SUMMARY— The protein malonaldehyde reaction was studied as a potential mechanism of protein denaturation in muscle, especially during frozen storage. Myosin, a structural protein of muscle, was reacted at pH 6.8 and ionic strength 0.5 with malonaldehyde, an oxidation product of polyunsaturated fatty acids. The rate of reaction with the s‐amino groups of myosin was greater at ‐20° than at 0° and was almost as great as that at +20°. The same relationship was observed when the decreasing malonaldehyde concentration was measured in the protein‐malonaldehyde reaction mixture. Amino acid analyses before and after reaction at 100° for 60 set showed that malonaldehyde reacted preferentially with histidine, arginine, tyrosine, and methionine. In frozen solution, malonaldehyde reacted with lysine, tyrosine, methionine, and arginine in decreasing order of intensity, but it did not react with histidine. The increased rate of reaction in the frozen system is explained as a concentration effect and as a catalytic effect involving the ice structure.
Rabbit and trout myosins, prepared under painstaking conditions to exclude all incidental heavy metal contamination from reagents, water, glassware, and other utensils, contained 42 free sulfhydryl (SH) groups per 5 × 105 g when the analysis was carried out with the Ellman reagent, 5,5′-dithiobis-2-nitrobenzoic acid, in the presence of 8 M urea which was 6 mM in ethylenediaminetetraacetic acid (EDTA). In the absence of EDTA, 40 free SH groups were determined, while performic-acid-oxidized myosin preparations yielded 42 cysteic acid (half-cystine) residues by amino acid analysis. The results indicated that no disulfide bonds were present in the native myosin molecules. The specific enzyme activity (ATPase) remained constant at a relatively high value of 1.38 to 1.45 μmoles P1 per milligram protein per minute over 4 weeks of storage at 0°. Such myosin preparations were also homogeneous in the ultracentrifuge up to 4 weeks.In myosins prepared with two times distilled water and "reagent grade" chemicals which had not been purified by passage over cation exchange resins, the SH content decreased after 1–2 days at 0°, to 37 SH per 5 × 105 g of protein, and this oxidized form of myosin showed aggregate peaks in the schlieren optical system of the ultracentrifuge after 3 days of storage at 0°.Trout myosin in the presence of traces of heavy metals was more susceptible to oxidation than rabbit myosin.
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°2020,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 (P3) and to a lesser degree by pyrophosphate (P2). 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.
Different malonaldehyde-amine condensation products were tested for their relative color contribution to the thiobarbituric acid (TBA) test, an indicator for oxidative rancidity of polyunsaturated lipids. The open chain mono-and disubstituted malonatdehyde (M) addition products (R-N=CH-CH=CHOH and R-N=CH-CH=CH-NH-R) gave complete (100%) recovery of M on a mole basis. When the __M residue was incorporated into cyclic products which formed between the ureido-or guanidino-substituents of a-amino acids such as citrulline or arginine and M, recovery of ~ by the TBA color test was 30% and 6%, respectively. Products, from imine-amine type interactions, containing the __M residue as in pyrazoline or pyrazole ring systems, released from 4% to no __M.
Extractability of the contractile proteins of muscle with salt solutions decreases during freezer storage or on heating. Studies on the insolubilized proteins with dissociating‐reducing solutions containing guanidine HCl (GuHCl) and mercaptoethanol or sodium borohydride (NaBH4) have previously suggested that the mechanisms of myosin aggregation in solution involve noncovalent interactions of hydrophobic and hydrogen bonds as well as the formation of disulfide (S‐S) bonds between peptide chains of different molecules. Further evidence is now presented which implies that these types of denaturation mechanisms are of a more general nature and are also responsible in the hardening, fuming or gelling of the protein structures of egg white, kamaboko and meats from fish and mammals during cooking. Methods are described by which foods whose structures are dependent upon these types of bonds can be dissolved nonhydrolytically, i.e., without excessive breaking of peptide chains and the destruction or racemization of amino acids which occur during acid or alkaline treatment at elevated temperatures.
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