Browsing by Author "Tsay, Yeou-Guang"

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Qualitative analysis of the fluorophosphonate-based chemical probes using the serine hydrolases from mouse liver and poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis
(2012-11-01)
Huang, Yi-Long
;
Chung, Tsai-Wen
;
Chang, Chia-Mao
;
Chen, Chih-Hau
;
Liao, Chen-Chung
The serine hydrolase family consists of more than 200 members and is one of the largest enzyme families in the human genome. Although up to 50 % of this family remains unannotated, there are increasing evidences that activities of certain serine hydrolases are associated with diseases like cancer neoplasia, invasiveness, etc. By now, several activity-based chemical probes have been developed and are applied to profile the global activity of serine hydrolases in diverse proteomes. In this study, two fluorophosphonate (FP)-based chemical probes were synthesized. Further examination of their abilities to label and pull down serine hydrolases was conducted. In addition, the poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis was demonstrated as an appropriate standard serine hydrolase, which can be applied to measure the labeling ability and pull-down efficiency of FP-based probes. Furthermore, mass spectrometry (MS) was used to identify the serine residue that covalently bonded to the active probes. Finally, these FP-based probes were shown capable of establishing the serine hydrolase profiles in diverse mouse tissues; the serine hydrolases pulled down from mouse liver organ were further identified by MS. In summary, our study provides an adequate method to evaluate the reactivity of FP-based probes targeting serine hydrolases.
The formation stability, hydrolytic behavior, mass spectrometry, DFT study, and luminescence properties of trivalent lanthanide complexes of H(2)ODO2A
(2012)
Chang, C. Allen
;
Wang, I-Fan
;
Lee, Hwa-Yu
;
Meng, Ching-Ning
;
Liu, Kuan-Yu
The trivalent lanthanide complex formation constants (log K-f) of the macrocyclic ligand H(2)ODO2A (4,10-dicarboxymethyl-1-oxa-4,7,10-triazacyclododecane) have been determined by pH titration techniques to be in the range 10.84-12.62 which increase with increasing lanthanide atomic number, and are smaller than those of the corresponding H(2)DO2A (1,7-dicarboxylmethyl-1,4,7,10-tetraazacyclododecane) complexes. The equilibrium formation of the dinuclear hydrolysis species, e.g. Ln(2)(ODO2A)(2)(mu-OH)(+) and Ln(2)(ODO2A)(2)(mu-OH)(2), dominates over the mononuclear species, e.g. LnODO2A(OH) and LnODO2A(OH)(2)(-). Mass spectrometry confirmed the presence of [Eu(ODO2A)](+), [Eu(ODO2A)(OH)+H](+), [Eu-2(ODO2A)(2)(OH2)(2)+H](+), [Eu(ODO2A)(OH)(2)](-) and [Eu-2(ODO2A)(2)(OH2)(3)](-) species at pH > 7. Density function theory (DFT) calculated structures of the EuODO2A(H2O)(3)(+) and EuDO2A(H2O)(3)(+) complexes indicate that three inner-sphere coordinated water molecules are arranged in a meridional configuration, i.e. the 3 water molecules are on the same plane perpendicular to that of the basal N3O or N-4 atoms. However, luminescence lifetime studies reveal that the EuODO2A(+) and TbODO2A(+) complexes have 4.1 and 2.9 inner-sphere coordinated water molecules, respectively, indicating that other equilibrium species are also present for the EuODO2A(+) complex. The respective emission spectral intensities and lifetimes at 615 nm (lambda(ex) = 395 nm) and 544 nm (lambda(ex) = 369 nm) of the EuODO2A(+) and TbODO2A(+) complexes increase with increasing pH, consistent with the formation of mu-OH-bridged dinuclear species at higher pH. Additional DFT calculations show that each Y(III) ion is 8-coordinated in the three possible cis-[Y-2(ODO2A)(2)(mu-OH)(H2O)(2)](+), trans-[Y-2(ODO2A)(2)(mu-OH)(H2O)(2)](+) and [Y-2(ODO2A)(2)(mu-OH)(2)] dinuclear complex structures. The first and the second include 6-coordination by the ligand ODO2A(2-), one by the bridged mu-OH ion and one by a water molecule. The third includes 6-coordination by the ligand ODO2A(2-) and two by the bridged mu-OH ions. The two inner-sphere coordinated water molecules in the cis- and trans-[Y-2(ODO2A)(2)(mu-OH)(H2O)(2)](+) dinuclear complexes are in a staggered conformation with torsional angles of 82.21 degrees and 148.54 degrees, respectively.