Site-Specific Bioconjugation of a Murine Dihydrofolate Reductase Enzyme by Copper(I)-Catalyzed Azide-Alkyne Cycloaddition with Retained Activity
Figure 5
THPTA-assisted CuAAC reaction and its effect on the enzymatic activity.
(A) In-gel fluorescence of reaction products from TBTA- and THPTA-assisted dye labeling via CuAAC. The mDHFR-pEthF (30 µM) was reacted at 25°C for 15 min with sulforhodamine-azide (60 µM) in the presence of 1 mM CuSO4, 1 mM TBTA, 2 mM ascorbate in a phosphate buffer (pH 8.0) containing 30% (v/v) DMSO or in the presence of 1 mM CuSO4, 1 mM THPTA, 2 mM ascorbate in a DMSO-free phosphate buffer (pH 8.0). (B) Effect of THPTA incubation on activity of the mDHFR-43pEthF. After incubation of 30 µM protein with (open circle) or without (close circle) 1 mM THPTA in 10 µl of DMSO-free phosphate buffer (pH 8.0) for 15 min at 25°C, the mixture was diluted 36-fold with the assay buffer for activity assay. Error bars represent standard errors (n = 3). (C) Effect of THPTA incubation on activity of the mDHFR-179pEthF (n = 3).