- Unfortunately, detergents may differentially affect the individual enzymes
and there is often a sharp concentration optimum for each preparation and
substrate. More recently, the pore-forming antibiotic alamethacin has been
widely used, which has a broader optimum concentration (typically 25–100mg/
mg protein) (Fulceri, 1994). Unfortunately, alamethacin is not very soluble and
may require small amounts of methanol or ethanol for solubilization. Organic
solvent concentrations should be kept at less than 0.5% in the final incubation.
Alamethacin is incubated with the microsomes and buffer on ice for 30–35 min
prior to addition of substrate and UDPGA. Addition of UDP-N-acetylgluco-
samine (UDP-GlucNAC) and ATP also stimulates activity (Fulceri, 1994), and
it has been suggested that this is due to transport of UDP-GlucNAC out of the
lumen in exchange for transport of UDPGA into the ER lumen by the
transporter (trans-stimulation) (Bossuyt, 1996). Other additives to incubations
may include a beta-glucuronidase inhibitor such as saccharo-1,4-lactone and
an esterase inhibitor. Esterase inhibitors may be important for substrates
containing carboxylic acids (acyl glucuronide formation). Addition of these
inhibitors can increase product formation especially if long incubation periods
are needed to increase assay sensitivity.
The choice of pH and buffers are also important considerations. Tris-
containing buffers are commonly used for glucuronidation reactions.
However, Tris has a relatively high pKa8.06 and a relatively narrow pH range
for adequate buffering capacity. Tris-maleate buffers have a broader pH
buffering range as maleate has a pKof 6.2, providing good buffering capacity
from 5.7 to 8.5. Tris buffers are temperature sensitive, and thus a pH 7.7 buffer
prepared at 25C has a pH of 7.4 at 37C. Phosphate (pKa7.2) is a good buffer,
but binds to Mg2+ions that are present in the incubation and interact with the
phosphate backbone of UDPGA in the active site. Final buffer pH values vary
widely in the literature. Owens and coworkers have consistently used a pH of 6.5
for bilirubin glucuronidation generating higher turnover rates whereas higher
activities may be demonstrated for weakly basic or phenolic substrates at pH
values of 8.0 or greater. The transferase reaction requires nucleophilic attack of
the high energy phosphate bond, so one would expect that amine substrates
(typical pKas of 8–10) would be better nucleophiles at higher pH.
Prior to conducting enzyme kinetic studies, it is important to determine
linearity of glucuronide production with respect to time and protein
concentration. This should be done at saturating substrate concentrations
(5 Km) or under conditions where less than 10% of the substrate is lost. The
Kmvalues for many substrates are in the low millimolar range, thus solubility
and solvent concentrations are important considerations. In general, a solvent
concentration of<1% is desirable, preferably0.1%. Unfortunately, many
drug substrates are hydrophobic and poorly water soluble. Uchaipichat et al.
recently studied the effects of different solvents on 4-methylumbelliferone and
1-naphthol glucuronidation by individual UGT enzymes. UGT2B15 (inhibi-
tion by0.5% acetonitrile) and UGT2B17 (inhibition by0.5% DMSO and
ethanol) appeared to be most affected by organic solvents.
UDP-GLUCURONOSYLTRANSFERASES 57