Enzyme immobilization in porous silicon: quantitative analysis of the kinetic parameters for glutathione-S-transferases.
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 (approximately 6.5 microm thick) with the enzyme in solution. Kinetic measurements were made by varying the glutathione concentration while maintaining a fixed saturating concentration of 1-chloro-2,4-dinitrobenzene. The reaction kinetics follow steady-state equilibrium behavior. The specific activity of the free enzyme in solution is approximately 4x higher than the immobilized enzyme, for which we measured an apparent K'(m)(GSH) value of 1.0 +/- 0.3. The maximum velocity, V'(max), is linearly proportional to immobilized enzyme concentration, but the magnitude is approximately 20 times lower than that in solution. Results suggest approximately 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.
Delouise
Lisa A
LA
Department of Dermatology and the Center for Future Health, University of Rochester Medical Center, Rochester, New York 14642, USA.
Miller
Benjamin L
BL
D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005982:Glutathione Transferase / Q000737:chemistry*; D007700:Kinetics; D012825:Silicon; D012996:Solutions
D004800:Enzymes, Immobilized; D012996:Solutions; D005982:Glutathione Transferase; D012825:Silicon