Polyphenol oxidase (PPO) from d'Anjou pears was purified and some
molecular properties were studied. The extraction of PPO was
accomplished in the presence of the phenolic binder AG 2X-8 and Triton
X-100. Chlorophyll pigment was removed by chromatography resulting in
a clear, colorless enzyme extract. Three fractions of pear PPO were
purified by hydrophobic interaction chromatography on Phenyl Sepharose
CL-4B, followed by chromatography on DEAE-cellulose and hydroxylapatite
(HA). The best resolution was achieved when the columns were
developed at room temperature. Reproducibility of the entire scheme
was excellent with chromatography on the hydrophobic resin as a key to
the procedure's success. The three fractions of pear PPO were
homogeneous when stained for protein with the silver stain after
polyacrylamide gel electrophoresis and corresponded to relative
mobilities of 0.41 (HA 1), 0.43 (HA 2), and 0.73 (DEAE 3). However,
other minor protein bands were noticed on lithium dodecyl sulfate,
LDS, electrophoresis.
Dimethylsulfoxide, DMSO, was found to significantly increase the
PPO activity over the control. An 80% increase in enzyme activity was
observed when 5% DMSO was added to the enzyme fraction. Chlorogenic
acid reacted with one of the purified fractions (HA 2) at pH 7 to
yield 5 additional PPO bands upon electrophoresis. However, no
electrophoretic changes were observed with another fraction (DEAE 3)
or when 2,3-dihydroxybenzaldehyde was reacted with the two PPO
fractions.
The molecular weights of the three multiple forms of pear PPO
were similar and were 45,300 ± 5% daltons by Sephacryl S-300 gel
filtration and 59,600 ± 3% daltons by LDS electrophoresis in 10% and
gradient polyacrylamide slab gels. The isoelectric points of the
three PPO fractions were: HA 1, pi 4.3 and 4.5; HA 2, pi 4.5 and 4.7;
and DEAE 3, pI 6.9. The three fractions differed in amino acid
composition with HA 1 having a higher proportion of hydrophobic amino
acids and less charged residues than either HA 2 or DEAE 3. All three
forms of PPO contained a high proportion of the acidic amino acids
and/or their amides, and glycine. / Graduation date: 1983
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/27448 |
| Date | 11 November 1982 |
| Creators | Wissemann, Kimberly Warner |
| Contributors | Montgomery, Morris W. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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