76D-15 |
The pro-oxidative activity of acid unfolded molten globular intermediates of flounder hemoglobin |
H. G. KRISTINSSON, Department of Food Science and Human Nutrition, University of Florida, Gainesville, FL 32611 Fish protein isolates of good functionality can be produced via a new process involving low pH (pH 2-3) treatment and subsequent isoelectric precipitation. The final protein isolate can contain sizable amounts of hemoglobin which can cause significant oxidation problems. The objective was to study how the structure and conformation of flounder hemoglobin is affected by low pH and subsequent protein refolding to neutral pH, and investigate the pro-oxidative activity of the unfolded and refolded hemoglobin. Hemoglobin was prepared from anesthetized flounder. Conformational changes of acid unfolded and refolded hemoglobin were monitored by changes in intrinsic tryptophan fluorescence, ANS binding, near and far-UV circular dichroism spectroscopy. Kinetics of heme group unfolding with time at low pH was monitored spectrally at 408nm. Hemoglobin dissociation at low pH was determined by native electrophoresis. Pro-oxidative activity of unfolded and refolded hemoglobins was determined on linoleic acid emulsion. Flounder hemoglobin co-operatively unfolded at low pH to a “molten globular” state with different levels of structure depending on how low the pH was and if NaCl (500 mM) was present, retaining large amount of secondary structure while loosing its tertiary structure. At low pH the heme lost its contact with the protein but was not released. Low pH fully dissociated the protein into its monomers. Longer unfolding time, lower pH and higher ionic strength made it more difficult to refold hemoglobin. The more unfolded or misfolded the protein was the more pro-oxidative it became, possibly as a result of more exposed heme, subunit dissociation and hemoglobin-micelle interactions. Low pH has a significant effect on flounder hemoglobin. Better understanding on the link between the conformational changes induced by pH and the pro-oxidative activity of hemoglobin will aid in developments of strategies to minimize oxidation in low pH processes utilizing muscle material.
Session 76D, Muscle Foods II
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