Basi Zehra, Turkoglu Vedat
Department of Chemistry, YüzüncüYıl University, Faculty of Science, Van, Turkey.
Biomed Chromatogr. 2018 May;32(5):e4175. doi: 10.1002/bmc.4175. Epub 2018 Jan 29.
In the present study, one-step purification of angiotensin-converting enzyme (ACE, peptidyldipeptidase A, EC 3.4.15.1), responsible for regulation of blood pressure, was achieved using affinity chromatography from human plasma. The enzyme was purified 12,860-fold with a specific activtiy of 5080 EU/mg protein. Optimum temperature and pH were determined for the enzyme as 35-40°C and pH 7.4-7.5, respectively. The purity of ACE was determined by SDS-PAGE and the enzyme showed two bands at 60 and 70 kDa on the gel. The native molecular weight of ACE was found to be 260 kDa by gel filtration chromatography, demonstrating that the enzyme has a heterodimeric structure. Natural fatty acids of Nigella sativa (Ranunculaceae) were isolated by means of column chromatography. The structures of these compounds were determined using NMR and GC-MS. The results showed that high concentrations of linoleic, oleic and palmitic acids were isolated from the plant. The effect of six fractions (Fr 1-6) on ACE activity was examined. Fraction 3 increased the ACE activity while the other fractions decreased the enzyme activity. The concentrations of the fractions inhibiting the half-maximum activity of the enzyme were calculated as 1.597 mg/mL for Fr 1, 0.053 mg/mL for Fr 2, 0.527 mg/mL for Fr 4, 0.044 mg/mL for Fr 5 and 0.136 mg/mL for Fr 6 using a Lineweaver-Burk graph.
在本研究中，利用亲和色谱法从人血浆中实现了对负责血压调节的血管紧张素转换酶（ACE，肽基二肽酶A，EC 3.4.15.1）的一步纯化。该酶纯化了12,860倍，比活性为5080 EU/mg蛋白质。确定该酶的最适温度和pH分别为35 - 40°C和pH 7.4 - 7.5。通过SDS - PAGE测定ACE的纯度，该酶在凝胶上显示出60和70 kDa的两条带。通过凝胶过滤色谱法发现ACE的天然分子量为260 kDa，表明该酶具有异二聚体结构。通过柱色谱法分离了黑种草（毛茛科）的天然脂肪酸。使用NMR和GC - MS确定了这些化合物的结构。结果表明，从该植物中分离出了高浓度的亚油酸、油酸和棕榈酸。检测了六个馏分（Fr 1 - 6）对ACE活性的影响。馏分3增加了ACE活性，而其他馏分降低了酶活性。使用Lineweaver - Burk图计算抑制该酶半数最大活性的馏分浓度，Fr 1为1.597 mg/mL，Fr 2为0.053 mg/mL，Fr 4为0.527 mg/mL，Fr 5为0.044 mg/mL，Fr 6为0.136 mg/mL。
