Natural actomyosin was extracted from frozen minced cod muscle stored
for up to 62 weeks at −20
°C with 0.6 M NaCl, and the insoluble aggregates, when formed, were
solubilized successively with
2% sodium dodecyl sulfate (SDS) and 2% SDS + 5%
β-mercaptoethanol (ME) solutions, giving
extracted fractions S1 (NaCl), S2 (SDS), and S3 (ME + SDS),
precipitates insoluble in 0.6 M NaCl
(P1) and 2% SDS (P2), and a precipitate not soluble in any of the
agents used (P3). SDS
polyacrylamide gel electrophoresis (SDS-PAGE) of fraction S1 showed
that the proportion of the
major proteins changed during frozen storage. Size exclusion
chromatography showed a decrease
in the peak containing myosin heavy chain (MHC) and actin (Ac).
Transmission electron microscopy
(TEM) of S1 showed at the outset a filamentous morphology associated
with globules interconnected
crosswise. As storage progressed, the number and size of
aggregates increased. In fractions S2
and S3, the major proteins detected by SDS-PAGE were MHC and Ac.
TEM showed a greater
abundance of ring-shaped structures than in S1. TEM of the
insoluble fractions showed a sarcomere-like structure, more pronounced the milder the solubilizing treatment
and the longer the storage
time.
Keywords: Aggregation; frozen storage; actomyosin; cod; mince; Gadus
morhua; microstructure
Natural actomyosin (NAM) extracted in 0.6 M NaCl from hake fillets stored at -20 and -30 degrees C for up to 49 weeks was studied. The extracted protein decreased as storage progressed and became poorer in myosin while the proportion of actin remained constant. Two major peaks composed of myosin plus actin and actin plus tropomyosin plus troponins were obtained by size exclusion chromatography. SDS-PAGE analysis of the protein retained in the precolumn filter showed that there was protein aggregated by covalent bonding. Surface hydrophobicity increased while Ca(2+)-ATPase activity, apparent viscosity, and SH groups decreased as storage progressed. The loss of Ca(2+)-ATPase activity was due mainly to denaturation of the extracted myosin, whereas the minimum viscosity values occurred earlier and were not directly due to the lower proportion of myosin in the extracts. Thus, the extracted NAM exhibited changes during frozen storage. The temperature-dependent difference was mainly quantitative due to a smaller amount of protein extracted at -20 degrees C.
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