Transcript Slide 1

Biochemical properties of collagen from skin of Gulf coast fish
M.
1
OGAWA ,
M.W.
1
Moody ,
R.J.
3
Portier ,
J.
1
Bell ,
M.
2
Schexnayder ,
and J.N.
1
Losso
1. Department of Food Science and 2. Fisheries Agent,
Louisiana State University Agricultural Center, Baton Rouge, LA 70803
3. Department of Environmental Studies, Louisiana State University,
Baton Rouge, LA 70803
EXPERIMENTAL
Acid-soluble collagen (ASC) was isolated from the skin of black drum
(Pogonias cromis) according to a conventional method. The molecular
properties of collagen were analyzed by SDS-PAGE and circular
dichroism (CD) spectroscopy. Denaturation temperature (Tm) of ASC
was determined based on the melting curve of collagen obtained by
monitoring [θ] at the wavelength of a positive extreme at 220 nm
which is characteristic of the CD spectrum for collagen triple helix.
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Molecular mass and Molecular type
•CD spectrum of the native ASC
sample showed that the ASC
formed collagen triple helices
(Fig. 2).
• Black drum skin ASC has at least two
different chains (1 and 2) and their
cross-linked chains (Fig. 1).
•The structure was lost at 45 ºC.
•Molecular mass of each chain was 126
kDa for 1 and 116 kDa for 2.
200kDa
•The molecular mass of ASC ([1] 22)
was 360kDa.
116kDa
97kDa
67kDa
55kDa
Amino acid composition
• Black drum ASC was rich in Pro,
Gly, and Ala, which are characteristic
of all collagens (Table 1).
• High levels of hydroxylysine (Hyl)
and hydroxyproline (Hyp), similar to
collagens from animal species, were
measured in black drum ASC.
0
205.0
215.0
225.0
235.0
245.0
-10000
Denatured collagen
(45 ºC, 30min)
-20000
-30000
-40000
Native collagen (15 ºC)
-50000
-60000
-70000

10000
Denaturation temperature
1
2
• Transition temperature Tm for
black drum ASC was 34.8 ºC,
which is similar to that of calf hide
ASC (36.3 ºC) (Fig. 3).
8000
6000
4000
2000
0
-2000
-4000
10
20
30
40
50
Temperature (ºC)
Fig. 3. Melting curve of black drum skin ASC
CONCLUSIONS
Table 1. Amino acid composition of skin collagens
Black drum
Coda
Calfb
Hyp
80.2±7.0
40.7
94
Asx
43.9±1.1
41.5
45
Thr
25.9±0.6
26.1
18
Ser
40.7±2.3
60.8
33
Glx
70.1±2.3
77.4
75
Pro
119.6±2.7
89.6
121
Gly
319.8±9.8
332.2
330
Ala
128.7±2.4
105.6
119
Cys
0.0±0.0
ND
ND
Val
19.6±0.5
25.2
21
Met
14.2±0.5
20.6
6
6.9±0.4
17.1
11
Leu
20.8±0.5
29.7
23
Tyr
4.0±0.3
5.0
3
Phe
13.8±0.8
13.8
13
Hyl
5.8±0.9
7.6
7
Lys
23.5±2.8
33.1
26
His
6.0±0.5
11.8
5
Arg
56.5±1.1
62.5
50
Ile
• The distribution patterns of amino
acid composition were closer,
especially for Pro and Hyp, to calf
ASC than to cod ASC.
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Fig. 2. CD spectra of black drum skin ASC
Fig. 1. SDS-PAGE pattern of skin collagen
using NuPAGE Tris-Acetate gel (3-8%)
(Invitrogen)
Black drum
Collagen isolated from land-based animal skin is presently used for
functional food, healthcare, and pharmaceutical applications.
However, in light of BSE and food-and-mouth disease crisis, and the
ban on European Union meat, the use of collagen and collagenderived products from land-based animal skin has been called into
question. Collagens from some other sources are needed. Fish skin
collagen is thought to be a good candidate. The objective in this study
was to isolate fish skin collagen and investigate its biochemical
properties in order to determine the suitability of fish skin collagen as
an alternate of land-based animal collagen.
Wavelength (nm)
[θ]220 (deg cm2 dmol-1)
Acid-soluble collagen (ASC) was isolated from the skin of black drum
harvested in the Gulf of Mexico Coastal waters. Analyses of molecular
weight profile, amino acid composition, and secondary structure
showed that the black drum ASC was typical type-1 collagen. The
molecular mass of 1 and 2 subunits, as determined by SDS-PAGE,
was 127 kDa and 116 kDa, respectively. The amino acid composition
of black drum ASC was closer to calf skin ASC than to cod skin ASC.
Thermal denaturation temperature, measured by melting curve using
circular dichroism, was 34.8 ºC. The literature value for the heat
stability of calf skin collagen is 36.3 ºC. The potentials of collagen
from black drum skin in the functional food, healthcare, and
pharmaceutical industries are discussed.
INTRODUCTION
Secondary structure
RESULTS and DISCUSSION
[θ] (deg cm2 dmol-1)
ABSTRACT
Total
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1000
aYamaguchi et al.(1976); bHerbage et al. (1977).
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• Black drum skin ASC had molecular mass and secondary structure of a
typical type I collagen.
• Black drum skin ASC was similar to calf skin ASC rather than cod skin
ASC in terms of thermal stability and amino acid composition.
• High stability of black drum ASC may be due to high Hyl and Pro contents.
• The ASC from Gulf coast fish skin may be useful for functional food,
healthcare, and pharmaceutical applications.
ACKNOWLEDGEMENTS
This work was supported by a grant from the US Department of
Commerce through Louisiana Sea Grant Project # 167-14-5114. We wish
to thank Mr. Harlon Pearce of LA Fish (Kenner, LA) for supplying the fish
skins used in this research.
REFERENCES
•Yamaguchi, K.; Lavéty, J.; Love, R.M. J. Fd. Technol. 1976, 11, 389399.
•Herbage, D.; Bouillet, J.; Bernengo, J.-C. Biochem. J. 1977, 161, 303312.