Haruo Negishi

The origin of the 30 kDa component appearing during post-mortem ageing of bovine muscle

The most predominant component appearing on SDS-PAGE of myofibrils prepared from bovine m. vastus intermedius (VI) during ageing for 31 days post mortem at 0-2 °C was a component with a molecular weight of 32 kDa (SDS-32 kDa). In this study, the origin of the SDS-32 kDa component, which was thought to correspond to the 30 kDa component already known, was investigated. On the SDS-PAGE of the crude troponins, both troponin T and the 34 kDa component were gradually degraded during ageing and then troponin T disappeared at 24 days post mortem, while the concentration of the 32 kDa component showed a tendency to increase during ageing. The 32 kDa component was prepared from troponin of bovine VI muscle stored for 17 days post mortem using CM-Toyopearl chromatography, and named the native 32 kDa component. Its mobility on SDS-PAGE agreed with that of the SDS-32 kDa component. The SDS-32, native 32 and 34 kDa components were recognized by the polyclonal anti-troponin T antibody, and furthermore, the patterns of amino acid composition of both the native 32 and 34 kDa components were very similar to troponin T. Thus, it was considered that these two components would be polypeptides from the degradation of troponin T. We concluded that the SDS-32 kDa component must be derived from the degraded poly-peptides of troponin T. However, it still remains a possibility that some degradation products from other myofibrillar proteins are included in the SDS-32 kDa component. Therefore, further studies about the identity of this component will be required.

Formation of acrylamide from glucans and asparagine.

Model foods consisting of carbohydrates, asparagine (Asn), albumin, and sodium chloride were heated at 180°C for various times, and the levels of acrylamide (AA) in these foods were determined by LC/MS/MS. When glucans such as β-cyclodextrin (β-CD), starch and cellulose were used as carbohydrates in the above model, the levels of AA formed were approximately the same as or much higher than those observed in the glucose model. Glucans were heated in the absence of Asn for one hour, and their degradation products were analyzed for sugar components by HPAEC-PAD and for volatile compounds by GC/MS. The amounts of glucose detected in the glucan models, however, were too low to consider that AA was formed from the glucans in these model foods via the intermediate production of glucose. By contrast, several carbonyl compounds such as acetaldehyde and acetone were detected in the glucan degradation products. Furthermore, AA was formed when acetaldehyde and Asn were heated together in sealed vials at 180°C. These results showed that AA was formed from glucans and Asn, not via glucose produced by glucan hydrolysis, but via volatile carbonyl compounds such as acetaldehyde produced by glucan pyrolysis.

Separation and amino acid composition of three troponin components from bovine muscle

Three troponin components were isolated from bovine skeletal muscle, and their molecular weights and amino acid composition were studied. Crude troponin prepared from bovine muscle was purified by DEAE-Toyopearl chromatography. The purified troponin was dissociated in the order of tropopins C, I and T by CM-Toyopearl chromatography in the presence of 6 M urea. The molecular weights of troponins C, I and T were 19,500, 23,300 and 40,400, respectively, as determined with SDS-PAGE. The separation of troponin into three components was also achieved using reverse-phase HPLC; however, the elution order of troponins T and C was contrary to that of the cation-exchange chromatography described above. In this study, the amino acid composition of the three troponin components from bovine skeletal muscle was first determined. The amino acid composition of the three troponin components among bovine, rabbit and chicken skeletal muscles showed stronger similarity than that between bovine skeletal and cardiac muscle with a different muscle type. We considered that this method of troponin preparation from bovine muscle must be a very useful technique for investigating the changes in troponin components, especially troponin T, during ageing of beef.