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378 Part 3: Meat, Poultry and Seafoodsendopeptidase, revealed differences between collagens from
skin and bone, and both were completely different from those of
calfskin collagen (Kittiphattanabawon et al. 2005).
Nalinanon et al. (2008) reported that threadfin bream skin
collagen (PSC) obtained with the aid of tuna pepsin showed
similar protein patterns compared with those found in ASC.
Nevertheless, pepsin from skipjack tuna caused the degradation
ofα- andβ-components. All collagens were classified as type I
with large portion ofβ-chain, consists of two identicalα1-chains
and oneα2-chain. However, proteins with MW greater than 200
kDa were abundant in ASC. Recently, Benjakul et al. (2010)
extracted PSCs from the skin of two species of bigeye snapper,
P. tayenusandP. macracanthus, with the aid of tongol tuna
(T. tonggol) pepsin and porcine pepsin. PSCs from the skin of
both species extracted using porcine pepsin had a higher content
ofβ-chain but a lower content ofα-chains compared with those
extracted using tuna pepsin.
The elution profiles of ASC and PSC from the skin of
arabesque greenling on the TOYOPEARL CM-650M column
after being denatured by heat treatment in the presence of 2 M
urea showed that the primary structure was modified to some de-
gree by albacore tuna pepsin digestion (Nalinanon et al. 2010).
β12/β22 dimer of ASC and PSC was the major component,
and high-MW component andγ-chain were also detected in the
same fractions. It was suggested that some ofα2-component
might either dimerize covalently intoβ-component and form
β12- orβ22-dimer or polymerize into higher MW components.
As a result, much lower band intensity ofα2-chain was detected
on SDS–PAGE and band intensity ratios ofα1/α2 more than
twofold were observed (Nalinanon et al. 2010). Collagens from
alkali–acid extraction and alkali direct extraction of great blue
shark skin were composed ofα-chains (α1 andα2) (Yoshimura
et al. 2000). Based on protein patterns and TOYOPEARL CM-
650M column chromatography, ASC and PSC from the skin of
brownbanded bamboo shark containedα-andβ-chains as their
major components and were characterized as type I collagen
with the cross-link ofα2-chain, which was different from type
I collagen from teleost fish species (Kittiphattanabawon et al.
2010a). The similar result was observed in collagens from the
skin of blacktip shark (Carcharhinus limbatus) (Kittiphattan-
abawon et al. 2010c). The chromatographic fractions indicated
by the numbers were subjected to SDS–PAGE. The fractions of
ASC (Fig. 20.6A) and PSC (Fig. 20.6B) were eluted as two ma-
jor peaks. Theα1-chain was found in the first peak (fraction nos.
28–40 and 23–36 for ASC and PSC, respectively), while theα2-
andβ-chains were found in the second peak (fraction nos. 41–59
and 37–55 for ASC and PSC, respectively). The results revealed
that both collagens might be type I collagen, although the band
intensity ofα1-chain was not twofold higher than that ofα2-
chain.α2-component might dimerize into theβ-component and
formβ 12 -dimer (Hwang et al. 2007). As a result, much lower
band intensity ofα2-chain was detected on SDS–PAGE. Similar
results have been reported for collagen type I from other elas-
mobranches (Hwang et al. 2007, Bae et al. 2008). Hwang et al.
(2007) purified PSC from skate skin by differential salt precip-
itation, followed by phosphocellulose column chromatography.
The collagen was purified as major collagen and minor collagen.The major collagen was defined as type I collagen, contained
α1andα2, with dimerization ofα2-chain intoβ-chain and
formβ12-dimer. This result was in accordance with other elas-
mobranch type I collagen obtained from other reports. For the
minor collagen, it containedα1 andα2 and was classified as
type V collagen.Fourier Transform Infrared SpectroscopyGenerally, the collagens from fish skin such as Nile perch (Muy-
onga et al. 2004), deep-sea redfish (Wang et al. 2007), channel
catfish (Liu et al. 2007), yellowfin tuna (Woo et al. 2008), carp
(Duan et al. 2009), arabesque greenling (Nalinanon et al. 2010),
brownbanded bamboo shark (Kittiphattanabawon et al. 2010a),
and blacktip shark (Kittiphattanabawon et al. 2010c) exhibited
similar FTIR spectra in which the absorption bands were situ-
ated in the amide band region including amides A, B, I, II, and
III. A slight difference in FTIR spectra between ASC and PSC
from deep-sea redfish (Wang et al. 2007) and arabesque green-
ling (Nalinanon et al. 2010) has been reported, indicating slight
differences in the secondary structure and functional groups of
collagens. Amide A band (3400–3440 cm−^1 ) is generally asso-
ciated with the N–H stretching vibration and shows the existence
of hydrogen bonds (Woo et al. 2008, Kittiphattanabawon et al.
2010a). When the NH group of a peptide is involved in a hy-
drogen bond, the position is shifted to lower frequencies. Amide
B band of collagens is observed at 2924–2926 cm−^1 , which
is related to CH 2 asymmetrical stretching vibration (Muyonga
et al. 2004). The amide I and II bands of several fish colla-
gens are observed at the wavenumber of 1640–1654 cm−^1 and
1531–1555 cm−^1 , respectively (Muyonga et al. 2004, Duan et al.
2009, Nalinanon et al. 2010, Kittiphattanabawon et al. 2010a,
2010c). The amide I band, with characteristic frequencies in the
range of 1600–1700 cm−^1 , is mainly associated with the stretch-
ing vibrations of the carbonyl groups (C=O bond) along the
polypeptide backbone and is a sensitive marker of polypeptide
secondary structure (Payne and Veis 1988, Wang et al. 2007,
Kittiphattanabawon et al. 2010c). The shoulder of amide I band
appearing at 1637 cm−^1 could be attributed to the triple helix
absorption of collagens (Petibois et al. 2006). Amide II (∼ 1550
cm−^1 ) is associated with N-H bending coupled with C-N stretch-
ing (Muyonga et al. 2004, Woo et al. 2008). Amide III band of
collagens is observed at 1234–1238 cm−^1 , which is related to C-
N stretching and N–H in plane bending from amide linkages as
well as absorptions arising from wagging vibrations from CH 2
groups from the glycine backbone and proline side chains (Mohd
Nasir et al. 2006) involved in triple-helical structure of collagen
(Muyonga et al. 2004, Wang et al. 2007, Woo et al. 2008). The
triple-helical structure of collagens is also confirmed from the
absorption ratio between Amide III and 1450 cm−^1 bands, which
was approximately equal to 1.0 (Plepis et al. 1996, Wang et al.
2007, Kittiphattanabawon et al. 2010a). A slight difference in
this ratio between ASC and PSC from deep-sea redfish (Wang
et al. 2007) and arabesque greenling (Nalinanon et al. 2010) has
been reported. The result suggested that pepsin might affect the
triple-helical structure of collagen to some degree. The differ-
ences in those ratios and wavenumbers of amide bands between