Oztürk Lokman, Bülbül Metin, Elmastas Mahfuz, Ciftçi Mehmet
Faculty of Science and Arts, Department of Biology, Gaziosmanpasa University, Tokat, Turkey.
Prep Biochem Biotechnol. 2007;37(3):229-38. doi: 10.1080/10826060701386711.
In this study, catalase (CAT: EC 1.11.1.6) was purified from parsley (Petroselinum hortense) leaves; analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps, including preparation of homogenate, ammonium sulfate fractionation, and fractionation by DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 9.5% and had a specific activity of 1126 U (mg proteins)(-1). The overall purification was about 5.83-fold. A temperature of 4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured at 240 nm. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acryl amide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for the enzyme. The molecular weight was found to be 183.29 kDa by Sephadex G-200 gel filtration chromatography. The stable pH, optimum pH, and ionic strength were determined for phosphate and Tris-HCl buffer systems. In addition, K(M) and V(max) values for H(2)O(2), at optimum pH and 25 degrees C, were determined by means of Lineweaver-Burk plots.
在本研究中，过氧化氢酶（CAT：EC 1.11.1.6）从欧芹（Petroselinum hortense）叶片中纯化得到；对该酶的动力学行为和一些特性进行了分析研究。纯化过程包括三个步骤，即匀浆制备、硫酸铵分级分离以及通过DEAE - Sephadex A50离子交换色谱进行分级分离。获得的酶产率为9.5%，比活性为1126 U（mg蛋白质）⁻¹。总体纯化倍数约为5.83倍。纯化过程中保持4℃的温度。酶活性通过在240 nm处进行分光光度法测定。为了监控酶的纯化情况，分别在4%和10%丙烯酰胺的凝胶中进行SDS - 聚丙烯酰胺凝胶电泳，用于积层胶和分离胶。SDS - 聚丙烯酰胺凝胶电泳显示该酶为单一一条带。通过Sephadex G - 200凝胶过滤色谱法测得分子量为183.29 kDa。针对磷酸盐和Tris - HCl缓冲系统测定了稳定pH值、最适pH值和离子强度。此外，在最适pH值和25℃条件下，通过Lineweaver - Burk作图法测定了H₂O₂的K(M)和V(max)值。
