Showing papers by "J. Bruce German published in 1995"

Effect of pH and temperature on protein unfolding and thiol/disulfide interchange reactions during heat-induced gelation of whey proteins

Abstract: In heat-induced whey protein isolate (WPI) gels, polymerization of the constituent whey proteins, via intermolecular disulfide (S-S) bonding, was dependent on both the pH of the WPI solution and the temperature to which the solution was heated. At pH 9 and 11, polymerization as determined by SDS-PAGE occurred at room temperature (22 o C), while at pH 3, 5, and 7, polymerization was only evident after heating to 85, 75, and 70 o C, respectively. Measurement of total sulfhydryl (SH) group content of gelling WPI solutions at each pH and temperature revealed that in the WPI solutions at pH 9 and 11 significant SH-SH oxidation to S-S occurred even at room temperature. In contrast, the total SH content of WPI solutions at pH 3 and 5 did not change with heating, indicating that polymerization reactions involving SH/S-S interchange rather than SH/SH oxidation predominated. Estimation of the degree of unfolding of the whey proteins by measuring the exposure of hydrophobic amino acid residues showed that at pH 9 and 11 extensive irreversible unfolding of the protein molecules had occurred at room temperature

Direct Characterization of Protein Adducts of the Lipid Peroxidation Product 4-Hydroxy-2-nonenal Using Electrospray Mass Spectrometry

Abstract: Oxidative stress and exposures to xenobiotic substances generate reactive substances including the cytotoxic aldehyde 4-hydroxy-2-nonenal. This aldehyde exhibits a variety of biological effects and has been reported as a marker of lipid peroxidation. The toxicity and atherogenicity of 4-hydroxy-2-nonenal have been attributed to the formation of covalent protein adducts. In the current study, two model proteins, beta-lactoglobulin B and human hemoglobin, were exposed to 4-hydroxy-2-nonenal, and the protein adducts were characterized using electrospray ionization mass spectrometry. Our findings provided clear and direct evidence that > 99% of protein modification occurred via Michael addition, and only trace amounts of Schiff base adducts were formed. Confirmation of this result was obtained via quantitative conversion of the modified proteins to oxime and pentafluorobenzyl oxime derivatives as demonstrated by electrospray ionization mass spectrometry, spectrophotometric protein carbonyl assays, and gas chromatography/mass spectrometry determination of 4-hydroxy-2-nonenal released upon treatment with hydroxylamine. These results further demonstrate the availability of the protein-bound aldehyde for subsequent reaction or as a site of molecular recognition. The preponderance of Michael addition products over Schiff base adducts also suggests that most methods for determining 4-hydroxy-2-nonenal in biological tissues or fluids are based on erroneous assumptions that hydrazines or hydroxylamines release 4-hydroxy-2-nonenal from proteins.

Crystal morphology of mixtures of tripalmitin and butterfat

Abstract: Blends of butterfat and tripalmitin are of interest for use as moisture barriers in edible films. Tripalmitin was blended with butterfat in the ratios 100:0, 90:10, 80:20, and so on through to 10:90 and 0:100. Crystal morphologies of squash and smear preparations were determined by polarizing light microscopy. Samples were stored at 19–22°C and re-examined after 6 d and after 30 d. Morphology was strongly dependent on composition and the presence of a coverslip. Morphology was less dependent on polymorphic form and age. Barrier properties depend more strongly on morphology than on polymorphic form.