Quantum dot labeling of butyrylcholinesterase maintains substrate and inhibitor interactions and cell adherence features

Abstract

Butyrylcholinesterase (BChE) is the major acetylcholine hydrolyzing enzyme in peripheral mammalian systems. It can either reside in the circulation or adhere to cells and tissues and protect them from anticholinesterases, including insecticides and poisonous nerve gases. In humans, impaired cholinesterase functioning is causally involved in many pathologies, including Alzheimer's and Parkinson's diseases, trait anxiety, and post stroke conditions. Recombinant cholinesterases have been developed for therapeutic use; therefore, it is important to follow their in vivo path, location, and interactions. Traditional labeling methods, such as fluorescent dyes and proteins, generally suffer from sensitivity to environmental conditions, from proximity to different molecules or special enzymes which can alter them, and from relatively fast photobleaching. In contrast, emerging development in synthesis and surface engineering of semiconductor nanocrystals enable their use to detect and follow molecules in biological milieus at high sensitivity and in real time. Therefore, we developed a platform for conjugating highly purified recombinant human BChE dimers (rhBChE) to CdSe/CdZnS quantum dots (QDs). We report the development and characterization of highly fluorescent aqueous soluble QD-rhBChE conjugates, present maintenance of hydrolytic activity, inhibitor sensitivity, and adherence to the membrane of cultured live cells of these conjugates, and outline their advantageous features for diverse biological applications.

Keywords: Anticholinesterases; bioconjugation; butyrylcholinesterase; confocal microscopy; quantum dots; transmission electron microscopy.

Figure 1

Characterization of the CdSe/CdZnS QDs. (A) Absorbance (red) and emission spectra (black, dashed line) of the QDs in toluene. The excitation and emission peaks were at 610 and 626 nm, respectively. (B) TEM image of the QDs with average size of 10 ± 1 nm in diameter.

Figure 2

UV fluorescence of slowly migrating electrophoretically separated QD-rhBChE conjugates. (A) QD-rhBChE conjugates prepared under conditions of rhBChE excess. Note slow migration compared to unconjugated QD controls. (B) QD-rhBChE conjugates prepared under QDs excess. Note smeared fluorescent band including both conjugates and free QDs.

Figure 3

TEM images of QD-rhBChE conjugates. (A) Positive staining with uranyl acetate of QD-rhBChE 1:1 conjugates. (B) QD-rhBChE conjugates in a different field on the grid. The inset is a magnification of one of the conjugates; the dark area is the QD, while the lighter area is rhBChE.

Figure 4

QD-rhBChE conjugates maintain catalytic activity. (A) Extract from the conjugate band but not the extract from a similarly migrating empty band of rhBChE sample or the QDs band showed catalytic activity. Y axis: Arbitrary units of spectrophotometric absorbance. (B) Catalytic activity of conjugates prepared with excess of QDs over rhBChE was 50% of that of free rhBChE subjected to the same process. Control samples with QDs alone did not show any catalytic activity.

Figure 5

Scheme of the QD-rhBChE conjugates. Adsorption of rhBChE dimers to CdSe/CdZnS core/shell QDs with MPA as a ligand. The carboxyl groups on the QDs attract the basic residues (blue) on rhBChE’s surface. Arrows mark the active site gorge where ACh gets hydrolyzed.

Figure 6

Paraoxon inhibits QD-rhBChE catalytic activity. (A) Ellman’s assay measurements showed a decrease of 90% in the catalytic activity of free enzyme after treatment with paraoxon (empty squares) in comparison to untreated preparation (full squares). (B) Ellman’s assay measurements showed a decrease of 80% in the catalytic activity of QD-rhBChE after treatment with paraoxon in comparison to untreated QD-rhBChE (full circles). Data shown is mean ± S.E. of triplicates.

Figure 7

QD-rhBChE conjugates adhere to the membranes of live cells. Shown are confocal microscopy cross section photographs of bEnd.3 microvasculature endothelial cells incubated with QD-rhBChE (red) and with the nucleus marker Hoechst 33342 (blue). Merging of transmission and luminescence images (upper row) and luminescence images of the QD-rhBChE conjugate (lower row) demonstrate rings of QD-rhBChE surrounding those cell membranes which remained exposed to the culture environment and converged as going up in the Z-axis. These results demonstrate that the QD-rhBChE conjugates interact with accessible cell membrane sites.