58 (in a 5:1 ratio) was added, vortexed, and left for 30 minutes on ice. Subsequently, the

buffer and water were added in that order and vortex-mixed. Immediately preceding a 20-

minute incubation at 37oC, UDPGA was added to initiate the reaction. The reaction was

terminated by the addition of 2 mL ice-cold methanol. Precipitated protein was pelleted

by centrifugation at 2000 rpm for 10 minutes. The supernatant, 2 mL, was then mixed

with 0.5 mL NaOH (lN) and the fluorescence of B[a]P-3-glucuronic acid measured at

excitation/emission wavelengths of 300/421 nm (Singh & Wiebel, 1979). The activity of

UGT (nmol/min/mg) was then determined.

 Preliminary studies established the conditions for linearity of reaction with respect

to time, protein and detergent concentrations, at the same time ensuring that substrate

consumption did not exceed 10%. The apparent Km for UDPGA was determined by

performing experiments at a fixed concentration of 3-OH-B[a]P (10 C1M). Saturating

UDPGA concentrations were used in order to determine 3-OH-B[a]P glucuronidation

kinetics.

Kinetic Analysis. Duplicate values for the rate of conjugate formation at each substrate

concentration were used to calculate kinetic parameters using Prism v 4.0 (GraphPad

Software, Inc., San Diego, CA). Equations used to fit the data were the Michaelis-Menten

hyperbola for one-site binding (eq. 1), the Hill plot (eq. 2), substrate inhibition for one-

site binding (eq. 3) (Houston and Kenworthy 2000), and partial substrate inhibition due to

binding at an allosteric site (eq. 4) (Zhang et al., 1998).

 v = Vmax[S] / (Km + [S]) (1)

 v = Vmax[S]h / (S50h + [S]h) (2)

 v = Vmax[S] / (Km + [S] + ([S]2/K,)) (3)