Pastor Isabel, Prieto Manuel, Mateo C Reyes
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202-Elche, Spain.
J Phys Chem B. 2008 Nov 27;112(47):15021-8. doi: 10.1021/jp805368s.
Immobilization of proteins in sol-gel glasses has allowed the development of a new generation of robust and sensitive analytical devices as well as contributes to the investigation of the effect of molecular confinement on the structure of proteins. The immobilized protein usually preserves its structural integrity and functionality, while interactions with the matrix and its surface seem to contribute to alter its dynamics and stability. With the aim of better understanding the nature of such interactions, we have encapsulated the enzyme bovine Cu,Zn superoxide dismutase (BSOD), negatively charged at physiological pH, in a sol-gel matrix and the photophysical properties of its single tyrosine have been determined using both steady-state and time-resolved fluorescence techniques. Fluorescence spectra, quenching experiments, fluorescence lifetimes, and anisotropy measurements indicate that immobilization does not lead to any major conformational change, at least in the region of protein where the tyrosine residue is located. In addition, fluorescence anisotropy decays recorded above and below the isoelectric point of the protein indicate that, at neutral pH, well above its isoelectric point, the entrapped BSOD freely rotates within the matrix pore, but showing a different rotational behavior as compared with that in the bulk aqueous solution. However, below the isoelectric point, the global motion of the protein is totally hindered upon entrapment. Electrostatic interactions with the gel matrix, changes in water viscosity, and protein-to-pore size ratio are discussed as possible factors responsible for this behavior.
将蛋白质固定在溶胶 - 凝胶玻璃中，不仅推动了新一代坚固且灵敏的分析装置的发展，还有助于研究分子限制对蛋白质结构的影响。固定化的蛋白质通常能保持其结构完整性和功能，然而与基质及其表面的相互作用似乎会改变其动力学和稳定性。为了更好地理解此类相互作用的本质，我们将在生理pH下带负电荷的牛铜锌超氧化物歧化酶（BSOD）封装在溶胶 - 凝胶基质中，并使用稳态和时间分辨荧光技术测定了其单个酪氨酸的光物理性质。荧光光谱、猝灭实验、荧光寿命和各向异性测量表明，至少在酪氨酸残基所在的蛋白质区域，固定化不会导致任何重大的构象变化。此外，在蛋白质等电点之上和之下记录的荧光各向异性衰减表明，在中性pH（远高于其等电点）下，被包封的BSOD在基质孔内自由旋转，但与在本体水溶液中的旋转行为不同。然而，在等电点以下，蛋白质的整体运动在被包封后完全受阻。讨论了与凝胶基质的静电相互作用、水粘度的变化以及蛋白质与孔径比作为造成这种行为的可能因素。
