Conformational and functional changes of hemoglobin and myosin induced by pH: Functional role in fish quality
Conformational and functional changes in trout hemoglobin and cod myosin were investigated at low and high pH and after subsequent refolding. Hemoglobin cooperatively unfolded at low pH to a “molten globular” state with different levels of structure depending on how low the pH was and if 500 mM NaCl was present. At low pH the heme lost its contact with the protein but was not released but the protein was fully dissociated. Alkaline pH had little effect on hemoglobin but the heme environment suggested a strong heme-distal histidine link at pH 10.5. Longer unfolding time, lower pH and higher ionic strength made it more difficult to refold hemoglobin. The more misfolded the protein was the more hydrophobic it became and the lower its solubility after refolding it to pH 5.5. The presence of salt greatly facilitated the hydrophobic aggregation. The more unfolded or misfolded the protein was the more pro-oxidative it became, maybe as a result of more exposed heme, heme and subunit dissociation and hemoglobin-membrane interactions. High pH completely suppressed pro-oxidative activity of hemoglobin, possibly due to a strong heme-distal histidine link preventing autoxidation.
Helical structure of myosin rods was little affected at low and high but the head group was substantially unfolded. The protein was possibly fully dissociated at low pH while at high pH only half of the light chains dissociated. The myosin head was misfolded on refolding with increased hydrophobicity, reactive −SH groups, loss in ATPase activity and lower conformational stability. The heavy chains may have fully reassembled on refolding from acid pH but light chains failed to reassemble to the protein. Refolded myosin had almost identical solubility as native myosin at pH 7.5 while cod myofibrillar proteins had increased solubility after acid and alkali treatment compared to the untreated proteins. Myosin and myofibrillar proteins exhibited improved emulsification activity and stability at pH 7.5 after pH treatment. Myosin gelled at a lower temperature and formed stronger gels at pH 7.5 in 600 mM KCl. The pH treated myofibrillar proteins also gelled at a lower temperature than native myosin but had a different gelling mechanism on heating.