Partial purification of latent persimmon fruit polyphenol oxidase.
Persimmon fruit polyphenol oxidase (PPO) was partially purified using a combination of phase partitioning with Triton X-114 and ammonium sulfate fractionation between 50 and 75%. The enzyme, which showed both monophenolase and diphenolase activities, was partially purified in a latent form and could be optimally activated by the presence of 1 mM sodium dodecyl sulfate (SDS) with an optimum pH of 5.5. In the absence of SDS, the enzyme showed maximum activity at acid pH. SDS-PAGE showed the presence of a single band when L-DOPA was used as substrate. The apparent kinetic parameters of the latent enzyme were determined at pH 5.5, the V(m) value being 15 times higher in the presence of SDS than in its absence, whereas the K(M) was the same in both cases, with a value of 0.68 mM. The effect of several inhibitors was studied, tropolone being the most active with a K(i) value of 0.45 microM. In addition, the effect of cyclodextrins (CDs) was studied, and the complexation constant (K(c)) between 4-tert-butylcatechol (TBC) and CDs was calculated using an enzymatic method. The value obtained for K(c) was 15580 M(-1).
Núñez-Delicado
Estrella
E
Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Campus de Espinardo, E-30071 Murcia, Spain.
Sojo
M Mar
MM
García-Carmona
Francisco
F
Sánchez-Ferrer
Alvaro
A
D000645:Ammonium Sulfate; D004156:Catechol Oxidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D005591:Chemical Fractionation; D029788:Diospyros / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005638:Fruit / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007980:Levodopa / Q000378:metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D004791:Enzyme Inhibitors; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D007980:Levodopa; D017830:Octoxynol; C010615:Nonidet P-40; D004156:Catechol Oxidase; D000645:Ammonium Sulfate