Inhibition of protease activity 2. Degradation of myofibrillar proteins, myofibril examination and determination of free calcium levels

The structure of muscle injected with specific cysteine protease inhibitors was examined to determine whether inhibitors cause denaturation and the degradation post-mortem of myofibrillar proteins was followed using SDS electrophoresis. Given the central role of calcium in theories of tenderisation the level of free calcium was measured during the early post-mortem period. The protease enzyme inhibitor E-64 was injected into the m. longissimus et thoracis lumborum (LTL) on the right side of 12 lamb carcasses within 15 min of death and in another 12 carcasses with the protease inhibitor Z-Phe-Ala-CHN(2). The left LTL (control) was injected with saline (0.25 M NaCl). Muscle samples were obtained at death, pH 6.2 and 6.0 and then at 1 and 2 days post-mortem (n=215). Muscle samples were selected from eight portions of the LTL (1-day post-mortem, from six different carcasses) for examination by transmission electron microscopy. Matching light images of myofibrils were obtained after determination of myofibrillar fragmentation. Free calcium concentration was determined for all samples (n=191) using an ion selective electrode excluding those 'at death'. Light images of myofibrils from treated samples showed normal striations and no evidence of denaturation or aggregation compared to control samples. This also applied to the samples processed for examination by electron microscopy. Appearance of the 30-kDa subunit increased with time (P<0.001) post-mortem. The interaction between ageing and stimulation had an effect (P<0.001) on the amount of a protein designated M1. The amount of M1 measured pre-rigor was greater for stimulated muscle, but the rate of decline was also greater through to day 2 post-mortem. Proteolysis was very rapid in the first 24 h post-mortem in ovine muscle. Ageing had an effect (P<0.001) on the free calcium concentration, which increased as muscle aged. As a covariate pH also had an effect (P< 0.05). Based on a non-linear model when the concentration of free calcium reached a plateau (～110 μM) the predicted pH was 5.5 (ultimate). From the qualitative observation of images and the levels of free calcium in injected muscle there is no support for the view that the inhibitors bind to sarcomere proteins, occupying sites to which calcium might bind. The levels of free calcium do not provide support for the view that m-calpain has a role in post-mortem tenderisation, but do suggest along with results of protein degradation that activation of μ-calpain is likely to occur before the pH drops to 6.2-6.1.