 12-h incubation, the reaction mixtures containing
Cu2+/H2O2 exhibited the fragments of BSA at 33, 40 and 52 kDa (data not shown), while
these fragments of BSA were not observed on SDS-PAGE in the other treatments.
Protein content examined by Bradford method also decreased with time to 70-80%
during the 24-h incubation, compared to the control. These corresponded with the results
from the free amino group analysis where the free amino groups increased 20-40% at the
end of the incubation.
Effect of Mannitol as Hydroxyl Scavenger in Metal-induced BSA Fragmentation
Mannitol is one of the most widely used hydroxyl radical scavengers. Therefore,
the fragmentation of BSA is inhibited if the hydroxyl radicals are involved in such
fragmentation. The data from BSA content determination by Bradford method and free
amino group analysis suggested that 10 mM mannitol mostly prevented the fragmentation
of BSA under the conditions studied (data not shown). However, the hydroxyl scavenger
mannitol did not completely protect the BSA fragmentation in the Cu2+/H2O2 system.
This is because BSA could bind tightly to copper ion and led to stronger oxidizing power
than the other systems.
Mechanisms of Metal-induced BSA Fragmentation
The results suggested that the pro-oxidative activity of spice extracts was
dependent on dosage and the transition metal. It is well known that metal-induced
oxidation catalyzes the fragmentation of proteins. Metal ion can promote hydroxyl radical
formation and further bring about protein degradation as observed in BSA and cartilage
(Kocha et al., 1997). In the Cu2+/H2O2 system, copper bound to BSA (Cu-BSA) on the
specific sites may participate in the Fenton reaction, and hydroxyl radicals would be
formed on the BSA molecule surface. This leads to specific pattern of fragmentation.
In case of the Fe2+/ascorbate and Fe3+/H2O2/EDTA systems, Fe2+ and Fe3+ weakly
bind to BSA molecules without specific binding site. Hence the damage of BSA
molecules by hydroxyl radicals occurred randomly on the protein surface. The presence
of spice extract would enhance the hydroxyl radical formation, which further promoted
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BSA fragmentation.
Literature Cited
Bagnati, M., Perugini, C., Cau, C., Bordone, R., Albano, E. and Bellomo, G. 1999. When
and why a water-soluble antioxidants becomes pro-oxidant during copper-induced
low-density lipoprotein oxidation. Biochem. J. 340:143-152.
Bradford, M.M. 1976. Protein determination by dye-binding method. Anal. Biochem.
72:248-254.
Habeeb, A.F.A.S. 1966. Determination of free amino groups in proteins by
trinitrobenzenesulfonic acid. Anal. Biochem. 14:328-336.
Kocha, T., Yamaguchi, M., Ohtaki, H., Fukuda, T. and Aoyagi, T. 1997. Hydrogen
peroxide-mediated degradation of protein: different oxidation modes of copper- and
iron-dependent hydroxyl radicals on the degradation of albumin. Biochim. Biophys.
Acta 1337:319-326.
Palozza, P. 1998. Pro-oxidant actions of carotenoids in biologic systems. Nut. Rev.
56:257-265.
Winterbourn, C.C. 1981. Hydroxyl radical production in body fluids: roles of metal ions,
ascorbate and superoxide. Biochem. J. 198:125-131.
Figures
200 kDa -116------97-----66-----
45-----
Lane: Marker 1
2
3
4
5
6
7
8
Fig. 1. SDS-PAGE of BSA in the presence of safflower extract, incubated at 37°C with
different metal-induced oxidation systems for 24 h.
Lane 1: 10 µg BSA
Lane 2: 10 µg BSA + 100 µM CuSO4/3 mM H2O2
Lane 3: 10 µg BSA + 50 µM FeSO4/50 µM Ascorbic acid
Lane 4: 10 µg BSA + 50 µM FeCl3/3 mM H2O2/100 µM EDTA
Lane 5: 10 µg BSA + 1 mg safflower extract
Lane 6: 10 µg BSA + 100 µM CuSO4/3 mM H2O2 + 1 mg safflower extract
Lane 7: 10 µg BSA + 50 µM FeSO4/50 µM Ascorbic acid + 1 mg safflower extract
Lane 8: 10 µg BSA + 50 µM FeCl3/3 mM H2O2/100 µM EDTA + 1 mg safflower
extract
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200 kDa -116------97-----66----45-----
Lane:
Marker 1
2
3
4
5
6
7
8
Fig. 2. SDS-PAGE of BSA in the presence of lemongrass extract, incubated at 37°C with
different metal-induced oxidation systems for 24 h.
Lane 1: 10 µg BSA
Lane 2: 10 µg BSA + 100 µM CuSO4/3 mM H2O2
Lane 3: 10 µg BSA + 50 µM FeSO4/50 µM Ascorbic acid
Lane 4: 10 µg BSA + 50 µM FeCl3/3 mM H2O2/100 µM EDTA
Lane 5: 10 µg BSA + 1 mg lemongrass extract
Lane 6: 10 µg BSA + 100 µM CuSO4/3 mM H2O2 + 1 mg lemongrass extract
Lane 7: 10 µg BSA + 50 µM FeSO4/50 µM Ascorbic acid + 1 mg lemongrass
extract
Lane 8: 10 µg BSA + 50 µM FeCl3/3 mM H2O2/100 µM EDTA + 1 mg
lemongrass extract
90
200 kDa -116------97-----66-----
45-----
Lane:
Marker 1
2
3
4
5
6
7
8
Fig. 3. SDS-PAGE of BSA in the presence of nutmeg extract, incubated at 37°C with
different metal-induced oxidation systems for 24 h.
Lane 1: 10 µg BSA
Lane 2: 10 µg BSA + 100 µM CuSO4/3 mM H2O2
Lane 3: 10 µg BSA + 50 µM FeSO4/50 µM Ascorbic acid
Lane 4: 10 µg BSA + 50 µM FeCl3/3 mM H2O2/100 µM EDTA
Lane 5: 10 µg BSA + 1 mg nutmeg extract
Lane 6: 10 µg BSA + 100 µM CuSO4/3 mM H2O2 + 1 mg nutmeg extract
Lane 7: 10 µg BSA + 50 µM FeSO4/50 µM Ascorbic acid + 1 mg nutmeg extract
Lane 8: 10 µg BSA + 50 µM FeCl3/3 mM H2O2/100 µM EDTA + 1 mg nutmeg
extract
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