Gençer Nahit, Arslan Oktay
Balikesir University, Science and Art Faculty, Department of Chemistry/Biochemistry Section, 10100 Balikesir, Turkey.
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Jan 15;877(3):134-40. doi: 10.1016/j.jchromb.2008.11.037. Epub 2008 Nov 30.
In this study, a new purification strategy for human PON1 enzyme was developed using two-step procedures, namely ammonium sulfate precipitation and sepharose-4B-L-tyrosine-9-aminophenantrene hydrophobic interaction chromatography. SDS polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent MW of 43kDa. Overall purification rate of our method was found 901-fold for R isoenzyme and 453-fold for Q isoenzyme. The V(max) and K(M) of the purified enzyme were determined for Q isoenzyme 55 EU and 0.599 mM and for R isoenzyme 50 EU and 0.492 mM, respectively. The in vitro effects of some heavy metals (Hg, Cd, Cu, Mn and Ni) were investigated on the purified human serum PON1Q and R isoenzyme, using paraoxon as substrate. Metals were more effective inhibitors on purified human serum PON1(R192) activity than PON1(Q192) activity. The kinetics of interaction of metals with the purified human serum PON1(R192) and PON1(Q192) indicated a different inhibition pattern. Kinetic constants K(M), V(max), and inhibition type were determined.
在本研究中，开发了一种用于人对氧磷酶1（PON1）的新纯化策略，采用两步法，即硫酸铵沉淀和琼脂糖-4B-L-酪氨酸-9-氨基菲疏水相互作用色谱法。该酶的十二烷基硫酸钠聚丙烯酰胺凝胶电泳显示一条表观分子量为43kDa的单带。我们方法对R同工酶的总纯化率为901倍，对Q同工酶为453倍。纯化酶的V(max)和K(M)，对于Q同工酶分别为55 EU和0.599 mM，对于R同工酶分别为50 EU和0.492 mM。以对氧磷为底物，研究了一些重金属（汞、镉、铜、锰和镍）对纯化的人血清PON1Q和R同工酶的体外作用。金属对纯化的人血清PON1(R192)活性的抑制作用比对PON1(Q192)活性更有效。金属与纯化的人血清PON1(R192)和PON1(Q192)相互作用的动力学表明了不同的抑制模式。测定了动力学常数K(M)、V(max)和抑制类型。
