The covalent immobilization of microsomal uridine diphospho-glucuronosyltransferase (UDPGT): initial synthesis and characterization of an UDPGT immobilized enzyme reactor for the on-line study of glucuronidation.
The microsomal fraction of rat liver containing uridine diphospho-glucuronosyltransferase (UDPGT; EC 2.4.1.17) has been covalently immobilized on a high performance chromatographic support. In this study Nucleosil Si-500 silica was converted into diol-bonded silica and subsequently converted into an aldehyde form through oxidation with sodium periodate. The microsomal fraction was immobilized via Schiff base formation followed by reduction with sodium cyanoborohydride. The resulting immobilized enzyme reactor (IMER) was placed in a multi-dimensional chromatographic system which utilized a mixed mode (C18 and anion exchange) column to trap the parent compound and glucuronide and a C18 column to separate the substrate and product. The IMER system was used for the online glucuronidation of 4-methylumbelliferone (4Me7OHC) and acetaminophen (APAP). The Michaelis-Menten kinetic parameters (Km and Vmax) associated with the formation of 4Me7OHC and APAP glucuronides demonstrated that the immobilization had not significantly affected the enzymatic activity of the UDPGT relative to the non-immobilized enzyme. The IMER retained enzymatic activity for more than 6 weeks. The results of this study demonstrate an easy and convenient way to identify compounds which may be glucuronidated and to synthesize and characterize the resulting products.
Kim
Hee Seung
HS
Bioanalytical and Drug Discovery Unit, National institute on Aging, National Institutes of Health, Gerontology Research Center, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825, USA.
Wainer
Irving W
IW
D000818:Animals; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D004800:Enzymes, Immobilized / Q000032:analysis* / Q000378:metabolism; D020719:Glucuronides / Q000302:isolation & purification / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000032:analysis* / Q000378:metabolism; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D015203:Reproducibility of Results; D012822:Silicon Dioxide; D000099148:UGT1A1 Enzyme
D004800:Enzymes, Immobilized; D020719:Glucuronides; D014453:Glucuronosyltransferase; D012822:Silicon Dioxide; D000099148:UGT1A1 Enzyme; C499946:Ugt1a1 protein, rat