Banana Polyphenoloxidase. Preparation and Properties

Abstract

Griffiths (13) has presented evidence in(licating that the browning reactions of banana fruit result from the enzymic oxi(lation of (lopamiiine (3.4 (lih-droxyphenylethylaminei) hy pIolyphenoloxi(lase. Al-thoughi (lopamine does occur in various fruits andl vegetables (23), it has not previously b)een imiplicat-ed as an important substrate in browning reactions. The present investigation was undertaken to develop metho(ds for preparation of banana polvphenoloxidcase (PPO) 3 an(l to determinie solile of its properties in comparison with phenol oxi(lases frolim othier sources. Materials and Methods Materials. The chemiiicals use(an 1 their suppliers were as follows: Dopamiline HCI: D.l,-ar-terenol HCI; L(-) arterenol bitartrate hydrate; chlorogeniic acid-, tyramine HCI: California Corporation for Biochemi cal Researchi. 3.4-dihydroxv-phenylalanine (DOPA): Nutritionial Bliochemicals Corp. L (-) tyrosine: Pfalistielil Laboratories. Inc. Catechlol; o-cresol: Fishier Scientific Co. p-Cresol (redistilledl); 8-hydroxyquinoline: L(+)'cysteine HCI: Matheson, Colemialn, and Bell. DE,AE-cellulose (Type 20, 0.82 mie(q jg): Schileicher-& Schuell Co. Assayv Procediures. The standard assay for banana PPOC was adaptedl fromil tile tyrosine assav of Fox and Burnett (10). The increase in optical (lensity at 470 mia after mixing enzyme and substrate wvas followed at 25° in a DK-2 sl)ectrophotomieter eqluippe(l with a time (Irive. The reactioin mlixture, in a final volume of 3 ml, contained 0.033 \) potassium phosphate , pH 7.0; 5 X 10-:-\ (lopaiiine and sufficient enzyme to cause an increase in optical density of 0.02 to 0.1 per minute. Rates were calculate(d from the initial slope of the curve alid were proportional (+ 10 %) to enzyme concentration. Some extracts vere assayed indirectly (8) by following spectrophotonmetricallv at 265 nm/L the rate of disappearance of ascorbic acid at 25° in a reaction mixture containing 0.05 l potassiunl phosl)lpate, pH 1 Revised manuscript received Jan. 28, 1963. 2 A preliminary report of this work lhas appeared in Planit Physiol. 36, Suppl. xxxviii, 1961. 3 Terminology used in this paper: Pheniol oxidase, generic term to include all enzymes which catalyze the oxidation of phenols; tyrosinase, enzyme which catalyzes the oxidation of both mono-and diphenols; polyphenol-oxidase, enzyme which catalyzes the oxidation of ortho dihydric phenols only. 7.0: 10-5 EDTA; 4.2 X 10-) i% ascorbic acid; 5 X 10-4 31 dopamline and enzyme in a final volumie of 3 ml. The reaction rate wN'as proportional to eiiz) iie concentrationi xNhen the decrease in optical lensitv was between 0.02 and 0.2 per minute. All extracts assayed by this method were free of interfering ascorbic acid oxidase. PPO was also assayed manometrically by stand-ardl \Varburg tecli(lue at 30°. Reaction mi.xtur-es are (lescribed for the individual experiments. One unit of PPO activity is expressed wherever lOxsible as that amilount of enzyme which ill catalyze the transformation of 1 Mmilole of substrate peri minute un(ler the con(litions of the assay'. Michaelis conistanits were obtai ne(l froml Lineweaver-Burk ploti (16). The protein conteIlt of the (letergenit extracts \\.as estimlate(l frolm nlitrogenl determinations (1 1)ill tile extraCIct after a 4 h1our d(ialsis agailnst 400 volumes of 0.02 M\ potassiumii phosphate, pH 7.0 in a stirrinig dialyzer. Removal of nonproteiin nitrogen wx as es-senitially coliiplete undler these con(litions. but little or n1o I'PO (or (letergent) wvas lost. Protein in the fractions frolim DEAE cellulose column1i1s w\as estilimt-ed by the miethio(d of Wkaddell (25). Egg albumileni MWNNIT = 40.000) an(l gamma globuliin (Bovine Fraction II, MWNV 150,000) gave i(lentical standilard curves. This teclniqlue could not be usedl onl tile (letergent extracts as the detergent interfered. The spectromiietric technique of Mason (17) as use(l to studly the reactioni mechanism. A series ot 1 yiumole saniples of (lopanmine in 10 ml of 0.05 Mr potassium phosphate. pH 6.0 wvere oxidizecl by 50 nlg of Ag.,O ith shaking for periods varying bet een 1.5 ancd 20 mlinutes. The solutionls were filtere(d quickly, and the resulting spectra were compared with those of the enzyimic oxidationl products. D, L-DOPA was oxidlize(l un(ler simililar-conldlitionis to chieck the procedlure. Dopamiline f8-oxidase activity was assayed by the techni(lue of Smlithi andl Kirshner (21). A one nil ali(luot of diluted (1: 50) detergent extract was ill-cubated with 1 Aimole of dopamine at 370 for 1 hour. The reaction was stopped with 1 nil of 10 9 tri-chloroacetic acidl an(l the nmixture centrifuge(d at low speed to sedimilent protein. A 1.2 mln, ali(quot was extracted 3 times with ether and thein assaye(h fluori-mietrically (9) to (letermine arterenol (Beckmain DK-2 spectrophotometer. No. 22850 fluorescence at-tachlimlent, 515 Illn). 508