Kinetics of HaloTag Protein Labeling

Labeling kinetics of HaloTag7 and HaloTag9 with TMR-CA were measured by recording fluorescence anisotropy changes over time using a BioLogic SFM-400 stopped-flow instrument (BioLogic Science Instruments) in single mixing configuration at 37 °C. Monochromator wavelengths for excitation was set to 555 nm and a 570-nm long pass filter was used for detection. Protein and substrates were mixed in a 1:1 stoichiometry in activity buffer supplemented with 0.5 mg ml⁻¹ BSA. Concentrations were varied from 0.125 µM to 0.5 µM. The anisotropy of the free substrate was measured to obtain a baseline. The dead time of the instrument was measured according to the manufacturer’s protocol (BioLogic Technical note no. 53) by recording the fluorescence decay during the pseudo first-order reaction of N-acetyl-l-tryptophanamide with a large excess of N-bromosuccinimide and fitting the data to the first-order reaction rate law. Recorded data were processed removing pretrigger time points and averaging replicates. The data were fit to a two-stage kinetic model (equations (4) and (5)) using the DynaFit software⁴⁵ . Baseline anisotropy of the free fluorophore, substrate concentrations and dead time of the instrument were taken into account. The s.d. (normal distribution verified) and confidence intervals of fitted parameters were estimated with the Monte Carlo method^{46 (link)} with standard settings (N = 1,000, 5% worst fits discarded). The derived parameters K_D (dissociation constant) and k_app (apparent second-order rate constant) were calculated according to equations (6) and (7). $P+S\begin{array}{c}\hfill \stackrel{k_1}{\leftrightarrow }\hfill \\ \hfill k_{-1}\hfill \end{array}P{S}^{*}$ $P{S}^{*}\stackrel{k_2}{\to }PS$ $K_{\mathrm{D}}=\frac{k_{-1}}{k_1}$ $k_{\mathrm{app}}=k_1\frac{k_2}{k_2+k_{-1}}$