Frozen storage aggregation

Electron microscopic analyses of isolated preparations of fish actomyosin denatured by frozen storage (68,72-74) showed that actomyosin filaments with arrowhead structures aggregated side-to-side and crosswise when thawed immediately after freezing. As time of frozen storage increased further aggregations formed network structures (Figure 3). 

Figure 2. Hypothetical mechanisms of aggregation of fish actomyosin during frozen storage. (A) King, 69 (B) Connell, 61 (C) Matsumoto ( proposal in the present paper). AM, actomyosin M, myosin MD1 and MDt, denatured myosin A, actin.

ATPase activity, another property of myosin related to its contractile function, as is the actin-binding property, is also decreased by frozen storage. The specific ATPase activity of fish actomyosin decreases with increased time of frozen storage (66,67,72,78-82). This decrease should be due to a decrease in the ATPase activity of myosin. The rate of decrease is slower than that of free myosin (80,82). Connell (78) and Kawashima et al. (83) have detected some ATPase activity in insoluble aggregated actomyosin. 

These results led to the conclusion that denaturation and/or insolubilization of actomyosin and myosin during frozen storage is a result of aggregation caused by the progressive increase in intermolecular crosslinkages due to formation of hydrogen bonds, ionic bonds, hydrophobic bonds and disulfide bonds. 

Data on myosin (50,51,82,91) and LMM (82) support side-to-side aggregation of molecules without appreciable change in conformation during frozen storage, as proposed by Connell (91). 

Electron Microscopy. Examination of fish proteins by electron microscopy conclusively shows that actomyosin aggregates during frozen storage (59,63,69). The change in structures of the extracted myofibrillar proteins and of the myofibril residues of frozen-stored cod muscle was studied by electron microscopy. The decrease in the number of actomyosin filaments and an increase in the number and size of large aggregate were found (69). Unfrozen carp actomyosin, either dissolved in 0.6M KC1 or suspended in 0.05M KC1, exists in a typical arrowhead... 

Subunits of Myosin. Matsumoto et al. (64) isolated H-meromyosin (HMM) and L-meromyosin (LMM) from carp muscle (15) and studied their stabilities at — 20°C. The ATPase activity of HMM decreased much faster than that of myosin and the capacity of HMM to bind with F-actin as determined by electron microscopy was lost. LMM also exhibited a decreased capacity to form well-ordered paracrystals. These results tend to indicate that frozen storage causes myosin molecules to aggregate side-by-side and myosin subunits to undergo conformational deformations. 

Findings in the authors laboratory (100) demonstrated that the number of cross bonds in carp actomyosin and myosin increases during frozen storage and that solubility of these proteins decreases. Based on the types of chemicals that resolubilized these proteins at various rates, it was concluded that ionic bonds, hydrogen bonds, covalent bonds (S-S), and hydrophobic associations all are involved in the aggregation process. 

FIGURE 9.1 Scheme for denaturation and aggregation of muscle proteins during freezing and frozen storage. 

Huidobro, A., Mohamed, G.F., and Tejada, M. 1998. Aggregation of myofibrillar proteins in hake, sardine and mixed minces during frozen storage. Journal of Agricultural and Food Chemistry 46 2601-2608. 

Tejada, M., Careche, M., Torrejon, R, Del Mazo, M.L., Solas, M.T., Garcia, M.L., and Barba, C. 1996. Protein extracts and aggregates forming in minced cod (Gados morhua) during frozen storage. Journal of Agricultural and Food Chemistry AA 3308-3314. 

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