Eissa Ahmed S, Puhl Christa, Kadla John F, Khan Saad A
Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695-7905, USA.
Biomacromolecules. 2006 Jun;7(6):1707-13. doi: 10.1021/bm050928p.
In this study, we use FTIR spectroscopy to probe the conformational changes of beta-lactoglobulin (beta-LG)-the main constituent of whey proteins-as subjected to enzymatic cross-linking by transglutaminase. We investigate both the amide I region (1600-1700 cm(-1)) and the C-H stretching region (2800-3100 cm(-1)). In the amide I region, spectra of denatured conformations of beta-LG, known to be necessary for cross-linking, differ according to the denaturation procedure, i.e., chemical or thermal treatment. Denaturation by chemical denaturants, dithiothreitol (DTT) or beta-mercaptoethanol, show no effect on the alpha-helix, while shifting the monomer dimer equilibrium toward higher monomer concentration. On the other hand, denaturing by thermal treatment dissociates the beta-sheets in the native structure, leading to new intermolecular beta-sheets being formed. Preheated then enzyme cross-linked beta-LG molecules show very similar spectra in the amide I region to the molecules with no cross-linking, indicating minimal effects of the cross-links on the carbonyl stretching mode. However, chemically denatured (using beta-mercaptoethanol) then enzyme cross-linked beta-LG molecules show noticeable diminution in the alpha-helix band and formation of strong hydrogen-bonded intermolecular beta-sheets. In the C-H stretching region, preheated then enzyme cross-linked beta-LG molecules exhibit a different degree of exposure of aliphatic amino acids due to the enzyme action. The same behavior is observed for DTT-treated then enzyme cross-linked beta-LG molecules. Generally, the changes in the C-H stretching region clearly indicate that hydrophobic interactions are altered upon enzymatic cross-linking.
在本研究中，我们使用傅里叶变换红外光谱（FTIR）来探测β-乳球蛋白（β-LG）（乳清蛋白的主要成分）在经转谷氨酰胺酶进行酶促交联时的构象变化。我们研究了酰胺I区域（1600 - 1700 cm⁻¹）和C - H伸缩区域（2800 - 3100 cm⁻¹）。在酰胺I区域，已知交联所必需的β-LG变性构象的光谱会因变性程序（即化学或热处理）而有所不同。用化学变性剂二硫苏糖醇（DTT）或β-巯基乙醇进行变性处理，对α-螺旋没有影响，但会使单体 - 二聚体平衡向更高的单体浓度方向移动。另一方面，热处理变性会使天然结构中的β-折叠解离，导致形成新的分子间β-折叠。预热后再进行酶促交联的β-LG分子在酰胺I区域的光谱与未交联的分子非常相似，表明交联对羰基伸缩模式的影响最小。然而，用β-巯基乙醇进行化学变性后再进行酶促交联的β-LG分子，其α-螺旋带明显减弱，并形成了强氢键结合的分子间β-折叠。在C - H伸缩区域，预热后再进行酶促交联的β-LG分子由于酶的作用表现出不同程度的脂肪族氨基酸暴露。对于DTT处理后再进行酶促交联的β-LG分子也观察到了相同的行为。一般来说，C - H伸缩区域的变化清楚地表明，酶促交联后疏水相互作用发生了改变。
