Graduate Thesis Or Dissertation
 

Purification and properties of d'Anjou pear (Pyrus communis, L.) polyphenol oxidase

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  • 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.
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