Soyut Hakan, Beydemir Sukru, Hisar Olcay
Department of Chemistry, Science and Arts Faculty, Atatürk University, 25240, Erzurum, Turkey.
Biol Trace Elem Res. 2008 Summer;123(1-3):179-90. doi: 10.1007/s12011-008-8108-9. Epub 2008 Feb 21.
Carbonic anhydrase (CA) enzyme was purified from rainbow trout brain by Sepharose-4B-L: -tyrosine-sulfanilamide affinity chromatography. The enzyme was obtained with a specific activity of 2,275 EU mg(-1) and a yield of 22.5%. The sample obtained from the affinity column was used for kinetic properties and inhibition studies. Both optimum and stable pH were found as 9.0 in 1 M Tris-SO(4) at 4 degrees C, respectively. To check the purity and subunit molecular weight of enzyme, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was performed, and MW was found as approximately 29.0 kDa. The molecular weight of native enzyme was estimated to be approximately 27.3 kDa by gel filtration chromatography. The purified enzyme had apparent K (m),V (max), and k (cat) as follows: 0.92 mM, 0.207 micromol.min(-1) and 43.6 s(-1) for p-nitrophenylacetate. The inhibitory effects of Co(II), Cu(II), Zn(II), Ag(I), and Cd(II) on CA enzyme activity were determined using the esterase method under in vitro conditions at low concentrations of the corresponding metals. The obtained IC(50) values, which cause 50% inhibition on in vitro enzyme activity, were 0.05, 30, 0.31, 159, and 82.5 mM for cobalt, copper, zinc, silver, and cadmium, respectively. K ( i ) values were also calculated from Linewaever-Burk plots for these substances as 0.014, 27.68, 2.15, 193.86, and 94.18 for cobalt, copper, zinc, silver, and cadmium, respectively; it was determined that cobalt, silver and cadmium inhibited the enzyme competitively, copper inhibited noncompetitively while zinc inhibited the enzyme uncompetitively.
通过琼脂糖-4B-L：-酪氨酸-磺胺亲和色谱法从虹鳟鱼脑中纯化碳酸酐酶（CA）。获得的酶比活性为2,275 EU mg(-1)，产率为22.5%。从亲和柱获得的样品用于动力学性质和抑制研究。在4℃下，1 M Tris-SO(4)中，最佳pH和稳定pH分别为9.0。为检查酶的纯度和亚基分子量，进行了十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶电泳，发现分子量约为29.0 kDa。通过凝胶过滤色谱法估计天然酶的分子量约为27.3 kDa。纯化后的酶对硝基苯乙酸的表观K(m)、V(max)和k(cat)如下：0.92 mM、0.207 μmol·min(-1)和43.6 s(-1)。在体外条件下，使用酯酶法在低浓度相应金属存在下测定Co(II)、Cu(II)、Zn(II)、Ag(I)和Cd(II)对CA酶活性的抑制作用。对体外酶活性产生50%抑制的IC(50)值，钴、铜、锌、银和镉分别为0.05、30、0.31、159和82.5 mM。还从Linewaever-Burk图计算出这些物质的K(i)值，钴、铜、锌、银和镉分别为0.014、27.68、2.15、193.86和94.18；确定钴、银和镉竞争性抑制该酶，铜非竞争性抑制，而锌反竞争性抑制该酶。
