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Troponin T (TnT) is a myofibrillar protein present in striated muscle that develops important structural and regulatory functions. This protein is easily degraded during post-mortem ageing but details of the TnT hydrolysis occurring during much longer processes, e.g. the dry-cured processing, as well as the specific sequences of the generated fragments, still remain undetermined. In the present study, the isolation and identification of 27 peptides, generated from TnT during dry-cured processing, are reported for the first time. This study has been developed using matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF) and it evidences the extensive degradation of TnT occurring during the processing of dry-cured ham. Some of the cleavage sites, corresponding to these sequenced fragments, confirm the sites reported by other authors both in vitro and during post-mortem ageing of meat.

Dipeptidyl peptidases (DPP) constitute a group of proteolytic enzymes able to release a good number of dipeptides from the N-terminus of both synthetic substrates and natural polypeptides. Specific sequences generated by the action of DPP purified from porcine skeletal muscle have been assayed to evaluate their capacity to inhibit the activity of angiotensin-I converting enzyme (ACE; EC 3.4.15.1). A fluorimetric assay based on the hydrolysis of the internally quenched fluorescent substrate o -aminobenzoylglycyl- p -nitrophenylalanylproline by the action of ACE was used for this purpose. The generated fluorescence of the product (the o -aminobenzoylglycine group) was continuously monitored in a microtiter-plate multiscan fluorometer. Among the assayed dipeptides, Arg-Pro showed the strongest ACE inhibitory activity, being able to suppress more than 60% of initial enzyme activity at a concentration of 25 μM. Dipeptides Lys-Ala, Gly-Pro and Ala-Ala also demonstrated to be effective ACE inhibitors, although at a lower degree. Dipeptides Ala-Arg and Gly-Arg caused a more moderate inhibition of ACE activity, and even lower was the inhibition exerted by Arg-Arg. From the data obtained, it is suggested that the proteolytic action of DPP along the ripening period of dry-cured meat products could contribute to the generation of antihypertensive peptides.

Dipeptidyl peptidase IV (EC 3. 4. 14. 5) from porcine skeletal muscle has been purified to homogeneity by selective protein fractionation with ammonium sulfate and HPLC separations with strong anion-exchange chromatography. Pure DPP IV showed a single band with Mr about 70 kDa by SDS-PAGE and optimum activity at pH 8.0 and 45°C. Substrates best hydrolyzed were those containing a proline residue in the penultimate position at the N-terminal, but it was also possible to hydrolyze (in lower amounts), X-Ala- synthetic and peptide substrates. The presence of diprotin A, puromycin, Co 2+ and Fe 2+ considerably inhibited DPP IV activity. A contribution of DPP IV action to the total proteolytic activity occurring in postmortem muscle during meat storage and ripening of meat products is feasible and could contribute to flavor generation in those products.

Activity of polyphenol oxidase (PPO) from "Rojo Brillante" persimmon (Diospyros kaki L.) fruits was characterized. Crude extracts were used for characterization of enzyme activity and stability at different temperatures (60, 70 and 80 °C), pHs (from 3.5 to 7.5) and substrate concentrations (catechol from 0 to 0.5M). Maximum enzyme activity was reached at pH 5.5 and 55 °C. Enzyme stability was higher than PPO activities found in other natural sources, since above pH 5.5 the minimum time needed to achieve an enzyme inactivation of 90% was 70 min at 80 °C. However, at pH 4.0 the enzyme stability decreased, reaching inactivation levels above 90% after 10 min even at 60 °C. Thus it was concluded that acidification can circumvent browning problems caused by PPO activity. Moreover, polyacrylamide gel electrophoresis of the enriched extract revealed the presence of at least four bands with strong oxidase activity, suggesting the existence of different PPO isoforms.