JOURNAL OF FOOD SCIENCE

CHEMISTRY/BIOCHEMISTRY

Desmin Degradation in
Postmortem Fish Muscle
V. Verrez-Bagnis, J. Noel, C. Sautereau, and J. Fleurence

ABSTRACT
The degradation of desmin was studied in postmortem white dorsal muscle from
sea bass, brown trout, turbot and sardine, stored at 0-4C. Based on desmin
specific antibody tests, results showed that the extent and rate of desmin degradation in postmortem fish muscle varied considerably among species. There was
no degradation of desmin during the first 4 days postmortem storage of sea bass
and brown trout, whereas 10 to 20% of muscle desmin was degraded during the
first 4 days of postmortem storage of turbot. Sardine muscle presented a very
complex pattern of desmin degradation products and aggregated desmin fragments detected within the first 24h. Most polypeptides were larger than the
nondenatured desmin. Desmin degradation would not be a suitable marker for
evaluation of postmortem changes of stored fish.
Key words : desmin, fish, proteolysis, degradation, white muscle

INTRODUCTION
P OSTMORTEM TENDERIZATION OF FISH
muscle is a major problem related to freshness and quality and many studies have been
undertaken on the deterioration of fish muscle during ice storage. However, little is
known about such changes. The changes vary
depending upon a range of factors such as
species, physiological condition, stress prior to death and temperature of postmortem
storage. Fish muscle contains cytoskeletal
proteins which form a continuous intracellular network spanning the muscle fiber.
Key cytoskeletal structure includes intermediate filaments with the main protein
desmin. Desmin polymerizes to form intermediate filaments surrounding each Z-line
and individual myofibrils interconnecting to
one another and to the cell membrane at the
level of their Z-discs (Granger and Lazarides,
1978 ; Lazarides, 1980). It is unclear whether the desmin filaments are interwoven into
the Z-disc (Yang and Makita, 1995). Desmin
intermediate filaments are essential in the
maintenance of myofibril, myofiber and
whole muscle tissue structural and functional integrity (Capetanaki et al., 1997). The
postmortem degradation of desmin seems to
be involved in the increased fragility of myofibrils during postmortem storage in land
animal meat, in beef muscle (Hwan and
Bandman, 1989; Taylor et al., 1995; Ho et
al., 1996), in pork muscle (Hortos et al., 1994;
Morrison et al., 1998) and in chicken (TakaAuthors Verrez-Bagnis, Nol and Fleurence are with
the Quality and Physico-Chimie Laboratory, Institut
Franais de Recherche pour lExploitation de la Mer
(IFREMER), rue de lIle dYeu, B.P. 21105, 44311
Nantes Cedex 03, France. Author Sautereau was a
student affiliated with this laboratory. Address inquiries to Dr. V. Verrez-Bagnis.

240

hashi, 1996). Intermediate filament degradation may tenderize meat by facilitating the
separation of myofibrils and thus, weakening the lateral strength of meat (Young et al.,
19801981).
Our objective was to evaluate the rate and
extent of desmin degradation in postmortem
white muscle of four fish species.

MATERIALS & METHODS

RABBIT ANTI-GIZZARD CHICKEN DESMIN
antibodies and phosphatase-coupled anti-rabbit IgG were obtained from Sigma Chimie
(LIsle dAbeau Chesnes, France). 4-(2-Aminoethyl)-benzenesulfonyl fluoride (Pefabloc-SC) was purchased from Interchim
(Montluon, France). Prestained molecular
weight markers and nitrocellulose membranes were from Biorad (Ivry sur Seine,
France). All other chemicals were analytical
grade.
Muscle samples

Live sea bass (Dicentrarchus labrax)

were obtained from the Socit Epinrine
dAquaculture (Noirmoutier, France), live
brown trout (Salmo trutta) were from the
SEMII fish station (IFREMER/INRA, Camaret, France) and live turbot (Psetta maxima) were from France Turbot Society (Noirmoutier, France). Sardine (Sardina pilchardus) were caught on the Atlantic coast (near
La Turballe, France) around 6h before arriving in the laboratory. Sea bass, turbot, and
sardine were beheaded and eviscerated, and
then stored in ice in a 4C room. Brown trout
had been filleted before storage at 4C. During storage, total protein extracts (proteins
soluble in 8M urea and 1 % sodium dodecyl
sulfate SDS) were prepared within 1 h after
slaughter to provide pre-rigor samples (ex-

JOURNAL OF FOOD SCIENCEVolume 64, No. 2, 1999

cept for sardine) and every 24h. A total protein extract was also prepared from chicken
gizzards purchased from a local supermarket. Total protein extracts prepared from prerigor and aged muscles stored at 04C were
prepared in triplicate (except for sardine
where they were prepared in quadruplicate)
and analyzed for desmin proteolysis by immunoblotting with a polyclonal antiserum
after SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
Total protein extracts

Total protein extracts were prepared according to the modified procedure of Hwan
and Bandman (1989). White dorsal muscle
(1g) was homogenized in 8 mL solution containing 8M urea, 0.5 mM Pefabloc-SC (instead of 1 mM phenylmethylsulfonylfluoride
PMSF) and 1% 2-mercaptoethanol. SDS and
Tris-HCl (pH 7.0) were added to the homogenate to a final concentration of 1%, respectively. The homogenate was boiled 3 min and
centrifuged at 15,000 g for 10 min.
Electrophoresis and blot

Protein extracts were added to 0.5 volumes of SDS sample buffer as described by
Wang (1982). Samples were heated to 100C
for no longer than 2 min and stored at -80C.
Samples were loaded onto a 10% SDS-PA
gel (70 mm 80 mm) (Laemmli, 1970) and
electrophoresis was carried out at 30 mA for
1h.
After electrophoresis, proteins were transferred at 25V overnight to a nitrocellulose
sheet in 20 mM Tris-HCl, 150 mM glycine,
20% methanol, 0.01% SDS. After blocking
with 8% non-fat dried milk in 0.9% NaCl,
the nitrocellulose sheet was incubated with
rabbit anti-gizzard chicken desmin antibodies for 1h and washed with phosphate buffered saline (PBS) 150 mM NaCl in 10 mM
Na2HPO4 / NaH2PO4 (pH 7.2) containing
0.05% Tween 20 (v/v) solution. Desmin and
related fragment bands were detected by incubating membrane with goat anti-rabbit IgG
antibodies labelled with phosphatase diluted
in 5% (w/w) bovine serum albumin in 0.9%
NaCl and using nitro blue tetrazolium / 5bromo-4-chloro-3-indolyl phosphate (NBT/
BCIP) as substrate.
To estimate the molecular weight of fragments from desmin proteolysis and to precisely evaluate the extent of proteolysis, all
bands in Western blotting lanes were inte 1999 Institute of Food Technologists

grated by means of a scanner coupled with

Bioimage software (Ann Arbor, MI). The
ratio of each band to the sum of all bands
(bp or bi / Sbp +bi) where bp=fragment bands
and bi=intact desmin band) was calculated
for each lane. Prestained molecular weight
markers were also run on the same sheet, giving precise information about the molecular
weight of each band.

RESULTS & DISCUSSION

THE METHOD OF WESTERN IMMUNOBLOTting was used for analyzing desmin degradation. Total protein extracts from aged
chicken gizzard and from pre-rigor and postrigor fish white muscle were tested with polyclonal rabbit antibodies against chicken gizzard desmin. The immunospecificities of
these antibodies were compared (Fig. 1).
They reacted only with desmin in fish muscle and they also reacted with smaller peptides which likely were degradation fragments of desmin in aged chicken gizzard.
For farmed fish, like brown trout or sea
bass, no degradation fragment was observed
even after 3 or 4 days storage at 0 or 4C.
These experimental data confirmed the results of Busconi et al. (1989) that no desmin
degradation occurred in white croaker stored
7 days at 0C. In turbot, a desmin fragment
with an apparent molecular weight of 49 kDa
was detected after 2 days storage (Fig. 2A).
The molecular weight of intact muscle
desmin depended on fish species and varied
from about 53 kDa to 57 kDa.
In sardine, known to lose freshness very
fast (Andrade et al., 1997), the polyclonal
antibodies reacted not only with intact
desmin, but also with many degradation fragments of desmin and also, unexpectedly, with
polypeptides bigger than desmin (Fig. 2B).
We hypothesized that these molecules were
aggregates between different degradation
fragments or between such fragments and
intact desmin. Hubbard and Lazarides (1979)
and OShea et al. (1981) had shown that purified desmin formed 10 nm filaments in vitro. But, it remains unclear why we observed
such self-assembled aggregates in the presence of urea. The fact that the 3-dimensional
matrix of desmin intermediate filaments was
degraded very early after death could contribute partly to disintegration of the muscle
and could explain the relative ease with
which the sardine skeletal muscle became
disorganized.
Whereas the coefficient of variation of the
percentage of proteolysis fragments related
to intact desmin was equal to 35% for turbot, these coefficients varied in a whole
range (70 to 135%) for sardine. This could
be due to the heterogeneity of the caught sardines in contrast with farmed fish, which are
more homogenous. Farmed fish are derived
from the same sires, farmed in the same manner and conditions of handling would be the
same. They may also be more heterogeneity
of degradation mechanisms that produced the

Fig. 1Western blotting analysis of desmin degradation and specificity of polyclonal antidesmin antibodies in aged (lane A) chicken gizzard, (lane B) turbot, (lane C) sea bass and
(lane D) brown trout. Lane ST corresponds to the molecular weight standards.

Fig. 2Proteolysis of desmin during fish storage at 0-4C. Histograms show percentages
of each class of proteolysis fragment (bp) with respect to total bands (Sbp +bi), including
intact desmin (bi) for each step of storage of (A) turbot and (B) sardine. Inset: molecular
weight ranges of desmin fragments and aggregates. Error-bars on histograms represent
means of variation coefficients.

desmin fragments in sardine muscle.

Results on bovine muscle (Takahashi,
1996; Hwan and Bandman, 1989; Ho et al.,
1996) have indicated desmin degradation was
found at the early postmortem storage and

the degradation of a-actinin was noted after

long storage at 4C. However in our results,
little or no degradation of desmin (except for
sardine) was observed in fish muscle. The
a-actinin, involved in the anchorage of actin

Volume 64, No. 2, 1999JOURNAL OF FOOD SCIENCE

241

Desmin Degradation in PM Fish Muscle . . .

filaments to Z-line, had been shown to be
partly released and proteolyzed from the Zline of sea bass and sea trout white muscle in
the first 24h postmortem (Papa et al., 1996).
The a-actinin release and proteolysis were
dependant not only on time/temperature conditions but also on fish species as was desmin
degradation. Results suggested that a-actinin was important in postmortem changes
of the Z line and cytoskeletal structure of fish
contrary to results on mammalian meat.
The mechanisms of desmin degradation
remain unclear. Takahashi (1996) demonstrated in bovine, porcine and chicken semitendinosus muscles that proteases did not
participate in the fragmentation of desmin.
Properties of desmin intermediate filaments
were hypothesized to be changed by the binding of calcium ions and that depolymerization of desmin intermediate filaments took
place prior to fragmentation of molecules.
However, Hwan and Bandman (1989) found
that both Ca2+-activated protease and acid
protease were responsible for degradation of
desmin during postmortem storage of bovine
semitendinosus muscle at 4C. Moreover,
Johnson and Guindon-Hammer (1987) described a desmin specific calpain I type enzyme (or desminase) that they identified from
smooth chicken gizzard muscle. We could not
suggest a mechanism for desmin degradation
in fish muscle because the rate of fish desmin
degradation seemed to be different according to species and quite different from mammals as had been reported for a-actinin (Papa

242

et al., 1996) and dystrophin (Papa et al.,

1997).
Desmin was not easily degraded during
the early phases of postmortem conditioning in fish, except for sardine. Further investigations on the desmin degradation mechanisms and particularly on whether the
calpain-like enzymes also function in fish
muscle could contribute to the understanding of differences in muscle tenderization
between fish species. These results indicate
this protein would not serve as a biochemical indicator for predicting general muscle
quality of fish species.

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Ms received 6/26/98; revised 11/8/98; accepted 11/16/
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We are grateful to the farmed fish stations: Socit Epinrine
dAquaculture, SEMII and France-Turbot for fish samples.