Human liver dihydrodiol dehydrogenase 1-catalyzed reaction generating 9alpha,11beta-prostaglandin F2 from prostaglandin D2 followed by micellar electrokinetic chromatography.
An enzyme reaction converting prostaglandin D2 (PGD2) into 9alpha,11beta-prostaglandin F2 (9alpha,11beta-PGF2) by a human liver-originated recombinant dihydrodiol dehydrogenase 1 (DD1) has been studied using CE. Four prostaglandins, viz. PGD2, 9alpha,11beta-PGF2, PGE2, and PGF2a (see Fig. 1, the latter two major PGs are possibly coexisting compounds in the assay mixtures), were completely separated by using SDS or PEG as buffer additives. Because analysis times in the SDS system were shorter than in the PEG system, SDS was employed in measurements of the enzyme activity of DD1. The pH dependence and the reaction temperature dependence of enzyme activity have been studied. The present method enabled us to detect all of the participants in the enzyme reaction: PGD2, 9alpha,11beta-PGF2, nicotinamide adenine dinucleotide phosphate (NADPH), and NADP+. Thus, direct, comprehensive, and reliable analysis of the enzyme reaction becomes possible. Enzyme activity has hitherto been estimated indirectly from the decrease of fluorescence derived from NADPH as an index of progress of the enzyme reactions in batch methods employed in conventional studies. In addition, the low sample consumption characteristic of CE should be a significant advantage of the present method in characterization of less commonly available enzymes such as the recombinant species used in this work.
Inagaki
Shinsuke
S
Laboratory of Pharmaceutical Analytical Chemistry, Gifu Pharmaceutical University, Gifu, Japan. Fax: +81-58-237-5979.
Esaka
Yukihiro
Y
Deyashiki
Yoshihiro
Y
Uno
Bunji
B
Hara
Akira
A
Toyo'oka
Toshimasa
T
D002021:Buffers; D002384:Catalysis; D020374:Chromatography, Micellar Electrokinetic Capillary; D015237:Dinoprost / Q000031:analogs & derivatives* / Q000096:biosynthesis*; D006801:Humans; D008099:Liver / Q000201:enzymology*; D010088:Oxidoreductases / Q000378:metabolism*; D011092:Polyethylene Glycols; D015230:Prostaglandin D2 / Q000378:metabolism*
D002021:Buffers; D011092:Polyethylene Glycols; D015237:Dinoprost; D010088:Oxidoreductases; C048338:trans-1,2-dihydrobenzene-1,2-diol dehydrogenase; D015230:Prostaglandin D2