Enzyme immobilization in porous silicon: Quantitative analysis of the kinetic parameters for glutathione-S-transferases

被引:82
作者
DeLouise, LA
Miller, BL
机构
[1] Univ Rochester, Med Ctr, Dept Dermatol, Rochester, NY 14642 USA
[2] Univ Rochester, Med Ctr, Ctr Future Hlth, Rochester, NY 14642 USA
关键词
D O I
10.1021/ac0486185
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
Porous silicon matrixes are attractive materials for the construction of biosensors and may also have utility for the production of immobilized enzyme bioreactors. In an effort to gain a quantitative understanding of the effects of immobilization on enzyme activity, we compared the activity of glutathione-S-transferase immobilized in electrochemically etched porous silicon films (similar to 6.5 mu m thick) with the enzyme in solution. Kinetic measurements were made by varying the glutathione concentration while maintaining a fixed saturating concentration of 1-cbloro2,4-dinitrobenzene. The reaction kinetics follow steady-state equilibrium behavior. The specific activity of the free enzyme in solution is similar to 4x higher than the immobilized enzyme, for which we measured an apparent K '(GSH)(m) value of 1.0 +/- 0.3. The maximum velocity, V '(max), is linearly proportional to immobilized enzyme concentration, but the magnitude is similar to 20 times lower than that in solution. Results suggest similar to 25% of the enzyme is bound with the catalytic site in an inactive conformation or in a hindered orientation. Finally, the effects of hydration and exposure to denaturants on the immobilized enzyme activity are presented.
引用
收藏
页码:1950 / 1956
页数:7
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