Mechanism of Production of Troponin T Fragments during Postmortem Aging of Porcine Muscle SHIN-ICHI KITAMURA, † SUSUMU MUROYA, ‡ SOICHI TANABE, † TOMOYUKI OKUMURA, § KOICHI CHIKUNI, ‡ AND TOSHIHIDE NISHIMURA* ,† Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan, Department of Animal Products, National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan, and Research and Development Center, Nippon Meat Packers, Inc., 3-3 Midorigahara, Tsukuba, Ibaraki 300-2646, Japan Troponin T (TnT) is one of the myofibrillar proteins that is easily degraded during postmortem aging of pork. In this study, we determined the N-terminal amino acid sequences of TnT degradation fragments produced during postmortem aging and by m-calpain hydrolysis. The N-terminal amino acid sequences of TnT fragments produced during postmortem aging were EVHEPEEKPRPKLTAP, EKPRPKLTAPKIPEG, and APKIPEGEKVDF. On the other hand, the N-terminal amino acid sequences of TnT fragments produced by the action of m-calpain were APPPPAEV, EVHEPEEK, and APK. These sequences of degradation fragments could be mapped on fast type TnT isoform 2. The pep- tide bonds of His 37 -Glu 38 and Thr 51 -Ala 52 in fTnT2 were cleaved during postmortem aging as well as by the calpain hydrolysis; therefore, calpain was concluded to have an important role in TnT degradation during postmortem aging. It was also found that the sourness-suppressing peptide APPPPAEVHEVHEEVH (Okumura et al. Biosci. Biotechnol. Biochem. 2004, 68, 1657-1662) derived from TnT degradation could be produced by the action of calpains on Glu 21 -Ala 22 and His 37 -Glu 38 sites. KEYWORDS: Troponin T (TnT); postmortem aging; calpain; porcine; degradation; N-terminal amino acid sequence INTRODUCTION It is well-known that muscle is converted to meat as food during postmortem aging. Due to the increase of peptides and free amino acids during postmortem aging, meat taste is improved (1-6). For example, we have previously reported that during postmortem aging of pork, a peptide APPPPAE- VHEVHEEVH was derived from troponin T (TnT) to contribute to improvement of taste (sourness-suppressing) (1). The postmortem degradation of skeletal muscle proteins and its relationship to meat tenderness and quality have been the subject of considerable research. TnT is well-known as one of the myofibrillar proteins to be easily degraded during postmor- tem aging of meat (7). In conditioned meat, a 30 kDa fragment was detected and immunologically identified as a TnT fragment (8-11). The degradation of TnT progresses simultaneously with the postmortem tenderization of meat, showing a good correla- tion between the two events (12). In addition, we found that the above-mentioned sourness-suppressing peptide was also useful as a conditioning indicator in pork loins (13). However, the details of TnT degradation and the relationship between TnT degradation and meat tenderization remain poorly understood. First, especially in pork, the cleavage sites of TnT are unknown because the amino acid sequence of the degrada- tion products had not been determined. Second, the mechanisms of the fragmentation of TnT proteins during postmortem aging have also not been analyzed. Therefore, although several candidate proteinases, such as calpains and cathepsins, can cleave TnT in vitro (14), how much each of them contributes to TnT degradation is not still clarified. Understanding of the mechanism of TnT degradation would lead to control of meat quality during postmortem aging. Recently, we have determined the amino acid sequence of porcine TnT isoforms. At least eight porcine fast-type TnT (fTnT1/16, fTnT1/17, fTnT2/16, fTnT2/17, fTnT3/16, fTnT3/ 17, fTnT4/16, and fTnT4/17) and two slow-type TnT (sTnT1 and sTnT2) isoforms have been found so far by our group; these hold the sequential Accession Nos. AB176595, AB176599, AB176596, AB176600, AB176597, AB176601, AB176598, and AB176602 (fTnT) and AB118908 and AB118909 (sTnT) in the DDBJ/EMBL/GenBank nucleotide sequence databases. These sequences suggested that the sourness-suppressing peptide APPPPAEVHEVHEEVH was produced from fTnT isoforms fTnT1, fTnT2, and fTnT3. * To whom correspondence should be addressed: telephone +81-82- 424-7984; fax +81-82-424-7984; e-mail toshixy@hiroshima-u.ac.jp. † Hiroshima University. ‡ National Institute of Livestock and Grassland Science. § Nippon Meat Packers, Inc. 4178 J. Agric. Food Chem. 2005, 53, 4178-4181 10.1021/jf047974l CCC: $30.25 © 2005 American Chemical Society Published on Web 04/16/2005