Wann-Yin Lin

Separation and migration behavior of structurally related phenothiazines in cyclodextrin-modified capillary zone electrophoresis

The influences of buffer pH and the concentration of beta-cyclodextrins (beta-CDs) on the separation and migration behavior of 13 structurally related phenothiazines in CD-modified capillary zone electrophoresis (CD-CZE) using a phosphate background electrolyte at low pH were investigated. We focused on the separation of these phenothiazines, including the enantiomers of chiral analytes, with the use of beta-CD and hydroxypropyl-beta-CD (HP-beta-CD) as electrolyte modifiers or chiral selectors at concentrations less than 8 mM. The results indicate that the interactions of phenothiazines with beta-CDs are very strong and that effective separations of 13 analytes can be achieved with addition of 0.3 mM beta-CD or 0.5 mM HP-beta-CD in a phosphate buffer at pH 3.0. Binding constants of phenothiazines to beta-CDs were evaluated for a better understanding of the interactions of phenothiazines with beta-CDs.

Stopped-flow study of the enhanced chemiluminescence for the oxidation of luminol with hydrogen peroxide catalyzed by microperoxidase 8.

The presence of carbonate or Tris causes a dramatic enhancement in the chemiluminescence (CL) for the oxidation of luminol with hydrogen peroxide catalyzed by microperoxidase 8 (MP8). A nearly constant enhancement in CL was observed over a wide range of H(2)O(2) and luminol concentrations. The enhancement in CL is strongly pH-dependent, varying from 1.3 to 22.2 for carbonate and 1.6 to 10.2 for Tris. The CL enhancement is much more prominent at pH 9-10 than at high pH (>10.5) because of the extremely weak CL emission at pH below 10 when no enhancer is present. The CL enhancement is attributed to an accelerated CL cycle and the existence of alternative routes for luminol CL, possibly involving the carbonate, or Tris radicals. The dramatic enhancement in CL of the MP8-luminol-H(2)O(2) system by the readily available reagents, sodium carbonate or Tris, will have general applications for sensitive CL assays. As an example, the presence of antioxidant results in a diminished and delayed CL emission, allowing the determination of its concentration at sub-micromolar level.

Effects of metal ions on the catalytic and thermodynamic properties of the aminopeptidase isolated from pronase

Abstract The effect of metal ions on the catalytic and thermodynamic properties of the aminopeptidase isolated from pronase has been investigated. A decrease in K M and enhanced activity were observed for most of the metal ions examined. Ca(II) exhibits the most prominent effect on enzyme activity. The observed stability constants for the enzyme-bound metal ions are in the range of 10 2 –10 6 M −1 , which is much smaller than that of a metalloenzyme, indicating that the metal ion is not an integral part of the enzyme. The complexation of l -leucine -p- nitroanilide and the transition metal ions causes a reduction in free substrate concentration and hence a concomitant decrease in enzyme activity. Therefore, care must be taken to account for this decrease in substrate concentration in order to obtain reliable kinetic parameters. Binding of E and S to form ES was accompanied by a decrease in Gibbs free energy, whereas a dramatic increase in the free energy was observed for the conversion of ES to ES ‡ . Both the enthalpy and the entropy were found to be crucial in destabilizing ES ‡ . In the presence of Ca(II), ES is stabilized by ∼ 1 kcal/mol and ES ‡ by ∼ 1.4 kcal/mol. The stabilization of ES by the presence of Ca(II) is reflected by a smaller K M value compared to that of the metal-free enzyme. The activation free energies for the process E + S → ES ‡ were 10.8 and 9.4 kcal/mol for the metal-free and the Ca(II)-activated enzymes, respectively. The difference of ∼ 1.4 kcal/mol in the activation free energy may account for enhanced activity by the presence of Ca(II).

Intense chemiluminescence from the oxidation of luminol with m-chloroperoxybenzoic acid catalyzed by Mn(III)-microperoxidase

Abstract We have studied the chemiluminescence (CL) of luminol oxidation with m-chloroperoxybenzoic acid (mCPBA) or hydrogen peroxide (H2O2) catalyzed by Mn(III)-microperoxidase 8 (Mn(III)-MP8) using the stopped-flow technique. The CL involving Mn(III)-MP8 is two to three times as intense as that of the corresponding Fe(III)-MP8, while using mCPBA in place of H2O2 as the oxidant enhances the CL by 12–18-fold at the optimum pH (∼12). The rapid formation of the intermediate, OMn(IV)-MP8, upon addition of mCPBA to Mn(III)-MP8 will lead to a fast CL cycle. This in turn, will produce an intense CL of very short duration. The ratio of the peak CL intensity (mCPBA:H2O2) for the Mn(III)-MP8 system remains essentially constant (∼20) regardless of the pH and the concentrations of oxidant and luminol used, provided that the supply of the oxidant and luminol is sufficient. The novel use of Mn(III)-MP8/mCPBA for the CL of luminol oxidation will have a great analytical potential for very sensitive assays.