Department of Biological Chemistry, Indian Association for the Cultivation of Science Jadavpur , Kolkata 700 032, India.
Langmuir. 2014 Mar 11;30(9):2448-59. doi: 10.1021/la403835h. Epub 2014 Feb 25.
This article delineates the formation and characterization of different enzyme-carbon dot conjugates in aqueous medium (pH = 7.0). We used soybean peroxidase (SBP), Chromobacterium viscosum (CV) lipase, trypsin, and cytochrome c (cyt c) for the formation of conjugate either with cationic carbon dot (CCD) or anionic carbon dot (ACD) depending on the overall charge of the protein at pH 7.0. These nanobioconjugates were used to probe the location of enzymes in water-in-oil (w/o) microemulsion. The size of the synthesized water-soluble carbon dots were of 2-3 nm with distinctive emission property. The formation of enzyme/protein-carbon dot conjugates in aqueous buffer was confirmed via fluorescence spectroscopy and zeta potential measurement, and the structural alteration of enzyme/protein was monitored by circular dichroism spectroscopy. Biocatalytic activities of protein/enzymes in conjugation with carbon dots were found to be decreased in aqueous phosphate buffer (pH 7.0, 25 mM). Interestingly, the catalytic activity of the nanobioconjugates of SBP, CV lipase, and cyt c did not reduce in cetyltrimethylammonium bromide (CTAB)-based reverse micelle. It indicates different localization of carbon dots and the enzymes inside the reverse micelle. The hydrophilic carbon dots always preferred to be located in the water pool of reverse micelle, and thus, enzyme must be located away from the water pool, which is the interface. However, in case of trypsin-carbon dot conjugate, the enzyme activity notably decreased in reverse micelle in the presence of carbon dot in a similar way that was observed in water. This implies that trypsin and carbon dots both must be located at the same place, which is the water pool of reverse micelle. Carbon dot induced deactivation was not observed for those enzymes which stay away from the water pool and localized at the interfacial domain while deactivation is observed for those enzymes which reside at the water pool. Thus, the location of enzymes in the microdomain of w/o microemulsion can be predicted by comparing the activity profile of enzyme-carbon dot conjugate in water and w/o microemulsion.
本文描述了在水相介质（pH = 7.0）中不同酶-碳点缀合物的形成和特性。我们使用大豆过氧化物酶（SBP）、粘质沙雷氏菌（CV）脂肪酶、胰蛋白酶和细胞色素 c（cyt c），根据蛋白质在 pH 7.0 时的总电荷，形成与阳离子碳点（CCD）或阴离子碳点（ACD）的缀合物。这些纳米生物缀合物用于探测酶在油包水（w/o）微乳液中的位置。合成的水溶性碳点的尺寸为 2-3nm，具有独特的发射特性。通过荧光光谱和zeta 电位测量证实了酶/蛋白质-碳点缀合物在水缓冲液中的形成，并通过圆二色性光谱监测了酶/蛋白质的结构变化。发现与碳点缀合的蛋白质/酶的生物催化活性在磷酸盐缓冲液（pH 7.0，25mM）中降低。有趣的是，SBP、CV 脂肪酶和 cyt c 的纳米生物缀合物的催化活性在 CTAB 基反胶束中没有降低。这表明碳点和酶在反胶束中的不同定位。亲水性碳点总是优先位于反胶束的水相中，因此酶必须位于远离水相的界面处。然而，对于胰蛋白酶-碳点缀合物，在碳点存在的情况下，其在反胶束中的酶活性明显降低，这与在水中观察到的方式相似。这意味着胰蛋白酶和碳点必须位于同一位置，即反胶束的水相。对于那些远离水相并定位于界面域的酶，没有观察到碳点诱导的失活，而对于那些位于水相的酶，则观察到失活。因此，可以通过比较酶-碳点缀合物在水相和 w/o 微乳液中的活性谱，预测酶在 w/o 微乳液的微区中的位置。
