Enzchek phospholipase a2 assay kit

Phospholipase A₂ represents a family of enzymes that hydrolyze the sn-2 ester linkage of phospholipids. The assay we used to detect aiPLA₂ activity of hPrdx6 is based on measuring distinct fluorescence resonance energy transfer (FRET) emission of the substrate prior to and after cleavage in the absence of Calcium. Samples (hPrdx6), positive controls (0–10 Units/ml of PLA₂ from honey bee venom) and negative control (no PLA₂) have been prepared as per manufacturer’s recommendations (EnzChek Phospholipase A2 Assay Kit, Invitrogen). For each reaction, 100 μg of purified enzyme (diluted in 10 mM base buffer) in a total volume of 50 μl is incubated with equal volume of substrate-liposome mix (prepared by mixing of 30 μl 10 mM Dioleoylphosphatidylcholine, 30 μl 10 mM Dioleoylphosphatidylglycerol, and 30 μl 1 mM PLA₂ substrate) for 10 min at room temperature. To test pH dependence, base buffers used were glycine (pH 2.0–3.0), sodium acetate (pH 4.0–5.0), sodium cacodylate (pH 6.0), and Tris–HCl (pH 7.0–10.0). Fluorescence emission is measured at ~ 515 nm and ~ 575 nm (excitation at 460 nm) using TECAN microplate reader. Enzyme activity is quantified by comparative ratiometric analysis of samples and positive control’s fluorescence emission at 515 nm/575 nm. The enzyme assay was carried out at least three times.

Phospholipase A2 enzyme activities was measured using the EnzChek Phospholipase A2 Assay Kit (Invitrogen, Carlsbad, CA, #E10217) per the manufacturer’s instructions. Baseline phospholipase A2 (PEDF-R) activity was measured from healthy and L-ORD-iRPE maintained at 37 °C in 5% serum-containing RPE media. Phospholipase A2 activity in response to elevated AMPK was measured after 24 h exposure to 0% serum-containing media in healthy-iRPE. Samples were collected in a native gel lysis buffer (150 mM NaCl, 1% Triton X-100, 1 M Tris-HCL). Samples were centrifuged at 13,200 rpm for 10 min at 4 °C. The supernatant was transferred to new tubes and frozen on dry ice and stored at −80 °C. A total volume of 100 µL was used following the standard assay protocol: 50 µL sample + 50 µL substrate-liposome mix.

The PLA2 activity of B. lanceolatus venom was investigated using an EnzChek^® Phospholipase A2 Assay Kit (Invitrogen, Paisley, UK), following the manufacturer’s instructions. Venoms from B. jararaca and C. durissus terrificus were used as the positive controls. The venom concentrations were chosen according to preliminary dose-response curves. Fluorescence measurements were performed at 37 °C using the spectrometer FLUOstar Omega (BMG Labtech, Offenburg, Germany) at the wavelength λ_EM = 515 nm, with an excitation at λ_EX = 485 nm every 30 s for 5 min. Specific activity was expressed as the units of free fluorescence of cleaved substrate/min/µg of venom.

The inhibitory activity towards phospholipase A2 was determined using the EnzChek Phospholipase A2 Assay kit (Invitrogen). The manufacturer’s protocol was followed. Samples (oleoresin, V1, V2, and V3) were in the same concentrations evaluated in the cell-based experiments and were incubated for 10 min at 25°C. Phospholipase A2 was used as the positive control, while DMSO 0.15% was used as the negative control. All assays were conducted in triplicates.

The PLA₂ activity of the purified protein from the B. lanceolatus venom (5 µg) was analyzed using EnzChek^® Phospholipase A₂ Assay Kit (Invitrogen, Eugene, OR, USA) following the manufacturer’s instructions. Saline and B. lanceolatus venom were used as negative and positive controls, respectively. Fluorescence measurements were performed at 37 °C using the spectrometer FLUOstar Omega (BMG Labtech, Offenburg, Germany) at the wavelength λEM = 515 nm, with an excitation of λEX = 485 nm every 30 s for 5 min. Specific activity was expressed as units of free fluorescence of cleaved substrate/min/μg of protein.

Determination of PLA₂ enzymatic activity was performed using the fluorometric EnzChek™ Phospholipase A₂ Assay Kit (Invitrogen) according to manufacturer’s protocol. Fluorescence was measured using a plate reader (VICTOR® Nivo™, PerkinElmer) at an excitation wavelength of 480 nm and an emission wavelength of 530 nm. Measurements were made immediately after substrate addition and then every 30 s for 10 min to verify the linearity of the kinetics. The enzymatic activity was defined as the relative fluorescence obtained 5 min after substrate addition.
Neutralization of enzymatic activity was assessed by incubating 0.1 mg/mL of purified PLA₂s or PLA₂-containing fractions from different elapid snake venoms (PLA₂N from the venom of M. fulvius, Nn19 from Naja nigricollis, and Hh3 from H, haemachatus) with a 1:20 toxin to V_HH molar ratio of each anti-PLA₂ V_HH for 30 min at RT. PLA₂ activity was determined for each of the mixtures in duplicate. The PLA₂ activity in the presence of the V_HHs was normalized by setting the activity of the toxin incubated with buffer only to 100%.