Polyphenol oxidase (PPO) from strawberries was purified,
partially characterized, and used to study its role in the degradation
of anthocyanin pigments. The extraction method included the use of a
buffered solution containing the phenolic binders Polyclar AT and
Amberlite XAD-4. Addition of Triton X-100 resulted in an increase of
the extracted activity. Higher levels of PPO activity were obtained
using citrate rather than acetate in the buffer. This was possibly due
to the capacity of citrate to chelate calcium ions, and, therefore, to
inhibit crosslinking of the pectic polymers which would trap the
enzyme. Using a Phenyl Sepharose CL-4B (PS) hydrophobic column two
fractions of the enzyme, Fl and F2, were separated from the pectic
material and the bulk of the 280 nm absorbing material.
PP0-F1 obtained from the PS column was less hydrophobic than F2,
and showed a molecular weight of 111,000 by gel filtration on
Sephacryl S-300. Form Fl eluted in the void volume of an anion
exchange column and did not penetrate into the running gel during
electrophoresis. The binding of PP0-F1 to a Concanavalin-Agarose
(ConA) column demonstrated the presence of carbohydrate associated
with the enzyme. PP0-F2 showed a molecular weight of 34,500 and could be resolved by anion exchange chromatography into one large peak and a
smaller one at its shoulder. Fraction F2 of the enzyme resolved into
two bands during electrophoresis, and did not bind to the ConA column,
Both fractions of the enzyme showed multiple banding on lithium
dodecyl sulfate -- polyacrylamide gradient gels.
Both fractions of the enzyme obtained from the PS column showed
very high activity in the presence of D-catechin. The Michaelis
constants for D-catechin for PPO fractions Fl and F2 were 0.50mM and
O.4lmM respectively, and the maximum velocity 82,700 and 18,800 nmoles
0₂ per min per ml respectively. D-catechin in the presence of
PP0-F1 was rapidly oxidized and the resulting solution had a maximum
absorption at 390 nm. When D-catechin and PP0-F1 were combined in
model systems with either pure cyanin or pelargonin, an absorbance
peak at 390 nm was noticed with increased absorbance in the region of
the anthocyanin pigments. After 24 hr, 50% of the pelargonidin and 60%
of the cyanin was destroyed and a brown precipitate was formed. PPO-Fl
seemed to oxidize cyanin at a very slow rate but did not oxidize
pelargonin. It was suggested that the anthocyanin pigments were
destroyed by either the direct oxidation by the quinones formed from
D-catechin by PPO, or that the anthocyanin pigments were
co-polymerized into the brown polymeric pigment, tannin, formed from
D-catechin-quinone polymerization. / Graduation date: 1984
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/27100 |
| Date | 30 August 1983 |
| Creators | Wesche-Ebeling, Pedro Alfredo E. |
| Contributors | Montgomery, Morris W. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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