Belloque Josefina, Chicón Rosa, López-Fandiño Rosina
Instituto de Fermentaciones Industriales CSIC, Madrid, Spain.
J Agric Food Chem. 2007 Jun 27;55(13):5282-8. doi: 10.1021/jf070170w. Epub 2007 Jun 2.
The unfolding of beta-lactoglobulin during high-pressure treatment and its refolding after decompression were studied by 1H NMR and 2H/1H exchange at pH 6.8 and 2.5 and at 37 and 25 degrees C. The extent of unfolding increased with the pressure level. The structure of beta-lactoglobulin required higher pressures to unfold at pH 2.5 than at pH 6.8. More flexibility was achieved at 37 degrees C than at 25 degrees C. Results indicated that the structural region formed by strands F, G, and H was more resistant to unfold under acidic and neutral conditions. The exposure of Trp19 at an earlier time, as compared to other protein regions, supports the formation of a swollen structural state at pH 2.5. Refolding was achieved faster when beta-lactoglobulin was subjected to 200 MPa than to 400 MPa, to 37 degrees C than to 25 degrees C, and to acidic than to neutral pH. After treatment at 400 MPa for 20 min at neutral pH, the protein native structure was not recovered. All samples at acidic pH showed that the protein quickly regained its structure. Hydrolysis of beta-lactoglobulin by pepsin and chymotrypsin could be related to pressure-induced changes in the structure of the protein. Compared to the behavior of the protein at atmospheric pressure, no increased proteolysis was found in samples with no increased flexibility (100 MPa, 37 degrees C, pH 2.5). Slightly flexible structures were associated with significantly increased proteolysis (100 MPa, 37 degrees C, pH 6.8; 200 MPa, 37 degrees C, pH 2.5). Highly flexible structures were associated with very fast proteolysis (>or=200 MPa, 37 degrees C, pH 6.8; >or=300 MPa, 37 degrees C, pH 2.5). Proteolysis of prepressurized samples improved only when the protein was significantly changed after the pressure treatment (400 MPa, 25 degrees C, 20 min, pH 6.8).
通过1H NMR以及在pH 6.8和2.5、37℃和25℃条件下的2H/1H交换，研究了β-乳球蛋白在高压处理过程中的展开及其减压后的复性。展开程度随压力水平的升高而增加。β-乳球蛋白的结构在pH 2.5时比在pH 6.8时需要更高的压力才能展开。在37℃时比在25℃时能获得更大的柔韧性。结果表明，由F、G和H链形成的结构区域在酸性和中性条件下更抗展开。与其他蛋白质区域相比，Trp19在更早的时间暴露，这支持了在pH 2.5时形成膨胀结构状态。当β-乳球蛋白承受200 MPa压力时比承受400 MPa压力时、在37℃时比在25℃时、在酸性pH条件下比在中性pH条件下能更快地实现复性。在中性pH条件下于400 MPa处理20分钟后，蛋白质的天然结构未恢复。所有酸性pH条件下的样品都表明蛋白质能迅速恢复其结构。胃蛋白酶和胰凝乳蛋白酶对β-乳球蛋白的水解可能与压力诱导的蛋白质结构变化有关。与蛋白质在常压下的行为相比，在柔韧性未增加的样品（100 MPa，37℃，pH 2.5）中未发现蛋白水解增加。略微柔韧的结构与显著增加的蛋白水解相关（100 MPa，37℃，pH 6.8；200 MPa，37℃，pH 2.5）。高度柔韧的结构与非常快速的蛋白水解相关（≥200 MPa，37℃，pH 6.8；≥300 MPa，37℃，pH 2.5）。只有当蛋白质在压力处理后发生显著变化时（400 MPa，25℃，20分钟，pH 6.8），预加压样品的蛋白水解才会改善。
