Pb^(2+)对核糖核酸酶活性及其结构的影响

Effect of Pb^(2+) on RNase Activity and Its Structure

通过各种光谱学手段研究了Pb2 +对核糖核酸酶活性的影响及其作用机制.结果表明低浓度的Pb2 +可提高酶活性,高浓度则严重抑制酶活性,这是因为在高浓度下Pb2 +能完全竞争出核糖核酸酶中的Ca2 +,荧光滴定显示核糖核酸酶可结合 3个Pb2 +.利用EXAFS表征出Pb2 +已结合到核糖核酸酶主链氨基酸残基上,与N或O发生了配位,Pb—N(或O)键长分别为 0.2 42nm和 0.312nm,配位数均为 2.圆二色谱测试进一步表明高浓度的Pb2 +结合使核糖核酸酶的二级结构遭到严重破坏,α 螺旋含量、β 折叠及 β 转角大量下降。

Activity of RNase from porcine pancreas was enhanced by treatment with Pb2+ at low concentration (0.17 X 10(-2) similar to 0.6 x 10(-2) mumol/L) but was inhibited by Pb2+ - at high concentration (over 0. 85 x 10(-2) mumol/L). Pb2+ at high concentration could competitively displace Ca2+ from RNase and the fluorescence titration showed that one molecule of RNase had three binding sites for Pb2+ the association constant k(sv) for its low-affinity Pb2+-binding site was 6.03 x 10(6)(mol/L) (-1). The extended X-ray absorption fine structure (EXAFS) spectrum demonstrated that Pb2+ coordinated nitrogen or oxygen atoms of the active site of RNase or other amino acid residue of RNase, Pb-N (or O) bond length was 0. 242 nm and 0. 312 nm, and coordination number was 2, respectively. The secondary structure of RNase was greatly damaged by Pb2+ at high concentration resulting in the decrease of alpha-helix, beta-sheet and beta-turn contents and the increase of random coil contents.