1246
L. VALGIMIGLI ET AL.
addition, the biphasic system was vigorously stirred at room presence of ADA. The assay mixture contained, in a final vol-
temperature for 1 h, and the blue adduct was extracted with ume of 2 mL, 400 µL of enzyme solution, 10 mM MC, and
dichloromethane. The organic solution was washed with 2mM ADA in 50 mM sodium citrate buffer, pH 5.5. After 5
water, dried over Na SO , and evaporated in vacuo to yield min, dichloromethane (2 mL) was added and the mixture vor-
2
4
the blue product, which was purified by preparative thin-layer texed for 15 s in a stoppered test tube, to avoid dichloro-
chromatography on silica gel (20 cm × 30 cm × 1 mm; Merck, methane evaporation. The upper aqueous layer was drawn
Darmstadt, Germany) by eluting with dichloromethane/ off, the organic solution transferred to a spectrophotometer
methanol (95:5). The purified product was extracted from the cuvette, and the absorbance read at 617 nm. An ε value of
−
1
−1
plate with isopropanol, which was evaporated in vacuo at 11,080 M cm (in dichloromethane) was used for calcula-
room temperature. Yield = 75% after purification.
tions (discussed in the Results and Discussion section).
1
13
H and C nuclear magnetic resonance spectra were
recorded on a Varian Gemini 300 spectrometer by using a 20-
mg sample of purified adduct dissolved in 0.75 mL of CDCl3
RESULTS AND DISCUSSION
(
Sigma, Diesenhofen, Germany). Fourier transform infrared ADA and MQ rapidly and quantitatively react with a
spectra of the purified adduct were recorded with a Nicolet 1:1 stoichiometry to give the intensely blue product,
Protégé 460 spectrometer (Madison, WI) from a solution of 4-(4′-diethylamino-phenylimino)-2-hydroxy-5-methyl-cyclo-
the sample in chloroform (Aldrich) by using a KBr cell with hexa-2,5-dienone. The structure of this has been determined
an optical path of 0.1 mm. Mass spectroscopy was performed (Scheme 1), in accordance to the following analytical data:
−
1
on a double-analyzer VG 7070E spectrometer (EI+ 70 EV), ηmax (cm ) 3386, 3040, 2964, 2929, 1720, 1630, 1600. δ
H
with direct probe-introduction of the sample. Ultraviolet- (300 MHz, CDCl ) 1.21 (t, 6H, J = 7.5), 1.60 (OH, broad s,
3
visible (UV-Vis) spectra were recorded on a double-beam 1H), 2.18 (s, 3H), 3.42 (q, 4H, J = 7.5), 5.31 (s, 1H), 5.32 (s,
Jasco V-550 spectrophotometer.
Sample preparation. Ground and kneaded olive pastes MHz, CDCl ) 13.7, 30.6, 45.6, 103.5, 112.5, 125.7, 126.1,
were treated with liquid nitrogen immediately after being 139.2, 147.7, 151.0, 153.7, 163.1, 183.4 ppm. m/z 284 (M ,
1H), 6.62 (d, 2H, J = 6.0), 7.01 (d, 2H, J = 6.0) ppm. δ (75
C
3
+
drawn from the olive mill and were kept at −80°C until use. 20%), 269 (32%), 255 (37%), 163 (14%), 119 (30%), 97
To 1 g of the paste, 0.1 g of crosslinked polystyrene (Bio- (25%), 83 (35%), 69 (38%), 55 (40%), 43 (100%). Anal.
Beads SM-2, Bio-Rad, Hercules, CA) and 0.25 g of cross- calcd. for C H N O : C 71.81%, H 7.09%, N 9.85%.
1
7
20
2
2
linked poly(vinylpyrrolidone) (Aldrich Chemie) beads were Found: C 71.83%, H 7.08%, N 9.81%. The UV/Vis spectrum
added, immediately followed by 8 mL of 10 mM sodium cit- (Fig. 1) of the compound showed a λ of 617 nm, ε 11,080
max
−1
−1
rate buffer, pH 5.5. The suspension was gently stirred for 10 M cm (in dichloromethane).
min, and subsequently centrifuged at 4°C for 20 min at The substance is immediately and completely extracted
1
5,000 × g. The supernatant was finally filtered through from aqueous solutions by dichloromethane, where it shows
Whatman 1 paper (Maidstone, England).
a remarkable stability. As MC is among the best substrates
Fresh olives were lyophilized. The fruitstones were then for olive PPO, which oxidizes it to the corresponding MQ,
removed, and stoned fruits (10 g) were suspended in 100 mL the described method is quite suitable for PPO determination.
of 10 mM sodium citrate buffer, pH 5.5, containing 1 g of
To check and validate the assay, olives, ground and
SM-2 Bio-Beads and 2.5 g of crosslinked poly(vinylpyrroli- kneaded olive pastes, and virgin oil were all tested for PPO
done) beads. The mixture was homogenized for 2 min at activity by using both the standard method and that described
1
6,000 rpm in an Ultraturrax (Ika-Werhe GmbH, Staufen, above. Generally speaking, these results, obtained by using
Germany). The homogenate was centrifuged at 4°C and the proposed colorimetric method, parallel those achieved
2
2,000 × g, and the supernatant was filtered through What- with the standard one (Table 1), although higher activity was
man 1 paper. Virgin oil samples were extracted as described found with the use of the ADA-based method (see below).
elsewhere (10).
Enzyme assay. All the enzyme assays were performed at
30°C, with the aid of a thermostated water bath and a thermo-
static device mounted within the cuvette chamber.
Standard activity measurements. PPO activity was deter-
mined photometrically by measuring the initial rate of oxida-
tion of MC at 400 nm by using a Pharmacia Ultrospec 4000
spectrophotometer. The assay mixture contained, in a final
volume of 2 mL, 400 µL of enzyme solution and 10 mM MC
in 50 mM sodium citrate buffer, pH 5.5. An ε value of 1300
−1
−1
M cm at λ = 400 nm was used for calculations (5).
ADA-based activity measurements. PPO activity was de-
termined photometrically by measuring the overall formation
of the blue adduct of the enzyme with its substrate MC in the
SCHEME 1
JAOCS, Vol. 78, no. 12 (2001)