Ashrafi Hajar, Amini Mohsen, Mohammadi-Samani Soliman, Ghasemi Younes, Azadi Amir, Tabandeh Mohammad Reza, Kamali-Sarvestani Eskandar, Daneshamouz Saeid
Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran; Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran.
Int J Biol Macromol. 2013 Nov;62:180-7. doi: 10.1016/j.ijbiomac.2013.08.028. Epub 2013 Aug 30.
The present study aims to develop a novel L-asparaginase fatty acid bioconjugates and characterize their applicability for intravenous delivery of L-asparaginase. These bioconjugates were achieved by covalent linkage of fatty acids having different chain lengths (C12, C16 and C22) to the native enzyme. To determine the optimum conditions of bioconjugation, the effect of lipid:protein ratios, reaction time and medium composition on enzyme activity and conjugation degree were evaluated. The native and bioconjugates have been characterized by activity, conjugation degree, particle size, and zeta potential. The results showed that bioconjugated L-asparaginase were more resistant to proteolysis, more stable at different pH, and had prolonged plasma half-life, compared to the native form. From partition coefficient study, the modified enzymes showed approximately 15-fold increase in hydrophobicity. Secondary structure analysis using circular dichroism revealed alteration after lipid conjugation. In addition, the Michaelis constant of the native enzyme was 3.38 mM, while the bioconjugates showed the higher affinity to the substrate L-asparagine. These findings indicate that new lipid bioconjugation could be a very useful strategy for intravenous delivery of L-asparaginase.
本研究旨在开发一种新型的L-天冬酰胺酶脂肪酸生物共轭物，并表征其在静脉注射L-天冬酰胺酶中的适用性。这些生物共轭物是通过将不同链长（C12、C16和C22）的脂肪酸与天然酶共价连接而实现的。为了确定生物共轭的最佳条件，评估了脂质与蛋白质比例、反应时间和介质组成对酶活性和共轭程度的影响。天然产物和生物共轭物已通过活性、共轭程度、粒径和zeta电位进行了表征。结果表明，与天然形式相比，生物共轭的L-天冬酰胺酶对蛋白水解更具抗性，在不同pH值下更稳定，并且血浆半衰期延长。通过分配系数研究，修饰后的酶疏水性增加了约15倍。使用圆二色性进行的二级结构分析显示脂质共轭后发生了变化。此外，天然酶的米氏常数为3.38 mM，而生物共轭物对底物L-天冬酰胺表现出更高的亲和力。这些发现表明，新的脂质共轭可能是静脉注射L-天冬酰胺酶的一种非常有用的策略。
