Mechanisms of Heme Protein-Mediated Lipid Oxidation Using Hemoglobin and Myoglobin Variants in Raw and Heated Washed Muscle

Abstract

The hemoglobin variant rHb 0.1, which possesses a decreased ability to form subunits, stimulated lipid oxidation in washed fish muscle less effectively as compared to wild-type hemoglobin (rHb 0.0). This could be due to the lower hemin affinity and more rapid autoxidation rate of subunits as compared to tetramers. To differentiate between hemin affinity and autoxidation effects, ferrous V68T Mb was compared to ferrous wild-type myoglobin (WT Mb). WT Mb has a more rapid hemin loss rate (25-fold) than does V68T, while V68T autoxidized more rapidly than did WT Mb (60-fold). Ferrous WT Mb promoted TBARS and lipid peroxide formation more rapidly than did ferrous V68T (p < 0.01). This indicated hemin loss rate was more critical in determining onset of lipid oxidation as compared to autoxidation rate. Hemin alone was capable of stimulating lipid oxidation. Albumin enhanced the ability of hemin to promote lipid oxidation. MetMb promoted lipid oxidation more effectively than did ferrous Mb, which could be due to the lower hemin affinity of metMb as compared to that of ferrous Mb. EDTA, an iron chelator, had no effect on the rate or extent of lipid oxidation mediated by Mb in the cooked system. Variants with a 975-fold range of hemin affinities promoted lipid oxidation with equivalent efficacy in cooked washed cod contrary to results in uncooked washed cod. The cooking temperatures apparently denature the globin and release hemin reactant to such an extent that the impact of hemin affinity on lipid oxidation observed in the raw state is negated in the cooked state. These studies collectively suggest released hemin is of primary importance in promoting lipid oxidation in raw and cooked washed fish muscle.

Effect of heating oxyhemoglobin and methemoglobin on microsomes oxidation

“…Mb and Hb have been reported to promote lipid oxidation in muscle based foods (Baron, Andersen 2002, Kanner, Harel 1985, Grunwald, Richards 2006a, Richards, Dettmann et al 2005. The ability of Mb and Hb to promote oxidation is attributed to their heme group so the oxidation mechanisms are common although subject to several conditions that may result in enhancement or decrease of their prooxidative activity (Carlsen, Moller et al 2005).…”

“…The increased heme moiety exposure reported after protein unfolding as a consequence of heating at a moderate/high temperatures (60-70 C) (Kristensen, Andersen 1997) and the release of hematin (Grunwald, Richards 2006a) and iron (Decker, Hultin 1992) from Mb and Hb at higher temperatures have been indicated as major causes of the increased susceptibility to oxidation in cooked meats. In our conditions, heme and non-heme iron content results indicated that the iron-porphyrin moiety was quite resistant to thermal treatments even when heated at 90 C (Table 1) which was also in agreement with other works (Bou, Guardiola et al 2008, Kristensen, Andersen 1997, Han, Mcmillin et al 1993).…”

“…To study the pro-oxidant activity of Hb, this was added into microsomes to an Hb / protein ratio similar to that found in meats. The ability of OxyHb in the presence of microsomes to promote oxidation showed no differences after 2 hr of incubation whereas the blank containing only OxyHb showed lower and steady TBARS values over time Some controversy exists about whether the heme or free iron from both Mb and Hb is the major responsible for lipid oxidation in cooked meats (Johns, Birkinshaw et al 1989, Grunwald, Richards 2006a, Han, Mcmillin et al 1995, Schricker, Miller 1983. In order to a better understanding we conducted a trial in which OxyHb and MetHb either native or heated were incubated for 8 hr at 37 C in the presence of microsomes with or without added EDTA.…”

“…Various factors such as the ability of the Hb to autoxidize and release of hematin, the heme-iron moiety non-bound to the protein, have also been reported to be crucial in promoting lipid oxidation (Richards, Dettmann 2003, Grunwald, Richards 2006a, Richards, Dettmann et al 2005). …”

“…Several factors such as the release of hematin from Hb and iron from the porphyrin moiety and from other proteins such as ferritin and transferrin have been reported in meats (Monahan, Crackel et al 1993, Grunwald, Richards 2006a, Han, Mcmillin et al 1995. However, heating treatments provoke protein precipitation an this loss of solubility has been reported to be determinant for Mb in inhibiting oxidation (Bou, Guardiola et al 2008).…”