MATERIALS & METHODSBeef plasma protein (BPP), egg white and potato extract were tested for their ability to inhibit proteolysis in fish mince and surimi made from Pacific whiting (Merluccius pxxfuctus). Strong inhibition resulted from all three compounds in fish mince when measured by autolysis. However, when tested in surimi significant differences occurred among the compounds. BPP showed strongest inhibition of proteolytic effect followed by egg white and potato extract when measured by autolysis, gel electrophoresis and torsion. BPP was an effective inhibitor in surimi at a concentration as low as 1%.Surimi production

Commonly used protease assays and substrates were compared for sensitivity and simplicity in analyzing proteolytic activity in Pacific whiting causing gel weakening of surimi during heat-setting. Assay based on detection of trichloroacetic acid (TCA)-soluble products, using azocasein as substrate, showed highest sensitivity. By that assay, optimal pH of the protease was 5.5, and optimal temperature, 55°C. The validity of the assay for measuring activity was confirmed by pH profiles of residual proteolytic and autolytic activities of uncooked surimi. These analyses showed pH profiles similar to those of fish juice with a pH optimum of 5.5.

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The solution 500-MHz 1H NMR spectral parameters for ferricytochrome b562, a soluble 12-kDa electron carrier from Escherichia coli with axial His/Met coordination, are shown to be strongly influenced by protein concentration and ionic strength at low pH and 25 degrees C in a manner consistent with significant aggregation at low ionic strength. At high ionic strength a well-resolved 1H NMR spectrum reveals over 40 hyperfine-shifted resonances which arise from two isomeric species in the ratio 2:1. 2D COSY and NOESY maps at 25 degrees C for the hyperfine-shifted resonances allow the assignment of a number of axial His resonances and all heme peripheral substituent peaks. The resulting asymmetric heme contact shift patterns, together with the halving of the number of lines when reconstituting with 2-fold symmetric hemin, demonstrate the molecular basis of the solution heterogeneity to be heme orientational disorder. The strongly upfield-shifted axial Met-7 resonances, characteristic of low-spin ferricytochromes c with His/Met ligation, appear upfield only at very low temperatures. At elevated temperatures, all resonances, in particular those of the axial Met, move strongly downfield. Detailed analysis of the deviation from Curie behavior for different functional groups demonstrates the presence of a low spin in equilibrium high spin equilibrium with an intact His-Fe-Met coordination. The weaker axial field in ferricytochrome b562, relative to the purely low-spin ferricytochromes c, is attributed to a perturbed iron-Met bond. The contact shifts for a coordinated Met in the high-spin state are estimated. A link between equatorial hemin and axial ligand interactions is indicated by a differential population of the high-spin form for the two hemin orientations.

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