Rangel-Yagui Carlota O, Lam Henry, Kamei Daniel T, Wang Daniel I C, Pessoa Adalberto, Blankschtein Daniel
Department of Chemical Engineering, Room 66-444, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Biotechnol Bioeng. 2003 May 20;82(4):445-56. doi: 10.1002/bit.10586.
The enzyme glucose-6-phosphate dehydrogenase (G6PD) plays an important role in maintaining the level of NADPH and in producing pentose phosphates for nucleotide biosynthesis. It is also of great value as an analytical reagent, being used in various quantitative assays. In searching for new strategies to purify this enzyme, the partitioning of G6PD in two-phase aqueous mixed (nonionic/cationic) micellar systems was investigated both experimentally and theoretically. Our results indicate that the use of a two-phase aqueous mixed micellar system composed of the nonionic surfactant C(10)E(4) (n-decyl tetra(ethylene oxide)) and the cationic surfactant C(n)TAB (alkyltrimethylammonium bromide, n = 8, 10, or 12) can improve significantly the partitioning behavior of G6PD relative to that obtained in the two-phase aqueous C(10)E(4) micellar system. This improvement can be attributed to electrostatic attractions between the positively charged mixed (nonionic/cationic) micelles and the net negatively charged enzyme G6PD, resulting in the preferential partitioning of G6PD to the top, mixed micelle-rich phase of the two-phase aqueous mixed micellar systems. The effect of varying the cationic surfactant tail length (n = 8, 10, and 12) on the denaturation and partitioning behavior of G6PD in the C(10)E(4) /C(n)TAB/buffer system was investigated. It was found that C(8)TAB is the least denaturing to G6PD, followed by C(10)TAB and C(12)TAB. However, the C(10)E(4)/C(12)TAB/buffer system generated stronger electrostatic attractions with the net negatively charged enzyme G6PD than the C(10)E(4)/C(10)TAB/buffer and the C(10)E(4)/C(8)TAB/buffer systems, when using the same amount of cationic surfactant. Overall, the two-phase aqueous mixed (C(10)E(4)/C(10)TAB) micellar system yielded the highest G6PD partition coefficient of 7.7, with a G6PD yield in the top phase of 71%, providing the optimal balance between the denaturing effect and the electrostatic attractions for the three cationic surfactants examined. A recently developed theoretical framework to predict protein partition coefficients in two-phase aqueous mixed (nonionic/ionic) micellar systems was implemented, and the theoretically predicted G6PD partition coefficients were found to be in reasonable quantitative agreement with the experimentally measured ones.
葡萄糖-6-磷酸脱氢酶（G6PD）在维持烟酰胺腺嘌呤二核苷酸磷酸（NADPH）水平以及为核苷酸生物合成产生戊糖磷酸方面发挥着重要作用。作为一种分析试剂，它也具有很高的价值，被用于各种定量分析中。在寻找纯化这种酶的新策略时，我们对G6PD在双水相混合（非离子/阳离子）胶束体系中的分配进行了实验和理论研究。我们的结果表明，使用由非离子表面活性剂C(10)E(4)（正癸基四（环氧乙烷））和阳离子表面活性剂C(n)TAB（烷基三甲基溴化铵，n = 8、10或12）组成的双水相混合胶束体系，相对于在双水相C(10)E(4)胶束体系中，能显著改善G6PD的分配行为。这种改善可归因于带正电荷的混合（非离子/阳离子）胶束与带净负电荷的酶G6PD之间的静电吸引，导致G6PD优先分配到双水相混合胶束体系富含混合胶束的上相中。研究了改变阳离子表面活性剂尾链长度（n = 8、10和12）对G6PD在C(10)E(4)/C(n)TAB/缓冲液体系中的变性和分配行为的影响。发现C(8)TAB对G6PD的变性作用最小，其次是C(10)TAB和C(12)TAB。然而，当使用相同量的阳离子表面活性剂时，C(10)E(4)/C(12)TAB/缓冲液体系与带净负电荷的酶G6PD产生的静电吸引比C(10)E(4)/C(10)TAB/缓冲液体系和C(10)E(4)/C(8)TAB/缓冲液体系更强。总体而言，双水相混合（C(10)E(4)/C(10)TAB）胶束体系产生的G6PD分配系数最高，为7.7，上相中G6PD的产率为71%，在所研究的三种阳离子表面活性剂中，在变性作用和静电吸引之间提供了最佳平衡。实施了一个最近开发的理论框架来预测蛋白质在双水相混合（非离子/离子）胶束体系中的分配系数，发现理论预测的G6PD分配系数与实验测量值在合理的定量范围内一致。
