1. Bilirubin glucuronide was synthesized in vitro in a system containing a rat liver microsomal fraction, UDP-glucuronic acid, Mg2+ and bilirubin. The enzymic synthesis was accomplished without the addition of a bilirubin carrier. 2. Azobilirubin and azobilirubin glucuronide were separated by t.l.c. and paper chromatography and the measurement of the conjugate provided a specific assay for bilirubin UDP-glucuronyltransferase (EC 3. This diazo compound was labelled when [U-14C]UDP-glucuronic acid was employed in the transglucuronidation reaction. 4. Identity of the glucuronide nature of the product was further confirmed by hydrolysis with β-glucuronidase prepared from limpets and Helix pomatia. In each instance azobilirubin and glucuronic acid were liberated. 5. There was a close correlation between the bilirubin glucuronyl-transferase activity as measured by two procedures, colorimetric and radioisotopic. The specific activities so measured were 19nmol of bilirubin ‘equivalents’ conjugated/h per mg of protein and 16.9–18.4nmol of UDP-glucuronic acid incorporated/h per mg of protein, respectively. On this basis, it was concluded that the major product formed in vitro was bilirubin monoglucuronide; this represents about 77% of the total products formed. 6. The Km values for bilirubin and UDP-glucuronic acid at pH8.2 are 3.3×10−4m and 1.67×10−3m, respectively. 7. The addition of Mg2+ at a final concentration of 5mm to the reaction mixture increased the rate of conjugation by 5.6-fold in the microsomal preparation that had been subjected to overnight dialysis against 10mm-EDTA (disodium salt). 8. Diethyl-nitrosamine at a final concentration of 1–20mm has no effect on the glucuronidation of bilirubin in vitro.

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