INFLUENCE OF DIFFERENT LETHAL CONDITIONS UPON FISH MUSCLE PROTEIN-II

DENATURATION OF CARP MYOSINS BY FREEZING AND FROZEN STORAGE

Abstract

Effect of freezing and cold storage upon the extractability and salting-out behavior of muscle protein was examined, using carp killed in two different ways; one was instantly put to death by beheading and the other was beheaded after allowed to struggle in air for several hours.
Fillets taken from these two groups of carp were frozen at -15°C and stored at this temperature for a period varying from 2 to 14 days. Before freezing and at each different time of cold storage, myosins were extracted from these fillets with 0.6M KCI (pH 6.6), precipitated by dilution. and the amount was measured. With isolated myosins salting-out behavior toward ammonium sulfate was determined at pH 6.9 and at the temperature of 0°C.
Extractability Myosins were always extracted greater in amount from death-after-struggle group than from instant death group throughout the period of the present experiment. No significant difference, however, in the ratio of amount of myosins (M) to salt extractable protein (L), was noticed between the two groups. After frozen storage of 10 days, a slight diminution in the values of L and M was observed in the muscle of instantly killed fish, but it was not appreciable for death-after-struggle group within the limited duration of the present test. Meanwhile, the ratio M : L seemed to decrease somewhat in both groups on prolonged storage (Table 1).
Salting-out Curves Around the range of 30-40% (v/v) saturation of ammonium sulfate in the salting-out curves for either group, there was found a dominant peak, which presumably pepresented myosin and actomyosin. With fresh fillet before freezing, this peak was more prominent for the instant death group than for the other.
However, by freezing and subsequent frozen storage, the pattern of the curves for instant death group turned out close to those of unfrozen fillet of death-after-struggle group, and inversely the pattern of the latter group became kin to those of the former group that was not frozen. This will be obviously perceived from the mode of slope and fall in the s-c curves in the range of 30-40% saturation of ammonium sulfate (Figs. 1 and 2).
In another series of experiments similarly conducted for isolated myosin gel, the results given were not quite the same as obtained for the fillets. The salting-out curve of the gel from fish killed after struggle became analogous to that of fresh, unfrozen gel from instant death group, just as seen in the case of the fillets. But the curves of frozen myosin gel from instant death group was not altered by freezing process and cold storage (compare Figs. 1. 2 and Figs. 3 and 4).
It is notable in Figs. 1-4 that there was found concentration range of ammonium sulfate in which components salted-out decreased in amount with increasing concentration as the result of freezing and subsequent storage. This means that a part of components which were saltedout at concentration c₁ remained in solution in the range of higher concentrations between c₁ and c₂. This effect will be valid for either fillet or isolated myosins gel.
Actin fraction in the salting-out pattern In the previous paper ??, it was shown with fresh fillets that actin fraction appeared in the salting-out pattern of myosin fraction from carp killed after struggle, but the corresponding fraction was absent in the case of carp put to an instant death. In addition, it was mentioned that this actin fraction would not appear, if the extraction of myosins from carp killed after struggle was carried out in the presence of ATP.
In the present work, it was found that when the fillets were frozen and kept for 2 days or 6 days, a prominent peak presumed as actin fraction, appeared also in the case of carp put to an instant death.