Tayefi-Nasrabadi H, Keyhani E, Keyhani J
Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, 13145 Tehran, Iran.
Biochimie. 2006 Sep;88(9):1183-97. doi: 10.1016/j.biochi.2006.04.001. Epub 2006 May 2.
Conformational changes induced by the binding of nickel to horseradish peroxidase C (HRPC) were studied by electronic absorption spectroscopy, fluorescence spectroscopy and circular dichroism spectroscopy. Incubation of HRPC with various concentrations of Ni(2+) for 5 minutes resulted in changes in the enzyme absorption spectrum, including variations in the intensities of the Soret, beta and charge transfer (CT1) bands absorption, shift in the Soret, beta and CT1 bands maxima and absorption increase at 275 nm. Increases in the enzyme's intrinsic fluorescence as determined by fluorescence spectroscopy, as well as changes in the alpha-helical content, as determined by circular dichroism spectroscopy, were also found. Correlatively, alterations of the enzymatic activity by Ni(2+) were studied by following the H(2)O(2)-mediated oxidation of o-dianisidine and 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) by HRPC. With both reducing substrates, it was found that in the presence of sufficient amount of enzyme, 1-10 mM nickel would enhance the enzymatic activity, while higher Ni(2+) concentrations (20-50 mM) would inhibit it. The enzyme was completely inhibited after 5 minutes incubation in 50 mM Ni(2+). Prolonged incubation would induce complete inhibition at lower Ni(2+) concentrations. Spectrophotometry investigations also showed that inhibitory concentrations of Ni(2+) altered compounds I and II formation, compound II being the first affected. Based on spectrophotometry, fluorescence and circular dichroism spectroscopy, and data on compounds I and II formation, a scheme is suggested for HRPC conformational changes in different Ni(2+) concentrations. HRPC was found to have four potential attachment sites for Ni(2+) which were sequentially occupied in a dose- and time-dependent manner by the metallic ion.
通过电子吸收光谱、荧光光谱和圆二色光谱研究了镍与辣根过氧化物酶C（HRPC）结合诱导的构象变化。将HRPC与不同浓度的Ni(2+)孵育5分钟导致酶吸收光谱发生变化，包括Soret、β和电荷转移（CT1）带吸收强度的变化、Soret、β和CT1带最大值的位移以及275nm处吸收增加。还发现了通过荧光光谱测定的酶内在荧光增加，以及通过圆二色光谱测定的α-螺旋含量变化。相应地，通过跟踪HRPC对邻联茴香胺和2,2'-偶氮双（3-乙基苯并噻唑啉磺酸）（ABTS）的H(2)O(2)介导的氧化反应，研究了Ni(2+)对酶活性的改变。对于这两种还原底物，发现在存在足够量酶的情况下，1-10 mM镍会增强酶活性，但更高的Ni(2+)浓度（20-50 mM）会抑制酶活性。在50 mM Ni(2+)中孵育5分钟后酶被完全抑制。长时间孵育会在较低的Ni(2+)浓度下诱导完全抑制。分光光度法研究还表明，抑制浓度的Ni(2+)会改变化合物I和II的形成，化合物II是首先受到影响的。基于分光光度法、荧光和圆二色光谱以及化合物I和II形成的数据，提出了不同Ni(2+)浓度下HRPC构象变化的方案。发现HRPC有四个Ni(2+)潜在结合位点，金属离子以剂量和时间依赖性方式依次占据这些位点。
