Yang J, Dunker A K, Powers J R, Clark S, Swanson B G
Department of Food Science and Human Nutrition, Washington State University, Pullman, Washington 99164-6376, USA.
J Agric Food Chem. 2001 Jul;49(7):3236-43. doi: 10.1021/jf001226o.
Beta-lactoglobulin (beta-LG) was treated with high hydrostatic pressure (HHP) at 600 MPa and 50 degrees C for selected times as long as 64 min. The intrinsic tryptophan fluorescence of beta-LG indicated that HHP treatment conditions induced a conformational change. HHP treatment conditions also promote a 3-fold increase in the extrinsic fluorescence of 1-anilinonaphthalene-8-sulfonate and a 2.6-fold decrease for cis-paraneric acid, suggesting an increase in accessible aromatic hydrophobicity and a decrease in aliphatic hydrophobicity. Far-ultraviolet circular dichroism (CD) spectra reveal that the secondary structure of beta-LG converts from native beta-sheets to non-native alpha-helices following HHP treatment, whereas near-ultraviolet CD spectra reveal that the native tertiary structure of beta-LG essentially disappears. Urea titrations reveal that native beta-LG unfolds cooperatively, but the pressure-treated molecule unfolds noncooperatively. The noncooperative state is stable for 3 months at 5 degrees C. The nonaccessible free thiol group of cysteine121 in native beta-LG became reactive to Ellman's reagent after adequate HHP treatment. Gel electrophoresis with and without beta-mercaptoethanol provided evidence that the exposed thiol group was lost concomitant with the formation of S-S-linked beta-LG dimers. Overall, these results suggest that HHP treatments induce beta-LG into hydrophobic molten globule structures that remain stable for at least 3 months.
β-乳球蛋白（β-LG）在600兆帕和50摄氏度的条件下接受高静水压（HHP）处理，处理时间长达64分钟。β-LG的内在色氨酸荧光表明，HHP处理条件诱导了构象变化。HHP处理条件还使1-苯胺基萘-8-磺酸盐的外在荧光增加了3倍，顺式对香豆酸的外在荧光降低了2.6倍，这表明可及的芳香族疏水性增加，脂肪族疏水性降低。远紫外圆二色性（CD）光谱显示，HHP处理后，β-LG的二级结构从天然的β-折叠转变为非天然的α-螺旋，而近紫外CD光谱显示，β-LG的天然三级结构基本消失。尿素滴定表明，天然β-LG协同展开，但经压力处理的分子非协同展开。这种非协同状态在5摄氏度下可稳定3个月。天然β-LG中半胱氨酸121的不可及游离巯基在经过适当的HHP处理后，对埃尔曼试剂有反应。有无β-巯基乙醇的凝胶电泳提供了证据，表明暴露的巯基在形成S-S连接的β-LG二聚体时丢失。总体而言，这些结果表明，HHP处理将β-LG诱导成疏水熔融球状结构，该结构至少可稳定3个月。
