Formation of Zinc Protoporphyrin IX from Myoglobin in Porcine Heart Extract

Abstract

The formation of zinc protoporphyrin IX (ZPP) in porcine heart extract was investigated by visible absorption and fluorescent spectral analysis. Characteristic absorption peaks of ZPP were observed at .+1, /.0, and /2. nm after anaerobic incubation of the extract with metmyoglobin and ZnCl, in the dark at -*ῌ for 1, h. In the fluorescent spectra (excitation : .+* nm), a strong peak due to ZPP was obtained at /23 nm. The time-course study showed that ZPP formation greatly increased after a lag period, during which metmyoglobin was reduced. ZPP formation was significantly facilitated by the use of oxymyoglobin. Zinc chelatase activity in the extract was estimated as +-2 mU/mL-extract and its specific activity was ,.3/ mU/mg-protein. The di#erence in activities with aerobic and anaerobic incubation was not significant. Measurements at pH /./ῌ1.* showed that activities were higher at lower pH. The activity of zinc chelatase was stimulated by the presence of ATP, and the highest activity was obtained at an ATP concentration of ,./ mg/mL.

Color Formation in Nitrite‐Free Dried Hams as Related to Zn‐Protoporphyrin IX and Zn‐Chelatase Activity

“…The broad range of activities found in raw muscles suggest that Zn‐chelatase is strictly dependent on individual muscles even if they belong to the same cut such as pig's hind leg. Data in Table 4 indicate a 3.3‐fold ratio between dark and light ST, in accordance with findings from Ishikawa and others (2006a, 2006b) who reported a 3.3‐fold increase of activity from light pork's loin to extremely dark porcine heart.…”

“…Finally, while this study does not exclude a nonenzymatic mechanism, results are supportive for the involvement of Zn‐chelatase in ZPP synthesis, in that (1) fluorescence in cold‐rested hams is negligible or barely detectable, fitting the typical behavior of an enzyme (Zn‐chelatase) reportedly favored by such mild temperatures (Taketani and others 2007) as those occurring in the maturing and aging stages of hams, and (2) ZPP forms in the oxygen‐free inside of the leg (muscles ST, BF) as well as in the air‐rich ham surface (muscle SM), in agreement with the described attitude of Zn‐chelatase to catalyze the reaction under both aerobic and anaerobic conditions (Ishikawa and others 2006b). …”

“…While the mechanism leading to ZPP in nitrite‐free dry‐cured hams is still to be elucidated, an enzyme such as Zn‐chelatase has been postulated to catalyze the key step of the reaction, that is the heme iron replacement by zinc (Wakamatsu and others 2004a, 2004b). Ishikawa and others (2006b) reported significant differences in Zn‐chelatase activities between a low‐myoglobin white muscle such as porcine Longissimus dorsi and a dark muscle (porcine heart), with the latter exhibiting greater chelatase activity. Therefore we expected differences to be found in enzyme activities of these muscles, which differed in their original redness hence myoglobin content.…”

“…Also, the enzyme appears to be released from the muscle rather than from the microbial flora naturally occurring in the meat, since pigment formation is not prevented when antibiotics are added to the reaction mixture (Wakamatsu and others 2004b). Zn‐chelatase activity has been reported in green meat (Ishikawa and others 2006a, 2006b) where it is able to promote ZPP even at high salt concentration (8%[w/w] NaCl) and in a broad range of pH (5.5 to 7.5) (Benedini and others 2008), fitting the chemical conditions typically encountered in meat after slaughter and along the curing process (Virgili and others 2007). …”

“…While these findings help describe the enzyme as key in color development, it remains unclear whether it remains active throughout the manufacturing stages of dried hams, where redness is known to steadily increase in intensity (Parolari 1996). Moreover, being related to the cell's mitochondria (Ishikawa and others 2006b), enzyme activities might differ in individual muscles according to their mitochondrial content which is known to vary between red and white muscles (Cassens and Cooper 1971). If so, major color differences might be expected between muscles in dried hams; however, this is not the case in Parma hams, whose final color is evenly distributed, as opposed to differences typically found in raw legs.…”