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Meat q ualit y Myoglobin Chemistry and Modifications that Influence (Color and) Color Stability Cameron Faustman Introduction Myoglobin is a heme-containing protein that participates in aerobic metabolism in antemortem muscle by storing and/or delivering oxygen. Several scholarly reviews have been devoted to the chemical characterization of myoglobin and its effect on meat color (Faustman and Cassens, 1990; Renerre, 2000; Mancini and Hunt, 2005). This review will re-confirm the criticality of heme properties as they relate to meat color and color stability but also remind the reader that myoglobin’s apoprotein, within which heme is located, plays an important role in the extent to which heme-related reactions may occur (Wang, 1962). Relevance of Myoglobin Structure The primary sequences of myoglobin are known for a variety of animal species including those that are used to produce meat (Joseph et al., 2010; Yin et al., 2011). As with all proteins, myoglobin’s constituent amino acids have chemical similarities but are distinguished from each other by their reactive side chains. Myoglobin contains considerable secondary structure and averages approximately 75-80% α-helix (Antonini, 1965). The primary sequence and chemistry (e.g., polarity) of myoglobin’s amino acid side chains drives the formation of its tertiary structure which in turn is critical for normal function. Alteration of myoglobin’s tertiary structure through application of energy including high pressure processing (Wackerbarth et al., 2009; Ma and Ledward, 2013; Chun et al., 2014) or irradiation (Brewer, 2004) affects the color of meat. Biological alteration of a protein’s structure may also be accomplished by substitution of one or more amino acids to form mutant proteins with compromised function. The apoprotein pocket within which heme resides in myoglobin is an important part of the protein’s ter- Cameron Faustman, Ph.D. Animal Science Department University of Connecticut Unit 4040, 3636 Horsebarn Road Extension Storrs, Connecticut 06269-4040 [email protected] American Meat Science Association tiary structure and affects redox stability (Livingston and Brown, 1981). The relevance of mutations for myoglobin is that a small structural change would alter the region around the heme prosthetic group and affect redox stability of the heme and/or the propensity for iron to be lost from the heme and increase lipid oxidation (Grunwald and Richards, 2006). Chemical alteration of the normal protein can also affect macromolecular properties. Reactive side chains of myoglobin’s amino acids may be accessible to chemical species that are naturally present in the sarcoplasm or that are added as a result of processing or experimentation. The extent of reactivity depends on the protein’s environment (pH, temperature), chemical nature of the amino acid side chain and the other chemical species present. Our laboratory has published extensively on the alkylation of myoglobin by α,β-unsaturated aldehydes and the destabilizing effect these have on oxymyoglobin stability (Faustman et al, 2010). The evolution of mass spectrometry and tools of proteomics have provided the basis for advancing studies related to myoglobin primary structure, and biological and chemical effects on redox stability (Suman and Joseph, 2011). Myoglobin and Meat Color/Color Stability The heme group of myoglobin contains an iron atom within a protoporphyrin ring. The color that myoglobin imparts to meat is determined by the redox status of its heme iron and the chemical species bound to heme. Four of the iron atom’s six coordination sites keep it anchored within the planar heme structure. One of the coordination sites binds heme to the apoprotein and the sixth coordination site is available to bind a variety of ligands; in meat these include oxygen, water, carbon monoxide and nitric oxide. The conditions for differing colors and their respective common names and their interconversions are presented in Table 1. Deoxymyoglobin (DeOxyMb) is most commonly observed in freshly cut beef slices. Skeletal muscles within postmortem, postrigor livestock carcasses destined for provision of meat are anoxic and DeOxyMb predominates. This ferrous myoglobin predominates until meat slice surfaces are exposed to an aerobic atmosphere 1 Table 1. Chemical basis for observed myoglobin-related colors in meat. Redox status of heme iron Ferrous, 2+ Ferrous, 2+ Ferric, 3+ Ferrous, 2+ Ferrous, 2+; Ferric, 3+ Ligand at 6th coordination site of heme ironNameColor Nothing Oxygen Water Carbon monoxide Nitric oxide where oxygen-binding and formation of oxymyoglobin (OxyMb) occur. Modified packaging atmospheres which contain oxygen in a proportion greater than that in the normal atmosphere have been used to saturate myoglobin and obtain a rich fresh meat color that persists during retail display (McMillin, 2008; Arvanitoyannis and Stratakos, 2012). Over time, the heme iron in ferrous myoglobins will oxidize to ferric status and this occurs over time in meat in a fairly predictable muscle-dependent manner (O’Keeffe and Hood, 1982; McKenna et al., 2005). The resulting molecule is known as metmyoglobin (MetMb) and is considered undesirable because of its brownish red color. The oxidation of ferrous to ferric myoglobin has been characterized thermodynamically and kinetically (Shikama, 1985). Under controlled conditions, MetMb accumulation occurs at different rates in different species (Chow, 1991; Gutzke et al., 2002; Yin et al., 2012; Nair DeoxyMb OxyMb MetMb CarboxyMb Nitric oxide Mb Purplish red Cherry red Brown red Cherry red Pink red et al., 2013) and metmyoglobin accumulates to a point at which brown coloration predominates and the meat is no longer considered acceptable. Each of these three redox forms of myoglobin display distinctive light absorption characteristics in the visible spectrum (Figure 1) and these have been used to estimate the relative proportions of the three redox forms in fresh meat (Tang et al., 2004). The relative concentration of oxygen to which myoglobin is exposed is critical to the redox form it will assume. There is an interesting and incompletely understood relationship between the partial pressure of oxygen and the redox form of myoglobin (Brooks, 1929; Ledward, 1970). When oxygen is completely absent, or constitutes the entire atmosphere within which myoglobin resides, the predominant redox form is DeOxyMb and OxyMb, respectively. When oxygen is present at approximately 4 to Figure 1. Absorption spectra for OxyMb, MetMb and their combination. Taken from Suman et al. (2006a) with permission. 2 67th Annual Reciprocal Meat Conference 6 mm Hg, the MetMb form will predominate (Ledward, 1970). This is a very important relationship and must be considered in light of natural oxygen-consumption that may occur from mitochondrial activity, lipid oxidation and/or bacterial growth. The reduction of ferric myoglobin to ferrous myoglobin has been observed in meat; its occurrence is attributed to MetMb reductase and/or related enzymes (Bekhit et al., 2005). It affects perceived color stability because it acts in opposition to the oxidation process. It is believed the MetMb reduction counters OxyMb oxidation to the extent made possible by “reducing equivalents” (e.g., NADH) present in meat. As these reducing equivalents are consumed and MetMb reduction wanes, MetMb accumulates and meat discoloration occurs. Two additional chemical species that readily react with myoglobin heme iron are carbon monoxide and nitric oxide. Carbon monoxide binds tightly to heme to form carboxymyoglobin (CarboxyMb) and yields an absorption spectrum (Suman et al., 2006a; Smulevich et al., 2007) and cherry red color that is very similar to that of OxyMb (Chow et al., 1997; Nam and Ahn, 2002). CarboxyMb is quite stable compared to OxyMb and can persist for long periods of time (Jayasingh et al., 2001); it has been identified as the source of pink color in irradiated poultry (Nam and Ahn, 2002). The inclusion of carbon monoxide-containing packaging atmospheres for the purpose of prolonging a desirable red color in fresh meat has been studied extensively (Luno et al., 1998; Hunt et al., 2004; Jeong and Claus, 2010). Nitric oxide myoglobin is typically formed in raw meat during the preparation of cured products (Moeller and Skibsted, 2002; Honikel, 2008). Sodium nitrite is chemically reduced through a series of complex chemical reactions to yield the highly reactive nitric oxide which binds to myoglobin at the 6th coordination site (Sebranek and Fox, 1985). Connolly et al. (2002) reported that peroxynitrite enhanced the oxidation of OxyMb. Other Chemical Modifications of Myoglobin Observed in Meat Several researchers have reported on the ability of hydrogen sulfide, presumably produced by contaminating bacteria (Nicol et al., 1970; Egan et al., 1989), to bind heme and form the green pigment, sulfmyoglobin (Morrell and Chang, 1967; Berzofsky et al., 1972). Interestingly, Smolander et al. (2002) reported on the intentional application of this reaction for detecting hydrogen sulfide in stored poultry meat via agarose-immobilized myoglobin. Fox et al. (1974) reported that the greenish heme protein, choleglobin, could be formed by reactions between hydrogen peroxide and heme at acidic pH. An interesting modification that does not apply to fresh meat but that is worth noting here is that of the displacement/replacement of iron in heme by zinc to form Znprotoporphyrin IX (Wakamatsu et al., 2004; Adamsen et American Meat Science Association al., 2006a). This occurs in dry-cured Italian and Spanish hams and is responsible for characteristic red colors associated with those traditional products; the addition of nitrite inhibits formation of the zinc complex (Adamsen et al., 2006b). Redox Stability as Affected by Modifications of the Myoglobin Apoprotein In 1989, Faustman et al. (1989ab) published the first reports of improved beef color stability obtained from vitamin E-supplemented cattle. The effect was subsequently confirmed by many others (Faustman et al., 1998) and was generally attributed to the fact that vitamin E, i.e., α-tocopherol, accumulated in the muscle membranes of cattle in a dose-dependent manner and acted as an antioxidant. The actual mechanism by which this fat-soluble antioxidant maintained greater redox stability of watersoluble myoglobin in meat was unclear. It was initially suggested that α-tocopherol protected unsaturated fatty acids of membrane phospholipids against oxidation and that this delayed the production of lipid oxidation breakdown products that were responsible for enhancing myoglobin oxidation. Subsequent investigations provided support for this hypothesis in vitro (Chan et al., 1997; Faustman et al., 1999; Lynch and Faustman, 2000). Faustman et al. (1999) demonstrated that α,β- unsaturated aldehydes, known secondary products of lipid oxidation, formed covalent complexes with myoglobin and that this was concomitant with enhanced oxidation of untreated (i.e., no aldehyde addition) controls. Joseph et al. (2009) reported that HNE can also alkylate CarboxyMb. The nucleophilic side chains of several of myoglobin’s constituent amino acids are targets of alkylation by α,βunsaturated aldehydes. Many investigators have used the α,β- unsaturated aldehyde, 2-hydroxy-4-nonenal (HNE), as a model secondary product of lipid oxidation (Poli et al., 2007); HNE has been identified in meat (Sakai et al., 1998). To date, histidine has been the only amino acid site confirmed for alkylation by HNE in myoglobin. HNE appears to decrease myoglobin redox stability and form more histidine adducts at neutral pH than at meat pH (Faustman et al., 1999; Alderton et al., 2003; Naveena et al., 2010). Multiple adducts can be formed depending on length of incubation and pH (Faustman et al., 1999; Joseph et al., 2009) and species origin (Suman et al., 2006b; Maheswarappa et al., 2009; Yin et al. 2011; Nair et al., 2014). Yin et al. (2011) investigated the relative effect of HNE on myoglobin from seven different meat-producing species and demonstrated that greater histidine numbers in the primary sequence predisposed those myoglobins to greater HNE-enhanced oxidation than those with a lower number of histidine residues. Suman et al. (2007) reported that histidine 36 in porcine myoglobin and histidine residues 81 and 88 in bovine myoglobins were preferentially alkylated by HNE and hypothesized that initial adduct formation might be sufficient to alter tertiary structure such 3 that subsequent alkylation could occur at previously inaccessible amino acid sites. The overall implication of these reactions would be that myoglobin’s heme pocket might be altered and expose heme iron to oxidation. The extent to which secondary products of lipid oxidation actually contribute to redox instability of myoglobin in meat has not been determined. The model studies noted previously provide a mechanism that could be a basis for the long known relationship between lipid oxidation and myoglobin oxidation. Additionally, the model was used by Suman et al. (2006b, 2007) to suggest a reason for different responses of pork and beef color stability to dietary vitamin E supplementation of pigs and cattle, respectively. That is, while many studies reported that beef color stability was improved, published support for enhanced pork color stability with vitamin E was considerably less consistent (Suman et al., 2007). This was unexpected because fatty acids in pork are more unsaturated and if vitamin E exerted its color-preserving effect by decreasing lipid oxidation, then pork rather than beef should demonstrate a greater response to increased muscle concentrations of α-tocopherol. An examination of the primary sequences of bovine and porcine myoglobins revealed that the number of histidine residues was 13 and 9, respectively. The in vitro redox stability of porcine myoglobin was greater than beef myoglobin when exposed to HNE. Not surprisingly, the extent of adduct formation was less in porcine myoglobin. Thus, Suman et al. (2007) concluded that bovine myoglobin’s primary structure predisposed it to greater HNE alkylation and concomitant myoglobin redox instability than that of porcine myoglobin. Subsequent support for the criticality of primary sequence in predicting/describing the bases for myoglobin susceptibility to HNE and redox instability, particularly as it related to the observations for porcine and bovine myoglobins, was provided by Tatiyaborworntham et al. (2012). Suman et al. (2006b) showed that bovine OxyMb was alkylated by HNE at 7 sites including histidine 88 and histidine 152, while porcine Mb was not alkylated at sites 88 (proline; P) or 152 (glutamine; Q). Tatiyaborworntham et al. (2012) hypothesized that the lack of histidine residues at sites 88 and 152 in porcine Mb contributed to its slower autoxidation than bovine myoglobin in the presence of HNE and they used wild type (WT) and mutant sperm whale myoglobins to test the hypothesis. WT sperm whale and porcine myoglobins contain proline and glutamine at sites 88 and 152, respectively. Mutant sperm whale myoglobins were developed in which histidine (H) was substituted at amino acid residues 88 and 152 (mutant P88H/Q152H). The oxidation of WT and P88H/Q152H sperm whale OxyMbs were similar, but the relative oxidation of P88H/Q152H appeared to be enhanced by HNE more than WT when each was compared to their respective controls (Figure 2). MALDI-TOF mass spectrometry revealed that each of the myoglobins contained mono- and di- adducts at 24 hr and 72 hr in- Figure 2. Effect of 4-hydroxy-2-nonenal (HNE) on relative metmyoglobin formation in wild type (WT) and mutant (P88H/Q152H) sperm whale myoglobins at pH 5.6 and 4°C. Taken from Tatiyaborworntham et al. (2012) with permission. 4 67th Annual Reciprocal Meat Conference cubation (pH 5.6, 4°C), respectively. Histidine residues 48, 88 and 152 were identified as HNE alkylation sites by MALDI-TOF-TOF tandem mass spectrometry. It is important to remember that the double mutation not only increased the number of nucleophilic histidine residues available for HNE alkylation, but also likely altered the tertiary structure which would have affected the potential for HNE adduct formation. Final Thoughts The chemistry of myoglobin is critical to understanding the basis for normal and anomalous color in fresh meat. Reactions critical to myoglobin redox stability can involve both the heme and apoprotein components, and future advancements for improving meat color stability will continue to require attention to the fundamental chemistry of this protein. Acknowledgements I would like to thank all of my colleagues worldwide that have engaged in meat color research and in particular those that have focused on the biochemistry of myoglobin. Appreciation is extended to Surendranath Suman and Joe Sebranek who provided helpful contributions to this manuscript. 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