Kinetically-controlled mechanism-based isolation of metabolic serine hydrolases in active form from complex proteomes: butyrylcholinesterase as a case study

Abstract

In this work an activity-based probe containing a carbamate group was designed to isolate human butyrylcholinesterase (hBChE), a metabolic serine hydrolase (mSH), from complex proteomes. The method took advantage of the native interaction mechanism of mSHs with carbamate pseudo-substrates for temporarily capturing the enzyme on a resin functionalized with the carbamate probe and releasing the enzyme in active form after removal of the contaminating proteins. The isolation relied on the possibility of manipulating the carbamylation and decarbamylation kinetics favoring the former during the capture and wash steps and the latter in the release step. The designed probe captured and released all the active hBChE isoenzymes present in plasma with high selectivity (up to ∼2000-fold purification) and reasonable yields (17% to 36%). The parameters affecting the performance were the incubation time used in the load and elution steps, the plasma to resin volumetric ratio, the elution temperature and the nature and concentration of the eluting agent. The carbamate resin could be prepared either by coupling a fully synthesized probe with an activated resin or by building the probe onto the resin by a step-by-step procedure, without major differences in performance between the two routes. The prepared resins allowed to process up to about 8.5 mL of plasma per g of resin with constant performance. Since the method was based on the general catalytic cycle of mSHs, we expect this approach to be applicable to other enzymes of the family, by selecting a suitable target-selective feature to link to the carbamate group.

Fig. 1. (a) Simplified schematic representation of the interaction between carbamates and metabolic serine hydrolases (mSHs). The formation of the enzyme-pseudo-substrate reversible complex and the following first-order carbamylation step are implicitally included in the carbamylation step. (b) Schematic representation of the method for the isolation of mSHs: in the load and wash steps the decarbamylation is the rate limiting step, therefore hBChE accumulates on the resin as a carbamate adduct. In the elution step, the carbamylation rate is reduced therefore the decarbamylation prevails. The sepharose bead is indicated with the letter “S”. (c) The structure of the probe used in this study. 6a and 6b were obtained following the syntheses depicted in Scheme 1 and S1 (ESI†), respectively.

Scheme 1. Synthesis of sepharose-6Ahx-TTD-TBT 6a (Strategy 1). Boc-6Ahx, Boc-6-aminohexanoic acid; CDI N,N′-carbonyldiimidazole; TTD, 4,7,10-trioxa-1,13-tridecanediamine; TBT, rac-terbutaline hemisulfate; 4NPCF, p-nitrophenyl chloroformate; DMAP, 4-(dimethylamino)pyridine.

Fig. 2. (a) Time- and concentration-dependent inhibition of hBChE after pre-incubation with various concentrations of compound 3. The one-phase exponential fit of the measured residual activity (solid lines) provided the k_obs values. (b) Linear correlation of the k_obs values vs. the concentration of compound 3. The slope of the linear regression corresponds to the bimolecular carbamylation rate constant, k_I. (c) Secondary plot obtained from the analysis the area under the inhibition–time curves (AUIC). The slope of the linear regression corresponds to the decarbamylation rate constant, k₃. (d) Recovery of hBChE activity after inhibition by compound 3 and dilution.

Fig. 3. (a) Left panel, non-denaturing PAGE showing the effect of active site inhibition (lane 2) and acid denaturation (lane 3) (before the load step), and the effect of the removal of the probe from the resin (lane 4) on the retention of hBChE; lane 1, untreated plasma loaded on resin containing the probe (6a); right panel, hBChE activity of the corresponding eluates; FT, flow-through fraction. (b) Non-denaturing PAGE showing the effect of denaturation (after load and wash steps) on the release of hBChE; the arrows indicate the position of hBChE tetramers.

Fig. 4. (a) Non-denaturing PAGE showing the effect of plasma incubation time on protein retention; (b) non-denaturing PAGE showing the effect of different concentration of 4MAC on protein release; (inset) hBChE activity of the isolates versus the concentration of 4MAC employed for the elution (apparent K_D = 36 mM); (c) non-denaturing PAGE showing the effect of 4MAC incubation time on protein release; (inset) comparison of the theoretical (solid line) and experimental (red circles) kinetics of the release of hBChE from the resin. The arrows indicate the position of hBChE tetramers.

Fig. 5. (a) Non-denaturing PAGE showing the lack of effect of additional pre-elution steps performed using 4MAC at 4 °C on the amount of proteins eluted with 4MAC at 37 °C; lane 1 and 1′, elution with 4MAC, pH 7 at 37 °C (no pre-elution); lane 2 and 2′, pre-elution with 4MAC, pH 5 at 4 °C followed by elution with 4MAC, pH 7 at 37 °C; lane 3 and 3′, pre-elution with 4MAC, pH 7 at 4 °C followed by elution with 4MAC, pH 7 at 37 °C. (b) hBChE activity of the eluates obtained in the three conditions; FT, flow-through fraction.

Fig. 6. Effect of repeated use of the resin on the yield and purity of hBChE isolated from plasma. The effect of repeated use on the isolation performance was evaluated by: (samples A1–A3) repeating three times the whole isolation protocol (i.e., each plasma incubation was followed by wash and elution steps); (sample B) repeating three times only the plasma incubation and the wash step followed by a single final elution (×3); (sample C) repeating three times only the plasma incubation followed by a single wash step (×10) and a single elution step (×3). (a) Non-denaturing gel electrophoresis of the eluates obtained with the three protocols; samples B and C were diluted 3-fold to match the amount of plasma loaded in samples A1–A3 (b) hBChE activity of the eluates obtained with the three protocols.

Fig. 7. Effect of plasma to resin volumetric ratio on the yield and purity of hBChE isolated from plasma. Non-denaturing gel electrophoresis of the eluates obtained the different conditions described in Table 5; the samples in lanes 4 and 5 were diluted 2.3-fold and 3.2-fold, respectively, to match the amount of plasma loaded in the other samples.