ACCELERATED DENATURATION OF MYOSIN IN FROZEN SOLUTION

SUMMARY The aggregation of rabbit and trout myosins was studied in frozen solution at high ionic strength μ= 0.50 KCl–potassium phosphate buffer pH 6.9 between 0 and –30°. During the initial stages of freezing, monomeric myosin S° 20,w = 6.5 S aggregated to form dimers and trimers with sedimentation rates s°20 20,w = 10 and 12 S, respectively. The higher aggregates sedimented at low centrifugal fields and were insoluble in molar salt solutions at pH 8. Solubilization was, however, achieved in solvents known to disrupt hydrophobic and hydrogen bonds in addition to conditions which will reduce disulfide bonds. The nature of the sulfhydryl groups of myosin was reinvestigated and, in accordance with their behavior, a mechanism for the aggregation reactions has been proposed which involves disulfide‐sulfhydryl exchange reactions between activated myosin molecules and aggregates. Previous kinetic and chemical data for myosin denaturation are in agreement with the proposed mechanism. The rate of formation of the insoluble, high molecular weight protein aggregates in myosin solutions increased as the temperature decreased below the freezing point and reached a maximum near the eutectic point of the myosin‐potassium chloride‐water solution (– 11°), due to concentration effects. Below the eutectic point, at –20 and –30° where only water bound to the protein remains unfrozen, aggregation and consequent insolubilization decreased again and approached the rate observed at 0° The general pattern of myosin solubility at different freezing temperatures was similar to the decreasing protein solubility during storage of whole muscle, with the difference that denaturation of purified myosin solutions was accelerated. At the most critical temperature, around ‐10°, the rate of denaturation of a 0.7% myosin solution was reduced by the enzyme substrates 0.02 M adenosinetriphosphate (ATP) or tripolyphosphate (P 3 ) and to a lesser degree by pyrophosphate (P 2 ). Substances such as glycerol or magnesium chloride which lowered the eutectic point also lowered the rate of denaturation at –10° N‐ethylmaleimide was not effective in reducing the rate of denaturation and mercapto‐ethanol led to the formation of gels. Electronphotomicrograph studies showed that aggregation of the monomeric myosin molecules proceeds in a side‐to‐side manner. Loss of solubility was also produced when myosin solutions contained a cross‐linking reagent such as malonaldehyde, or detergents which interfere with the hydrophobic bonding of the native molecule. The different mechanisms responsible for the denaturation or insolubilization of myosin and its increasing molecular weight are briefly discussed.