Sensitive Assay for the Lactonase Activity of Serum Paraoxonase 1 (PON1) by Harnessing the Fluorescence Turn-On Characteristics of Bioorthogonally Synthesized and Geometrically Controlled Chemical Probes

Abstract

The lactonase activity of paraoxonase 1 (PON1) has a crucial antiatherogenic function, and also serves as an important biochemical marker in human blood because the aberrant lactonase activity of PON1 is a key indicator for a number of diverse human diseases. However, no sensitive fluorescence assays that detect PON1 lactonase activity are available. We report the synthesis of two fluorescence turn-on chemical probes 16a and 16b (16) able to quantify PON1 lactonase activity. The chemical probes were constructed utilizing a disulfide-containing bicyclononyne, derivatives of rhodamine B and carboxyfluorescein, and reactions including copper-free azide-alkyne cycloaddition. Fluorescence quenching in 16 was characterized by spectroscopic studies and was mainly attributed to the effect of contact quenching. Kinetic analysis of 16b confirmed the outstanding reactivity and specificity of 16b with thiols in the presence of general base catalysts. The 16b-based assay was employed to determine PON1 lactonase activity, with a linear range of 10.8-232.1 U L^-1 and detection limit (LOD) of 10.8 U L^-1, to quantify serum PON1 activity in human sera, and to determine the K_i of 20.9 μM for the 2-hydroxyquinoline inhibition of PON1 lactonase. We are employing 16b to develop high-throughput assays for PON1 lactonase activity.

Keywords: bicyclononyne; chemical probe; fluorescence turn-on; lactonase; paraoxonase 1.

Scheme 1

Use of the chemical probe 16b to quantitatively analyze catalysis and inhibition of PON1 lactonase activity. The PON1-catalyzed hydrolysis of thiobutyl butyrolactone (TBBL, a thiolactone) would provide a labile hemithioacetal, which spontaneously cleaves to give 1-butanethiol (nBuSH) as one of the products. The thiolate of nBuSH is highlighted in blue. Subsequently, the thiolate nucleophilically attacked (the pink arrow) the disulfide bond in 16b to obliterate the fluorescence quenching effects (the green arrow) and to liberate the 6-carboxyfluorescein (6-FAM) fluorescence. Increments in the 6-FAM fluorescence (the light green arrow) are, therefore, proportional to increases in PON1 lactonase activity (the light blue arrow). a: (TBBL), Tris, Ca²⁺, pH 8.0. R¹ and R², H or the thiolate of nBuSH.

Scheme 2

Synthesis of azido-rhodamine B (9).

Scheme 3

Reaction sequence for separating 5(6)-carboxyfluorescein (FAM) into geometric isomer-pure 5-carboxyfluorescein (5-FAM, 13a) and 6-FAM (13b), which were further derivatized to provide the corresponding NHS esters 14a and 14b, respectively.

Scheme 4

Synthesis of the fluorescence turn-on chemical probes 16 from exo-6. a: 14a/14b, DIPEA, rt, O/N; b: 9, dioxane, reflux.

Figure 1

Contact quenching characteristics of the chemical probes 16 revealed by the UV-Vis absorption and the fluorogenic property of one of the chemical probes 16b when reacting with a thiol. (a) The UV-Vis spectra for 50 μM of 5(6)-FAM (the blue curve), rhodamine B (the red curve), and the fluorescence chemical probes 16 (the light green curve for 16a; the purple curve for 16b) in phosphate buffer (PB; 10% dimethylformamide (DMF), pH 7.4). (b) The time-dependent increase in 6-FAM fluorescence in the reaction of 16b (1 μM) with L-cysteine (50 mM) in PB.

Figure 2

Differential reactivity of 16b toward reactants as revealed by direct visualization and kinetic analysis. (a) Visualization of the 6-FAM fluorescence to characterize the fluorescent emission properties of 16b in the presence of various reactants. Each vial contained 25 μM of 16b in the Tris buffer (50 mM Tris, 1 mM Ca²⁺, pH 8.0). In addition, samples 2–12 also included the reactants (50 mM) of L-glutamate, glycine, L-methionine, L-lysine, L-serine, glutathione (GSH), L-cysteine, 2-mercaptoethanol (2-ThioEtOH), 2-aminoethanethiol (2-AET), DL-dithiothreitol (DTT) and 1-butanethiol (nBuSH), respectively. The reactions were carried out in the dark at rt for 1.5 h before the photograph was taken under UV illumination (365 nm). (b) Determination of the pseudo-first order rate constant k₁ from each of the reactions of 16b with various reactants. Please see Section 3.3 for the experimental details. Each reactant was analyzed at least three times in order to acquire the averaged k₁ values and standard deviation (the error bar). The error bars were too short to be visible in some reactions. The symbol * indicates that 16b had no measurable reactivity, meaning that the values of averaged k₁ and standard deviation could not be determined.

Figure 3

Titration studies to decrypt the reaction mechanism of 16b with 2-AET. (a) Kinetic analysis of 2-AET titration on the 2-AET–16b reaction was performed in the presence of 0.4 μM of 16b. (b) Kinetic analysis of pH titration on the 2-AET–16b reaction was carried out in the presence of 0.4 μM of 16b and 5 mM of 2-AET, while varying the pH. Please see Section 3.3 for the experimental details. Each reaction was analyzed at least three times in order to acquire the averaged k₁ values and standard deviation (the error bar).

Figure 4

The chemical probe 16b used for accurate determination of PON1 lactonase activity. (a) Each vial contained 2 μM (in dimethyl sulfoxide (DMSO), 0.5%) of 16b in the Tris buffer, and various combinations of PON1 (7.79 U L⁻¹, 50% glycerol) and TBBL (5 mM in acetonitrile (ACN), 0.2%). The reactions were carried out at rt for 15 min and visualized and photographed under UV lamp illumination. (b) Catalysis of a PON1-TBBL–16b reaction system was monitored spectrofluorometrically; the reaction contained 16b (0.8 μM in DMSO, 1.6%), rePON1 (232.1 U L⁻¹, 50% glycerol), and TBBL (10 mM in ACN, 1%) in the Tris buffer at 25 °C. The normalized FL intensity at 525 nm was acquired by subtracting a background fluorescence of 16b at the same wavelength from the original fluorescence intensity data.

Figure 5

The 16b-based assay for the determination of PON1 lactonase activity in sera. (a) PON1 standards obtained from E. coli were analyzed using the 16b-based method that afforded the linear regression calibration curve of PON1 lactonase activity vs. initial velocity (v_i), and gave an LOD of 10.8 U L⁻¹ and a linear detection range of 10.8–232.1 U L⁻¹. (b) PON1 lactonase activity in serum samples from three healthy males was measured using the 16b-based assay. Please see Section 3.4 for the experimental details. Each reaction was analyzed at least three times in order to acquire the averaged values and standard deviation (the error bar) of v_i or PON1 lactonase activity.

Figure 6

2-Hydroxyquinoline (HQ) inhibition of PON1 catalysis analyzed by the fluorescence assay based on 16b. (a) Time-course kinetic analysis of HQ inhibition of PON1 (250.9 U L⁻¹, 1.25% glycerol) catalysis was performed in the presence of TBBL (5 mM in ACN, 1%) in the Tris buffer. Each PON1 reaction contained 0, 50, 100 or 200 μM of HQ (in 1% ACN). (b) Dixon plot of the inhibitory kinetics of HQ on PON1 catalysis was derived from the v_i in (a) and Figure S5. The HQ inhibitor constant K_i was determined from the minus x-axis value at which the two extrapolated lines intersected, which, in this study, was 20.9 μM. Please see Section 3.4 for the experimental details. Each reaction was analyzed at least three times in order to acquire the averaged values and standard deviation (the error bar) of v_i.