Williams Dominique E, Basnet Kanchan, Grant Kathryn B
Department of Chemistry, Georgia State University, P.O. Box 3965, Atlanta, GA 30302-3965 (USA).
Chembiochem. 2015 Jul 6;16(10):1474-82. doi: 10.1002/cbic.201500041. Epub 2015 Jun 10.
With the goal of designing a lysosomal phospholipase mimic, we optimized experimental variables to enhance Ce(IV) -assisted hydrolysis of phosphatidylcholine (PC) liposomes. Our best result was obtained with the chelating agent bis-tris propane (BTP). Similar to the hydrolytic enzyme, Ce(IV) -assisted hydrolysis of PC phosphate ester bonds was higher at lysosomal pH (∼4.8) compared to pH 7.2. In the presence of BTP, the average cleavage yield at ∼pH 4.8 and 37 °C was: 67±1 %, 5.7-fold higher than at ∼pH 7.2 and roughly equivalent to the percent of phospholipid found on the metal-accessible exo leaflet of small liposomes. No Ce(IV) precipitation was observed. When BTP was absent, there was significant turbidity, and the amount of cleavage at ∼pH 4.8 (69±1 %) was 2.1-fold higher than the yield obtained at ∼pH 7.2. Our results show that BTP generates homogenous solutions of Ce(IV) that hydrolyze phosphatidylcholine with enhanced selectivity for lysosomal pH.
为了设计一种溶酶体磷脂酶模拟物，我们优化了实验变量，以增强铈（IV）辅助的磷脂酰胆碱（PC）脂质体水解。我们使用螯合剂双三羟甲基氨基甲烷（BTP）获得了最佳结果。与水解酶类似，与pH 7.2相比，在溶酶体pH（约4.8）下，铈（IV）辅助的PC磷酸酯键水解率更高。在BTP存在的情况下，在约pH 4.8和37°C时的平均裂解产率为：67±1%，比在约pH 7.2时高5.7倍，大致相当于小脂质体金属可及外小叶上发现的磷脂百分比。未观察到铈（IV）沉淀。当不存在BTP时，会出现明显的浊度，并且在约pH 4.8时的裂解量（69±1%）比在约pH 7.2时获得的产率高2.1倍。我们的结果表明，BTP生成铈（IV）的均匀溶液，该溶液水解磷脂酰胆碱时对溶酶体pH具有更高的选择性。
