A Spectrophotometric Assay of Polyphenoloxidase Activity
Rodney F. Boyer Hope College Holland, Michigan 49423
A special project in enzyme characterization Biochemistry laboratory texts are an excellent source of experiments in enzymology, most often allowing the student to either extract an enzyme and assay for its presence or t o study the kinetics and inhibition of commercially available enzymes. Few experiments offer the opportunity for the student to apply all of these techniques to the study of a single enzyme. Since an experiment of this type would require 2-3 laboratory periods, we have developed a special project around the enzyme polyphenoloxidase or tyrosiuase,' studying its extraction, activity, inhibition, cofactor reauirements. and kinetics. We have chosen this enzyme for several reasons: (1) it can be readilv extracted from various nlant sources such as mushrooms, hananas, and potatoes, (2j a convenient spectrophotometric assay of enzyme activity is available, (3) the metal cofactor allows for inhibition studies, and (4) it is of importance in food chemistry, pigmentation, and clinical medicine. The enzyme polyphenoloxidase is present in both plant and animal cells. Although its specific biological function in plants is unknown, i t has the ability to catalyze the hydroxylation of various monophenols and the aerobic oxidation of diphenols (eqns. (1) and (2)).
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Monophenol + 0 2 o-quinone + H20 (1) 2 o-diphenol 0 2 2 o-quinone + 2H20 (2) These reactions are readily observed whenever ~ l a n~t r o d u c t s such as potatoes or apples-darken upon injury. This darkening is due to the formation and further oxidative reactions of the quinones. Tyrosinase in mammalian cells appears to play a specific role in tyrosine metabolism (eqn. (3)).
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PH
Cuwf Numb4 Reaoent
Buffer ( I I I D o ~ Solution a
Enzyme Extract
1
2
3
d
2.90b 0.00 O.lOC
2.40 0.50 0.10
1.90 1.00 0.10
1.65 1.25 0.10
a ~ h final e volume in each cuvet is 3.0 ml b unit. are m ~ . " ~ n amo.nt e of e n z y m e extract t o be c.soa a.11aeoeno l o o n rne oreoarat on. E n z y m e levels fin ch g w e an aorcvoancy change o f 0.05 man w n o m m.m SJbSrraie c o n c e n t r a x ~ na r e c o n e n e n t .
Tyrosinase from both plants and animals is found to contain copper which can be complexed with various chelators therefore rendering the enzyme inactive. The use of phenylthiourea as inhibitor will be descrihed here, although many others may be used: diethyldithiocarbamate, azide, cyanide, or cvsteine. 1; this experiment a crude extract of polyphenoloxidase is prepared from potatoes ( 2 ) .Althoueh several assav methods arewailable, the most convenient I'nvolves the e&yme-catalyzed oxidation of 3,4-dihydroxyphenylalanine(dopa) to 2,3-dihydroindole-5,6-quinone. By observing the change in absorhancy a t 475 nm, the initial rate of quinone formation can he determined (3). Parts (a) and (h) of the experimental section describe enzyme preparation and the general assay procedure. The necessary details are given here so the student can follow these directions and can apply them to parts (c, and (d) where the effects of enzyme concentration and phenslthiourea are investigated, Experimental
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melanin pigments This reaction sequence is localized in melanocvtes or dament cells, where its final outcome is the produciion of melanin substances which impart thr coloration roskin, hair, and eyes ( 1 ). Irradinr~onof the skin by ultraviolet r a y s d the sun acrivates the tyrosinase, therefore causing greater melanin production and suntanning. 1 EC
Measurement of PolyphenoloxidareActivity
1.10.3.1; o-dipheno1:oxygen oxidoreductase.
Reagents, Materials, and Equipment 1) Small potato 2) Blender 3) Sodium phosphate buffer (I),0.1 M, pH 6.8, containing 0.1 M sodium fluoride 4) Sodium phosphate buffer (II),0.1 M, pH 6.8 5) DL-0-3,4-Dihydroxyphenylalanine(DL-dopa), 4 mg/ml in buffer (11). Prepare fresh each dey. Obtained from Sigma Chemical Co., St. Louis, MO. M 6 ) Phenylthiourea solution in buffer (II), 7) Recording spectrophotometer or calorimeter 8) Constant temperature bath Procedure (a) Crude Tyrosinose Extract-Peel a cooled potato and cut into small pieces. Rapidly weigh about 25 g of potatoand mix with 50 ml of ice-cold phosphate buffer (I). Grind the mixture in a blender far about I min. Filter the mixture through several layers of cheesecloth or glass wool. For further clarification of the filtrate it may be centrifuged. This crude extract should be prepared immediately before use and stored in an icebath during the entire experiment. ( b ) Determination of KM and V,.,-During the experiment the reagents, buffer(II),and dopa solution should be stored in a constant temperature bath (around 30%). The table is a typical protocol for measurement of K M and V,,. In each case buffer and dopa are mixed in a cuvet. The reaction is initiated by the addition of enzyme extract. The cuvet is mixed well and olaced in a soectro~hotometerto determine rhr absorbnnrv change at 475 nm. Ila recurding spectromcwr rr not nrn~lahle,ohsurbnner rnmaurcrnenrs cat, he taken ever" :tu I Volume 54. Number 9. September 1977 / 585
in a colorimeter. The rate, AAlmin,can he converted to velocity, v Imol/l/min~hv use of the absorhancv coefficient.. loe.. e = 3.7 (.4 .) . A hlnnk consmi& of huller 1111and du;a rhwld Ire analyed rororrecr hsr aut
