A. Abe et al.
Analytical Biochemistry 549 (2018) 164–170
LPLA2 activity. A truncated oxidized phosphatidylethanolamine (PE)
derivative conjugated with a BODIPY fluorophore at the amino group,
PGPE-BODIPY, was chosen due to the broad preference of LPLA2 to a
polar group of glycerophospholipids. Based on the acyl chain specificity
of LPLA2 against the truncated oxidized phospholipids, LPLA2 should
convert PGPE-BODIPY to palmitic acid and 1-lyso-PGPE-BODIPY. 1-
lyso-PGPE-BODIPY is a more polar lipid than either PGPE-BODIPY or
palmitic acid. Therefore, the lyso- PGPE-BODIPY and PGPE-BODIPY/
palmitic acid would be recovered into the aqueous phase and the or-
ganic phase, respectively, after phase partitioning by chloroform and
methanol. In addition, the emission of BODIPY fluorophore is in-
dependent of pH. Therefore, the LPLA2 activity assay could be attained
by a simple measurement of fluorescence intensity of the aqueous phase
as previously required.
In this study, recombinant mouse LPLA2 and mouse serum were
used as a source of LPLA2 enzyme. We first investigated whether the
fluorogenic truncated oxidized phospholipid properly acts as a sub-
strate for LPLA2 using recombinant LPLA2. We then established a
specific fluorescence measurement method of LPLA2 activity using the
serum prepared from LPLA2 deficient mice.
under ultraviolet (UV) light. In order to detect non-fluorescent products
formed in the reaction, the plate was soaked in 8% (w/v) CuSO
4 2
, 5H O,
6.8% (v/v) H PO , 32% (v/v) methanol. The uniformly wet plate was
3
4
briefly dried using a hair dryer and charred for 15 min in a 150 °C oven.
The charred plate was scanned and the content of the product was es-
timated by NIH-ImageJ 1.37v. For the aqueous phase, each sample was
directly applied on an HPTLC plate and then treated as described above.
Fluorescence measurement
The aqueous phase containing the BODIPY product was kept in a
brown tube. The BODIPY emission spectrum of the aqueous phase was
recorded in the scanning range 500–600 nm using a fluorometer
(Hitachi F-2300, Japan). The excitation wavelength was constant at
490 nm. Emission and excitation slits were 5 nm. The fluorescence in-
tensity at the emission peak (506.5 nm) was defined as (the fluores-
cence intensity obtained from the study in the presence of LPLA2 or
serum) - (the fluorescence intensity obtained from the study in the
absence of LPLA2 or serum) at each indicated time period in the re-
action.
The BODIPY-derivative formed in the reaction is not available as a
standard. To measure quantitatively the BODIPY-product, a standard
curve was created using a known amount of PGPE-BODIPY, which was
dispersed in the aqueous phase solution obtained by the phase partition
used in this study. PGPE-BODIPY was freely dispersed in the aqueous
phase solution and emitted BODIPY monomer fluorecence as the
BODIPY-product. In the present study, we assumed that the quantum
yield of PGPE-BODIPY in the aqueous phase solution is not different
from that of the BODIPY-product in the same solution.
Materials and methods
Materials
1-Palmitoyl-2-glutaroyl-sn-glycero-3-phosphoethanolamine-N-[4-
(
dipyrrometheneboron difluoride) butanoyl] (PGPE-BODIPY), 1,2-O-
octadecyl-sn-glycero-3-phosphocholine (DODPC), 1,2-dioleoyl-sn-gly-
cero-3-[phospho-rac-(1-glycerol)] (DOPG) and N-acetylsphingosine
(
NAS) were obtained from Avanti Polar Lipids Corp. (Alabaster, AL);
Results
recombinant mouse LPLA2 was from Proteos (Kalamazoo, MI); HPTLC
silica gel plates, 10 × 20 cm, were from Merck (Darmstadt, Germany);
Degradation of fluorogenic truncated oxidized phospholipid by LPLA2
4-(2-aminoethyl)benzenesulfonyl fluoride was from Sigma-Aldrich (St.
Louis, MO).
The molecular structure of PGPE-BODIPY, the fluorogenic truncated
oxidized phospholipid tested, is shown in Fig. 1A. DODPC liposomes
containing PGPE-BODIPY were incubated with recombinant mouse
LPLA2 to confirm whether LPLA2 is able to degrade the fluorogenic
phospholipid.
LPLA2 activity on PGPE-BODIPY
For the preparation of PGPE-BODIPY/DODPC liposomes, those li-
pids (molar ratio of PGPE-BODIPY to DODPC: 1:2.4) were mixed in a
glass tube and dried down under a stream of nitrogen gas. DODPC is a
non-hydrolyzable phosphatidylcholine. The dried lipid mixture was
dispersed into 50 mM sodium citrate (pH 4.5) using a probe-type so-
nicator for 8 min in an ice water bath.
The reaction solution containing PGPE-BODIPY/DODPC liposomes
(molar ratio of 1–2.4) prior to the addition of the enzyme showed red
orange color and was not fluorescence emitting. However, upon in-
cubation with the LPLA2 a change in the color of the reaction mixture
was observed. The color turned from red orange to light green (Fig. 1B-
1, right tube). No color change was observed in the reaction mixture in
the absence of LPLA2 (Fig. 1B-1, left tube). When the reaction mixtures
were exposed under UV light, the reaction mixture containing LPLA2
emitted fluorescence (Figs. 1B-2, right tube). In addition, the fluor-
escent product formed in the presence of LPLA2 was recovered in the
aqueous phase when the reaction mixture was partitioned into two
layers using chloroform/methanol/aqueous solution (2:1:0.8, v/v)
under acidic conditions (Figs. 1B-3, right tube). By contrast, PGPE-
BODIPY was recovered in the organic phase (Figs. 1B-3, left tube).
Thin layer chromatography (TLC) was used to identify the reaction
products (Fig. 2). A reaction product was recovered in the organic
phase that increased with time and was identified as palmitic acid when
compared with an authentic palmitic acid (Fig. 2B and C). Con-
currently, PGPE-BODIPY was gradually decreased in the presence of
LPLA2 (Fig. 2A). Also, the fluorogenic BODIPY reaction product re-
covered in the aqueous phase displayed a slower mobility than PGPE-
BODIPY and increased with time (Fig. 2D). In addition, the BODIPY-
product was hydrolyzed by treatment with diluted alkaline solution
(data not shown). One of the resultant products produced by alkaline
treatment was a fluorescent compound with mobility between the
BODIPY-product and PGPE-BODIPY on TLC. This product had the same
mobility as one of the products produced from PGPE-BODIPY by the
same alkaline treatment (data not shown). Because weak alkaline
The reaction mixture consisted of 49 mM sodium citrate (pH 4.5),
10 μg/ml BSA, 38 μM PGPE-BODIPY incorporated into 92 μM DODPC
liposomes, and LPLA2 or mouse serum in 500 μl of total volume. The
reaction was initiated by adding recombinant LPLA2 or mouse serum,
kept for a specific time period at 37 °C and terminated by adding 3 ml of
chloroform/methanol (2:1, v/v) plus 0.3 ml of 0.9% (w/v) NaCl (the
final ratio of chloroform/methanol/aqueous solution: 2:1: 0.8, v/v). In
other cases, one hundred μl of the reaction mixture was taken at a
specific time point and mixed with 3 ml of chloroform/methanol (2:1,
v/v) plus 0.4 ml of 50 mM sodium citrate (pH 4.5) and 0.3 ml 0.9%
NaCl. The mixture containing organic solvents was centrifuged at 800g
for 5 min at 20 °C. The resultant aqueous phase (upper layer) was col-
lected into a small brown glass vial with a screw cap and kept at room
temperature until the fluorescence was measured. The lower organic
layer was transferred into another glass tube and dried down under a
stream of nitrogen gas.
TLC assay
The dried lipid from the organic phase was dissolved in chloroform/
methanol (2:1, v/v), applied to an HPTLC plate and developed in a
solvent system consisting of either chloroform/methanol/water
(
60:35:8, v/v) or chloroform/methanol/pyridine (98:2:0.5, v/v). To
visualize the BODIPY fluorophore, the plate was dried and then exposed
165
Abe, Akira
