Browsing by Author "Chiang, Han-Ping"

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Dimerization Is Responsible for the Structural Stability of Human Sulfotransferase 1A1
(2009-05-01)
Lu, Lu-Yi
;
Chiang, Han-Ping
;
Chen, Wei-Ti
;
Yang, Yuh-Shyong
;
生物科技學系
Cytosolic sulfotransferases (SULTs) are responsible for the metabolism of a variety of drugs, xenobiotics, and endogenous compounds. Most cytosolic SULTs are found to be homodimers. However, transformation between monomeric and dimeric SULTs can be achieved by a single amino acid mutation. The importance of quaternary structure for cytosolic sulfotransferase was investigated using recombinant human SULT1A1, a homodimer, and its monomeric mutant (V270E). The differences between dimeric and monomeric SULT1A1 were examined by size-exclusion liquid chromatography, enzyme kinetics, substrate binding affinity, thermal inactivation, conformational stability, and circular dichroism. Variations, especially on their secondary structures and stability, between homodimer and monomer of human SULT1A1 were observed. It was found that the active site of SULT1A1 was not significantly perturbed after the change of its quaternary structure according to SULT1A1 kinetics and substrate binding affinity. However, the stability of monomeric SULT1A1 is significantly decreased. We proposed that the importance of human SULT1A1 as a homodimer was to maintain its structural stability, and the change of secondary structure was responsible for alternating its quaternary structure.
Surface Effect of Assembling Enzyme and Modulation of Surface Enzyme Activity with Electric Potential Stress
(2012-06-01)
Chao, Cheng-Han
;
Li, Kun-Lin
;
Wu, Chung-Shu
;
Lee, Cheng-Che
;
Chiang, Han-Ping
The fluorescent marker of rhodamine B amine is successfully used to evaluate the immobilization capability onto silicon-based patterns fabricated by semiconductor manufacturing. Only the silicon dioxide surface, by means of fluorescent observation, can immobilize the rhodamine molecule by the sequential linkage of (3-aminopropyl) triethoxysilane (APTES) and glutaraldehyde. The phenol sulfotransferase enzyme is also successfully immobilized onto the silicon dioxide surface by the linking molecules of APTES and sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohezane-1-carboxylate in the home-made apparatus. The enzyme activity of the sulfotransferase is determined from the absorbance of 4-nitrophenol at 400 nm wavelength. The surface immobilized enzyme remains its activity for catalytic reaction at least 120-min duration. The surface saturation effect on the activity of immobilized enzyme is explained and ascribed to the surface diffusion effect of electric double layers. We can success control the surface immobilized enzyme by electric potential stress. The activity of enzyme is reduced under negative potential, while is enhanced under positive potential. The electric potential can induce the enzyme structure variation and modulate the enzyme activity due to the electrostatic effect.
Surface Reaction Limited Model for the Evaluation of Immobilized Enzyme on Planar Surfaces
(2009-04-01)
Lee, Cheng-Che
;
Chiang, Han-Ping
;
Li, Kun-Lin
;
Ko, Fu-Hsiang
;
Su, Chien-Ying
Analysis of immobilized enzyme in situ is a crucial step to embed an enzyme onto the planar technology of standard integrated circuit (IC) and microelectromechanical systems (MEMS) for a bioreactor or enzyme-coupled biosensor. A surface reaction limited model, based on a systematized and standardized approach, mathematically derived from mass transfer dynamics and the Michaelis-Menten equation for the measuring the apparent K(m)(star) (Michaelis-Menten constant) and V(max)(star) (maximum reaction rate per unit surface area of catalyst) of an immobilized enzyme on a planar surface was developed. The derived equations for the kinetic model were simulated and experimentally confirmed. A platform of a microflow bioreactor with a one-sided planar catalytic surface that contained immobilized enzyme was constructed. The microfluidic bioreactor was designed to possess a channel height less than that of the diffusion layer thickness in a semi-infinite diffusion process, and K(m)(star) and V(max)(star) of rat phenol sulfotransferase (PST) immobilized on the silicon oxide surface were successfully determined in situ. Variation in kinetic constants and the possible differences in performance between free and immobilized PST are discussed.