Proteolytic Activity at Quantum Dot-Conjugates: Kinetic Analysis Reveals Enhanced Enzyme Activity and Localized Interfacial “Hopping”

Abstract

Trypsin from bovine pancreas (MW 23.8 kDa, 12 705 BAEE units/mg protein, 12 705 BAEE units/mg solid, foreign chymotrypsin activity 0.67 BTEE units/mg protein; EC number 3.4.21.4; derived from New Zealand sourced pancreas, ethanol precipitate) was purchased from Sigma-Aldrich (St. Louis, MO). Note: BAEE is N α -benzoyl-L-arginine ethyl ester; BTEE is N α -tyrosine-L-arginine ethyl ester. InstrumentsPL measurements were made using either a Tecan (Durham, NC) Sapphire or Tecan Infinite M1000 fluorescence multifunction plate reader. Peptide LabelingA mass of ca. 1 mg of peptide was dissolved in 1 mL of phosphate buffered saline (PBS; 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4), and combined with excess Cy3maleimide monoreactive dye (GE Healthcare, Piscataway, NJ) or A594-maleimide (Invitrogen by Life Technologies, Carlsbad, CA) at room temperature for 1-2 h and then left overnight at 4°C. Excess unreacted dye was initially removed by loading the reaction onto three consecutive 0.5 mL columns of Ni 2+ -nitrilotriacetic acid (Ni-NTA) agarose media (Qiagen Valencia, CA). Columns with bound peptide were washed with PBS, and the labeled peptide eluted with 300 mM imidazole in PBS. Cy3-labeled peptide was desalted and the imidazole removed using a reverse-phase oligonucleotide purification cartridge (OPC; Applied Biosystems, Foster City, CA) and washes with 0.1 M triethylamine acetate buffer. Labeled peptide was eluted using 1 mL of 70% acetonitrile in doubly-distilled deionized H 2 O. Purified Cy3-peptide was quantitated by UV-visible spectroscopy using the Cy3 (ε = 150 000 M −1 cm −1 at 550 nm) and A594 (ε = 73 000 M −1 cm −1 at 590 nm) absorbance peaks, then aliquoted, dried, and stored at −20 °C prior to use. This protocol has been described in detail elsewhere. 1 Preparation of 2-naphthylamine calibration samples2-Naphthylamine samples were prepared from the hydrolysis of the corresponding concentration of BANA using 1.4 µM trypsin at 37 ˚C for 4 h, and then left at 22 ˚C for 1.5 h. Preparation of pre-digested peptide (product fragment) samplesEqual volumes of Cy3/A594-labeled peptide substrate (50 µM) and trypsin (8.6 µM) were mixed and left overnight at room temperature. These solutions were used for calibrations with only digested product fragments. For the mixed calibration with both A594-labeled substrate and digested product fragment (vide infra), residual trypsin activity in the solution (800 µL, 25 µM)

Increasing the activity of immobilized enzymes with nanoparticle conjugation

“…When compared to enzyme in free solution, both QDs showed significantly enhanced enzymatic activity rates; enzymatic efficiency improved 30-40% overall [58]. Although the focus here has specifically been on enzyme activity when immobilized to NPs, it is also important to note that when substrate is attached to NPs such as QDs, marked improvements in enzymatic performance have also been reported [31].…”

“…Additionally, the mobility of the NPs themselves enhances substrate-to-enzyme interactions via Brownian motion [30] while secondary interactions at the NP-enzyme interface, due in part to substrate-NP attraction through forces such as electrostatic attraction, can also increase the activity of NP immobilized enzymes [31]. In other words, enhanced activity can be attributed to the fact that with each collision between NP-immobilized enzymes and free-floating substrate, the weak association between the substrate and the NP interface results in multiple binding occurrences on one NP before the substrate moves elsewhere [31].…”

“…In other words, enhanced activity can be attributed to the fact that with each collision between NP-immobilized enzymes and free-floating substrate, the weak association between the substrate and the NP interface results in multiple binding occurrences on one NP before the substrate moves elsewhere [31]. This proposed substrate-NP association or attraction would subsequently lead to a higher concentration of substrate near the periphery of the NP as opposed to the bulk environment which would further enhance the activity of enzymes immobilized on NPs than floating free in solution [32].…”

Materials for FRET Analysis: Beyond Traditional Dye–Dye Combinations

“…Algar and coworkers in particular have developed a series of time-gated FRETrelays, demonstrating the use of QDs as simultaneous acceptors and donors in bioassays for monitoring protease activity and nucleic acid hybridization [365,366]. In the case of the protease activity bioassays, the authors were able to demonstrate multiplex protease activity detection using a single QD color (Figure 6.20) [368]. Here, the QD (which acts as both a donor and an acceptor) was functionalized with peptide substrates for trypsin (labeled with a luminescent Tb complex donor) and chymotrypsin (labeled with an AlexaFluor dye acceptor).…”

Interesting Developments at the nanoparticle–protein interface: Implications for Next Generation Drug Delivery

“…For this, dye-labeled peptidyl substrates were ratiometrically attached to luminescent semiconductor quantum dots (QDs) to monitor trypsin proteolytic activity by FRET as a model system [11]. The only variable modified here was the ratio of peptide-substrate attached to the QDs.…”