Department of Bioengineering, Santa Clara University, Santa Clara, California, United States of America.
PLoS One. 2014 Jan 21;9(1):e86785. doi: 10.1371/journal.pone.0086785. eCollection 2014.
Research over the past few decades has attempted to answer how proteins behave in molecularly confined or crowded environments when compared to dilute buffer solutions. This information is vital to understanding in vivo protein behavior, as the average spacing between macromolecules in the cell cytosol is much smaller than the size of the macromolecules themselves. In our study, we attempt to address this question using three structurally and functionally different model enzymes encapsulated in agarose gels of different porosities. Our studies reveal that under standard buffer conditions, the initial reaction rates of the agarose-encapsulated enzymes are lower than that of the solution phase enzymes. However, the encapsulated enzymes retain a higher percentage of their activity in the presence of denaturants. Moreover, the concentration of agarose used for encapsulation had a significant effect on the enzyme functional stability; enzymes encapsulated in higher percentages of agarose were more stable than the enzymes encapsulated in lower percentages of agarose. Similar results were observed through structural measurements of enzyme denaturation using an 8-anilinonaphthalene-1-sulfonic acid fluorescence assay. Our work demonstrates the utility of hydrogels to study protein behavior in highly confined environments similar to those present in vivo; furthermore, the enhanced stability of gel-encapsulated enzymes may find use in the delivery of therapeutic proteins, as well as the design of novel strategies for biohybrid medical devices.
过去几十年的研究试图回答，与稀缓冲溶液相比，蛋白质在分子受限或拥挤的环境中是如何表现的。了解细胞胞质溶胶中大分子之间的平均间隔远小于大分子本身的大小，这对于理解体内蛋白质行为至关重要。在我们的研究中，我们尝试使用三种结构和功能不同的模型酶，将其包埋在不同孔隙率的琼脂糖凝胶中，来解决这个问题。我们的研究表明，在标准缓冲条件下，琼脂糖包埋酶的初始反应速率低于溶液相酶。然而，在变性剂存在的情况下，包埋酶保留了更高比例的活性。此外，用于包埋的琼脂糖浓度对酶功能稳定性有显著影响；与琼脂糖浓度较低的酶相比，琼脂糖浓度较高的酶更稳定。通过使用 8-苯胺基-1-萘磺酸荧光测定法对酶变性进行结构测量，也观察到了类似的结果。我们的工作证明了水凝胶在研究与体内存在的高度受限环境相似的蛋白质行为方面的实用性；此外，凝胶包埋酶的增强稳定性可能在治疗性蛋白质的递送上找到用途，以及用于生物混合医疗设备的新型策略的设计。
