Extensive studies have been done on beta-lactoglobulin (beta-Lg) fibrils in the past decade due to their potential as functional food ingredients, gelling agents, and encapsulation devices etc. (van der Goot, A. J.; Peighambardoust, S. H.; Akkermans, C.; van Oosten-Manski, J. M. Creating novel structures in food materials: The role of well-defined shear flow. Food Biophys. 2008, 3(2), 120-125 and Loveday, S. M.; Rao, M. A.; Creamer, L. K.; Singh, H. Factors affecting rheological characteristics of fibril gels: The case of beta-lactoglobulin and alpha-lactalbumin. J. Food Sci. 2009, 74 (3), R47-R55). However, most of the studies focus on the formation and mechanism of the fibrils. Little is known about fibril digestibility to date. In this work, in vitro pepsin digestion of bovine beta-lactoglobulin (beta-Lg) fibrils in simulated gastric fluid was investigated using thioflavin T fluorescence photometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, size-exclusion chromatography, matrix-assisted laser desorption/ionization mass spectrometry, and transmission electron microscopy (TEM). The fibrils were formed by heating beta-Lg solutions at 80 degrees C and pH 2.0 for 20 h. The fibrils were found to be digested completely by pepsin within 2 min, when long, straight fibrils were no longer observed by TEM. The peptides in the fibrils (2000-8000 Da) could be digested to smaller peptides (mostly <2000 Da) by pepsin. The peptides in the fibrils were believed to be more susceptible for pepsin to access and attack because of their hydrophobic nature. For comparison purposes, solutions of beta-Lg heated at neutral pH (pH 7.4) were also studied under the same conditions.

A H52Q variant of yeast cytochrome c peroxidase (CcP), in which the distal histidine is replaced by glutamine, catalyzes oxidation of H(2)O(2) instead of reduction. This redirection of catalytic action is detected by protein film voltammetry. In the presence of H(2)O(2), wild-type CcP, adsorbed on a graphite electrode, shows a strong catalytic reduction wave commencing at about 0.8V (pH 5.4); by contrast, H52Q does not exhibit this activity but instead shows a catalytic oxidation current at potentials in the region of 0.9 V. The oxidation current is partly suppressed in the presence of tetranitromethane (a superoxide scavenger) and is not observed for other mutants studied, including H52A. The only significant structural change in the H52Q variant is that the Q-52 side chain occupies the space vacated by the H-52 imidazole; specifically, the N-epsilon atom that is believed to transfer a proton and induce O--O cleavage is replaced, to within 0.75 A, by the carbamide-O. Thus, while the weakly basic amide functionality is unable to serve in the reorganization of bound H(2)O(2), it is able to facilitate its oxidation, most obviously by serving as a H-bond acceptor to assist formation of a labile superoxide intermediate.

In this study, in vitro digestion of β-lactoglobulin (β-Lg) fibrils and the re-formation of fibril-like structures after prolonged enzymatic hydrolysis (up to 48 h) were investigated using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), thioflavin T fluorescence photometry, and transmission electron microscopy (TEM). Pure β-Lg fibrils that had been formed by heat treatment at pH 2.0 were rapidly hydrolyzed by pepsin in the simulated gastric fluid (pH 1.2), and some new peptides that were suitable for further fibril formation were produced. TEM showed that the new fibrils were long and straight but thinner than the original fibrils, and both TEM and MALDI-MS indicated that the peptides in the new fibrils were shorter/smaller than the peptides in the original fibrils. The formation of new fibrils was found to be affected more by pH than by enzyme activity or temperature.