39 Polyphenol Oxidase

Edna C. Ramrez and John R. Whitaker

University of California, Davis, Davis, California, U.S.A.

Victoria M. Virador
National Institutes of Health, Bethesda, Maryland, U.S.A.

I.

INTRODUCTION (2)

Polyphenol oxidase, also known as tyrosinase, phenolase, catechol oxidase, catecholase, o-diphenol oxidase, monophenol oxidase, and cresolase, was discovered in mushrooms in 1856 by Schoenbein (1). The problem with naming the enzyme is that it may act on two general types of substrates, a monohydroxyphenol (such as p-cresol) to hydroxylate it in the o-position with respect to the original hydroxyl group (monophenol, L-dopa:oxygen oxidoreductase; EC 1.14.18.1 (2) [Eq. (1)]), and on o-dihydroxyphenols, such as catechol, oxidizing them by removal of the hydrogens of the hydroxyl groups, forming benzoquinones (1,2-benzenediol:oxygen oxidoreductase; EC 1.10.3.1 (2) [Eq. (2)]).

(1)

In this chapter, the rst enzyme activity will be referred to as a monophenol oxidase and the second

as an o-diphenol oxidase. The monophenol oxidases generally also act as o-diphenol oxidases, often at a faster rate (3). Therefore, they are sometimes classied either as monophenol oxidases, o-diphenol oxidases, or both depending on substrates used. But not all odiphenol oxidases can act as monophenol oxidases (4, 5). This view is not shared by all researchers in this eld. The benzoquinones formed by o-diphenol oxidases are very reactive nonenzymatically with O2 , sulfhydryl compounds, amines, amino acids, and proteins, so a variety of compounds, including melanin of the skin, are formed with colors including yellow, red, brown, and black. A third type of polyphenol oxidase reaction occurs with the enzyme laccase (benzenediol:oxygen oxidoreductase EC 1.10.3.2 (2) [Eq. (3)]), acting on pdihydroxy compounds, but not exclusively (Chapter 40), to give colored compounds. The laccases are also copper-containing enzymes, but the mechanistic oxidation pathway differs from that of the o-diphenol oxi-

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dases. The two types of enzymes can be distinguished by use of p-phenylene diamine or syringaldazine (these are substrates for laccase only). Salicylhydroxamic acid and cinnamic acid inhibit only o-diphenol oxidases (6), while p-diphenol oxidases are selectively inhibited by quaternary ammonium compounds such as cetyl tetra-ammonium bromide (CTAB) (7). See also Flurkey et al. (8) and Chapter 40 on laccase in this handbook. Polyphenol oxidases are found in many plant tissues (9, 10); in some fungi, especially those that produce brown laments including edible mushrooms (11); and in many animals, including insects (12), mice (13), and humans (14). There are numerous genes in humans that affect pigmentation (15, 16). According to Witkop (14), there are at least 40 clinical manifestations of hypopigmentation and 27 of hyperpigmentation in humans, some leading to cancerous skin melanoma.

II.

IMPORTANCE TO FOOD QUALITY AND FOOD PROCESSING

Polyphenol oxidases are very important enzymes in determining the quality and economics of fruit and vegetable harvesting, storage, and processing (1719). Bruises, cuts, and other mechanical damage during harvest, storage, and processing that allow O2 penetration result in rapid browning in many fruits and vegetables. Up to one-half of some tropical fruits are lost for consumer consumption owing to browning, since the off-color, off-taste, and loss of nutritional quality are unacceptable to consumers. Apricots, apples, peaches, grapes, strawberries, and bananas (among others) and several tropical fruits and juices therefrom become brown, as do Irish potatoes and some lettuces

and other leafy vegetables. Black spot development in shrimp is a major economic problem. Heat processing to inactivate polyphenol oxidase, especially in juices, is standard practice with certain fruits and vegetables. Some acceptable compounds such as ascorbic acid, thiol compounds, and sultes may be added to prevent browning of cut fruits and vegetables (11). Cinnamic, p-coumaric, and ferulic acids can be added to apple juice (sometimes at < 0:01%) to prevent browning due to polyphenol oxidase (20). 4-Hexyl-resorcinol is a safe and effective inhibitor of enzymatic browning, is used in shrimp processing (21, 22), and prevents apple slice browning (23) among other fruits and vegetables. Browning of foods can also be prevented by removal of O2 , acidication (if acceptability of the food permits), and removal of polyphenols by complexing with cyclodextrins and polyvinylpyrrolidone (2426). Polyphenol oxidase levels in fruits and vegetables can be decreased by breeding and through biotechnology. An example of the use of biotechnology is the use of a specic antisense RNA to turn off expression of polyphenoloxidase in grape tissues (27). Exclusion or decrease of O2 and separation of polyphenol oxidase and substrate (phenols) provide excellent prevention from browning. Fruits and vegetables have skins (waxes and other O2 impermeable compounds) on the surface that prevent O2 absorption. As long as this protective skin is intact and the cellular tissue is undamaged, browning does not occur. In food processing and packaging O2 can be excluded or reduced by use of gaseous N2 , use of O2 -impermeable coatings and lms, and controlled atmospheric storage (O2 reduced to the minimum needed to maintain cellular integrity at reduced temperature ($ 5 C)). Color development due to polyphenol oxidase activity is desirable in the processing of tea, coffee, cocoa,

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apple cider, prunes, black raisins, Black Mission gs, and zapote. III. A. PROPERTIES OF POLYPHENOL OXIDASES AS PROTEINS Primary Sequences

The amino acid sequence homologies among the nine polyphenol oxidases (Fig. 2) are much higher when compared only in the active-site regions A and B (28). In active-site region A, of 26 amino acids, there is a range from 100% to 31% homology (Table 2). In active-site region B of 56 amino acids, the homology ranges from 96% to 34% (Table 3). B. Molecular Weights

Several amino acid sequences of propolyphenol oxidases and polyphenol oxidases are known, primarily from sequencing the gene. Table 1 shows the similarities of nine plant PPO sequences. Another group of plant PPO sequences are depicted by a dendrogram (Fig. 1). Whitaker (28) has compared the amino acid sequence relationships among polyphenol oxidases of higher plants, fungi, and higher animals known to 1995. The homologies (relative to potato 1) among the higher plant polyphenol oxidase gene sequences from potato 1, potato 2, tomato, and broad bean were 96.6% (potato 1 and 2), 92.2% (potato 1 and tomato), and 38.1% (potato 1 and broad bean). Among the fungi S. glaucescens, S. antibioticus, and N. crassa, the homolgies were 87.5% (S. glaucescens with S. antibioticus) and 17.0% (S. glaucescens and N. crassa). Comparison between human and mouse polyphenol oxidases showed 41.0% homology. Overall, when potato 1 sequence was compared with those of S. glaucescens, S. antibioticus, N. crassa, and human and mouse polyphenol oxidases, there was 19.4%, 18.8%, 13.0%, 10.4%, and 10.9% homology, respectively. Only the primary sequences of N. crassa and S. glaucescens polyphenol oxidases were determined by the Edman degradation method on the mature protein.
Table 1 Similarity of the Aligned Plant PPO Sequences Tomato Tomato Tobacco Grenache grape Sultana grape Apple Bean Pokeweed Spinach Sugarcane 100 Tobacco 90.48 100 Grenache grape 59.52 58.01 100

The presumed mature molecular weights of the 12 polyphenol oxidases listed in Table 4 range from 30.7 kDa for S. antibioticus polyphenol oxidase to 128.0 kDa for mushroom polyphenol oxidase (with four subunits). The potato, tomato, and broad bean polyphenol oxidases have molecular weights in the range of 56.558.1 kDa while grape polyphenol oxidases have molecular weights of 40.7 kDa. Three of the fungi polyphenol oxidases have molecular weights of 30.9 46.0 kDa, while mushroom polyphenol oxidase is 128.0 kDa. The human and mouse polyphenol oxidases are 62.6 and 57.8 kDa, respectively. With the exception of the polyphenol oxidases from N. crassa, S. glaucescens, and edible mushroom (Agaricus bisporus) (29, 30), the molecular weights are based on the gene sequence translated to amino acid sequence. Nothing is known about posttranslational modications. C. Secondary, Tertiary, and Quaternary Structure X-ray crystallography data have been published only for the sweet potato polyphenol oxidase (31, 32). There are two isozymes of polyphenol oxidase in sweet potatoes of molecular masses 39.0 and 40.0

Sultana grape 60.03 58.52 99.18 100

Apple 56.97 56.49 67.17 67.34 100

Bean 56.8 56.83 64.42 64.42 69.7 100

Pokeweed 55.2 53.83 64.22 64.4 63.03 62.69 100

Spinach 53.74 51.43 57.83 57.73 54.88 56.84 57.58 100

Sugarcane 43.2 43.68 43.16 43.09 45.29 43 43.44 40.42 100

The GCG program OldDistances was used to calculate percent similarity among the plant PPOs from a complete PileUp alignment; part of this alignment is depicted in Figure 1. Genbank accession numbers are as follows: tomato, Z12837 S61013; tobacco, Y12501; Grenache grape, U83274; Sultana grape, Z27411; apple, D87670; bean, Z11702 S45506; pokeweed, D45385; spinach, X90869; sugarcane, U46014.

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protein. There are two disulde bridges (Cys11Cys28 and Cys27Cys89). Each of the two active-site coppers is coordinated to three histidine residues on -helices. Copper of site A is coordinated to His88 of helix 2 and His109 and His118 of helix 3. The copper of site B is coordinated to His240 and His244 of helix 6 and His274 of helix 7. As noted above, mushroom tyrosinase is thought to have quaternary structure (29, 30), probably composed of four subunits. The similarity of the subunits is uncertain. Based on gene sequences, there appear to be at least two isozymes of tyrosinase in mushrooms (33, 34).

IV.

ENZYMATIC PROPERTIES OF POLYPHENOL OXIDASES

Figure 1 Dendrogram illustrating the degree of similarity between the protein sequences of reported plant PPO genes. The Genbank accession numbers are apple (L29450), bean (Z11702), grape (Z27411), potato-1 (M95196), potato-2 (U22921), tomato-a (Z12833), tomato-b (Z12834), tomato-c (Z12834), tomato-d (Z12836), tomato-e (Z12837), tomato-f (Z12838), pokeweed (D34385), spinach (Z6655), and sugarcane (U846014). Sequences were aligned using the GCG programs PILEUP and FIGURE.

kDa (determined by MALDI-MS) (20). The sweet potato polyphenol oxidase molecular weight of 39.0 kDa has 345 amino acid residues, is a monomeric, ellipsoid molecule with dimensions of 55 45 45A. The secondary structure is primarily -helical in nature, with seven -helices and probably four short sheets with - and - turns and random coils in the structure (Fig. 3). It appears to be an   globular

Some polyphenol oxidases oxidize both monophenols such as p-cresol, tyrosine etc. (monophenols), and diphenols such as catechol and o-dihydroxyphenylalanine [Eqs. (1) and (2)]. Lerch and Ettlinger (3) investigated the activity of pure Streptomyces glaucescans polyphenol oxidase (tyrosinase) on a large number of mono- and o-diphenols. In all cases, the o-diphenolase activity was greater than the monophenolase activity. The ratio of kcat for activity on o-diphenol to that of kcat for activity on the analogous monophenol ranged from 222 for homocatechol/p-cresol to 2.89 for 3,4-dihydroxyphenylacetic acid/p-hydroxyphenylacetic acid. The Km values for the analogous o-diphenol to monophenol ranged from 1.00 to 7.88. Therefore, Km and Kcat are both responsible for the large differences in activity on mono- and o-diphenols. Table 5 shows some activity results of plant polyphenol oxidases on a limited number of mono- and odiphenols. Polyphenol oxidase from peaches was not able to hydroxylate p-cresol or p-coumaric acid, and polyphenol oxidase from pears was not able to hydroxylate p-coumaric acid. The highest activities on monophenols were the 5.5% and 4% on p-cresol compared to that of catechol for potato and broad bean, respectively. Note also the variable relative activities on odiphenols compared to that on catechol. There is some disagreement by researchers on whether or not all polyphenol oxidases can perform the hydroxylation step [Eq. (1)]. As shown in Eq. (1) a reducing compound, BH2 , is required in the reaction. BH2 is an o-diphenol such as catechol. If no BH2 is present, some polyphenol oxidases appear to be able to slowly produce BH2 during a lag period in the initial reaction with a monophenol (Fig. 4). The in vivo nat-

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Figure 2 Amino acid sequence relationships in and near the active sites of potato (Pot 1 and Pot 2), tomato (Tom A and Tom B), broad bean (BB), S. glaucescens (S.g.), S. antibioticus (S.a.), Neurospora crass (N.c.), Homo sapiens (H.s.), Mus musculus (M.m.), putative mouse transcript 4 (pMT 4) polyphenol oxidases, hemocyanin E (spider, Eurypelma californicum) and hemocyanin D (spider, E. californicum). Shown are regions A and B containing the presumed copper-binding histidine residues. The Cu indicates histidines that ligand to copper in N. crassa polyphenol oxidase, and presumably in the other polyphenol oxidases and hemocyanins. The - indicates that the sequence is the same as for POT1. (From Ref. 28.)

ure of BH2 is not known. Lerch and Ettlinger (3) reported lag periods of 0.3216.0 min for S. glaucescens polyphenol oxidase acting on N-acetyl-L-tyrosine hydrazide and p-hydroxyphenylacetic acid, respectively. Whitaker (unpublished data) showed that 1 107 M 4-methylcatechol added to mushroom polyphenol oxidase, along with p-cresol, was able to eliminate the 9-min lag period entirely. Equation (4) shows that the BH2 acts on the Cu(II) met form to reduce the two coppers to Cu(I) (deoxy form), which can then bind O2 and oxidize the monophenol substrate to the o-diphenol. Would similar experiments with other polyphenol oxidases show that all polyphenol oxidases can do both types of reactions?

Kinetically, the mechanism of peach polyphenol oxidase (5) and S. glaucescens polyphenol oxidase (3) followed an ordered BiBi mechanism (Fig. 5). The O2 must bind rst to the deoxy form (Cu(I) state) of the enzyme followed by phenol. The ordered BiBi mechanism assumes that the products come off in the order of the benzoquinone followed by the hydrogen peroxide. This author does not know of published experimental data to support this assumption. The overall proposed mechanism of action of polyphenol oxidase is shown in Figure 6. The top part shows the pathway for oxidation of o-diphenols. The met form (at the No. 1 position in the A cycle) is thought to be the resting form of the enzyme when

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Table 2 Amino Acid Sequence Homologies Among Nine Polyphenol Oxidases in Active Site Region A Source Pot 1 Pot 2 Tom B BB S.g. S.a. N.c. H.s. M.m.
a

Table 4 Mature Protein Molecular Weights of Some Polyphenol Oxidases Source Pot 1 Pot 2 Tom BB GPO1 GPO2 S.g. S.a. N.c. Mushroom H.s. M.m.
a

AA sequence region 110135 110135 110135 110135 5476 5477 96121 190215 191216

Identical AA 26a 26 26 25 13 11 11 8 8

% Homology 100 100 100 96 50 42 42 31 31

MW (kDa) 56.5 56.6 56.5 58.1 40.7 40.7 (29.0)a 30.9 30.7 46.0 128.0 62.6 57.9

The number 26 amino acid residues includes amino acid residues spaces for alignment. (Taken from Figure 2.)

no substrate is present. Addition of o-diphenol (catechol; BH2 ) binds to the met Cu(II) form (#2) producing the deoxy [Cu(I)] form of the enzyme (and obenzoquinone). The deoxy form binds O2 (#3) to give the oxy form [Cu(II)] which then binds a molecule of odiphenol (#4) to give the enzyme CuII O2 diphenol ternary complex. Two hydrogens are removed to give the benzoquinone (#5) and the met form of the enzyme in a complete cycle. Depending on substrate available, the oxy form of the enzyme (produced as above from the met form) can bind a monophenol (Pathway B) (#1 0 ) which is oxidized to the o-diphenol (#20 ) and recycles through the B pathway via the diphenol intermediate to the deoxy form (#40 ) which can bind O2 to form the oxy form (#50 ), etc. The met form, reduced to the deoxy form by an o-diphenol (Fig. 6), can also be reduced to the deoxy form by other reducing compounds (ascorbic acid,

There are two peaks of polyphenol oxidase activity from a Sephadex G-100 column (40.7 and 29.0 MW). The 40.7-kDa peak has the most activity. Source: Whitaker, 1998, unpublished data.

Table 3 Amino Acid Sequence Homologies Among Nine Polyphenol Oxidases in Active Site Region B Source Pot 1 Pot 2 Tom B BB S.g. S.a. N.c. H.s. M.m.
a

AA sequence region 241296 241296 241296 241295 189229 189229 277320 350390 352393

Identical No. 56a 54 54 35 20 19 20 19 21

% Homology 100 96 96 62 36 34 36 34 38

The No. 56 includes amino acid residues spaces for alignment.

hydroxylamine, dithionite) and by H2 O2 in the presence of O2 (39). The overall mechanism indicates there are three different steps in Figure 6 leading to oxidation of o-diphenols to benzoquinone. Polyphenol oxidases are irreversibly inactivated during the oxidation of substrate to product. GolanGoldhirsh and Whitaker (40) calculated that inactivation of mushroom polyphenol oxidase occurs at the rate of approximately one in 5000 turnovers of the substrate to product (an efciency of $ 0:02%). But complete inactivation of the enzyme can occur in 23 min reaction with substrate (with no added reductant such as ascorbic acid). The inactivation is due to a free radical-catalyzed fragmentation of one or more of the six histidine residues that bind the two coppers at the active site. The fragmentation leads to loss of histidine and release of copper (4042). Reaction inactivation of N. crassa polyphenol oxidase is due to loss of His306 in the active site (42). Golan-Goldhirsh et al. (43) later showed that ascorbic acid, copper, and O2 mimic the above reaction not only with polyphenol oxidase but also with ovalbumin, Kunitz trypsin inhibitor, bovine serum albumin, and small histidine-containing peptides, leading to loss of most of the histidine residues in 24 h at 25 C. In the case of mushroom polyphenol oxidase, the histidine residues of the protein are converted by the free radical process in the absence of mono- or diphenols, but require O2 , to several products including aspartic acid (major product), glycine, alanine, and urea (stoichiometric with the aspartic acid formation). Free radi-

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Figure 3 Ribbon drawing of sweet potato catechol oxidase, showing the front view of 39.0-kDa single polypeptide enzyme. (From Ref. 32.)

Table 5 Relative Substrate Specicities of Four Polyphenol Oxidases Activity relative to catechol Substrate Di- or triphenolic compounds Catechol 4-Methylcatechol d-Catechin Chlorogenic acid Caffeic acid Protocatechuic acid 3,4-Dihydroxy-L-phenylalanine Dopamine Gallic acid Pyrogallol Monophenolic compounds p-Cresol p-Coumaric acid
a b c d

Potatoa 100

Peachb 100 51.5 31.8 22.2 0 16.3 40.5 45.6 25.7

Broad bean leafc 100 200225 8 12.5 0.11 50 0.22 8595 4 0.05

Peard 100 72.3 7.79 71.8 4.41

140 76.5 54.3

15.6

5.5 nil

0 0

Ref. 35; pH 7.0. Ref. 4. For isozyme A of clingstone peach at pH 6.8 and 30 C. Ref. 36. Ref. 5; 35 C and pH 6.2; isozyme B.

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Figure 4 Theoretical graph for the velocity of product formation from oxidation of monophenols by polyphenol oxidases in the absence of BH2 . There is an initial lag period followed by maximum velocity and then slowing of the velocity as the enzyme undergoes reaction inactivation. The negative velocity phase is due to melanin formation, which absorbs at a different wavelength.

cals due to semiquinone formation of the substrate can be detected during polyphenol oxidasecatalyzed reactions (44, 45; Sugumara et al., cited in 46) in insect cuticle formation by polyphenol oxidase. Free radical formation (11) requires O2 , a reducing agent (we used ascorbic acid only), a histidine residue (histidine in small peptides are also fragmented), and Cu(II) (we could not detect Cu(I) formation by bathocuproine disulfonate in the reaction). The specic free radical formed has not been identied yet, to the authors knowledge.

Figure 5 (Top) Effect of O2 and chlorogenic acid concentrations on initial velocity, v0 , of pear polyphenol oxidase-Bcatalyzed reactions. The reactions were followed with an oxygen electrode at pH 4.0 and 30:0 C, permitting initial v0 to be determined. (Bottom) Kinetic mechanism for pear polyphenol oxidase-B catalysis, diagrammed according to the Cleland nomenclature for an Ordered Sequential Bi-Bi Mechanism. P is an o-diphenol; B is an o-benzoquinone; E O2 is a binary enzyme O2 complex; E O2 P is a ternary enzyme O2 o-diphenol complex, and E H2 O B is a ternary enzyme H2 Oo-benzoquinone complex. (From Ref. 5.)

V.

MEASUREMENT OF POLYPHENOL OXIDASE ACTIVITY

(4)

The two main methods of measuring polyphenol oxidase activity are spectrophotometry and polarography. There are strong advocates of each method, including statements that both methods are unreliable (47, 48). The basis of the unreliability is caused by the further nonenzymatic oxidation of the benzoquinone formed. As shown by Mayer et al. (47), results from the two are identical very early on in the reaction (Table 6), but by 54 sec the difference is 28%. Note that O2 consumption measurements contribute primarily to the difference because of the continued nonenzymatic oxidation of benzoquinone and other intermediate products. For example, the oxidation of 4-methyl catechol to 4methyl-2,3-benzoquinone requires 1.0 equivalent of O2 , while further reactions (generally nonenzymatic) require 1.4 additional equivalents of O2 . But, as shown by Whitaker (28), there is also a decrease in

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Figure 6 Proposed kinetic scheme depicting the mechanisms of oxidation of o-diphenol (catechol [A]) and monophenol (phenol [B]) by Neurospora crassa polyphenol oxidase. (From Refs. 37, 38.)

absorbance after $ 2 min as the benzoquinone is converted to other products that do not absorb maximally at the wavelength of benzoquinones. Therefore, it is critical to obtain initial velocity (v0 ) early on in the reaction. As shown by Eqs. (1) and (2), the oxidation of monophenols to benzoquinone plus H2 O requires two equivalents of oxygen, while oxidation of o-diphenols to benzoquinone requires one equivalent of oxygen. In reactions involving monophenols, a lag phase is generally observed (see Fig. 4). As shown in Eq. (1) and Figure 6, BH2 is required to reduce the met form of polyphenol oxidase to the deoxy form that can bind O2 . Addition of as little as 1 107 M catechol to the reaction can eliminate the lag period.

In the measurement of monophenol activities, the rate of conversion of the intermediate product (an odiphenol) to the benzoquinone is generally measured. But in some cases, kcat values for the monophenol and product o-diphenol can be very similar (3). Examples are shown in Table 7. The monophenol oxidation can be measured uniquely by use of 18 O-labeled O2 or tritium-labeled substrate. A. 1. Measurement of Monophenolase Activity Use of Tritium-Labeled Substrate

As shown in Eq. (1), one atom of oxygen (from O2 ) is covalently attached to the 2 position of p-cresol, along

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Table 6 Comparison of the Spectrophotometric and Polarographic (O2 electrode) Methods of Measuring Apple Catechol Oxidase Using 4-Methyl Catechol as Substrate Time (sec) 18 54 93.6 198 480
a

(1)a Quinone Formed (mol) 0.256 0.512 0.768 1.024 1.408

(2)b O2 consumption (mol) 0.261 0.653 1.133 1.568 2.464

Ratioc (2)/(1) 1.02 1.28 1.48 1.53 1.75

label. The unincorporated 18 O2 can be ushed from the solution by N2 , or the water can be distilled from the solids, and the water collected to measure the increase in 18 O level of the water, or the 4-methyl catechol, in the presence of a reducing agent such as ascorbic acid, can be obtained. The increase in 18 O in the product will give a measure of the velocity of the monophenol oxidase reaction. 3. Spectrophometric Method at 280 nm

Measured spectrophotometrically. b Measured polarographically. c o-Benzoquinone formation from 4-methylcatechol consumes 1 gram-atom of O2 per mole of catechol, whereas conversion of 4methylcatechol to melanin consumes 2.4 gram-atoms O2 per mole of catechol oxidized. Source: Ref. 47.

An easier method than those in 1 and 2 above is to measure the increase in absorbance at 280 nm due to the formation of 4-methyl catechol from p-cresol. Again, the measurements must be made within the rst 3090 sec of beginning of the reaction. 4. Use of Diphenol Conversion Rates to o-Benzoquinone

with a H to give 4-methyl catechol. By the use of the 2-tritiated p-cresol, tritium will be released into the aqueous phase. During the initial phase of the reaction (between 0 and 60 sec), the rate of release of tritium should give a good measure of the initial velocity of the hydroxylation step. The remaining titriated p-cresol must be removed by chromatography or by an adsorption method. 2. Use of
18

O-Labeled O2

As shown by Eq. (1), one atom of oxygen (from 18 O2 ) will be covalently attached to the 2 position of p-cresol, along with a H to give 4-methyl catechol with an 18 O

Km values for monophenols are in the range of 4:5 105 M (N-chloroacetyl-L-tyrosine ester ester) to 5:34 103 M (glycyl-L-tyrosine) for the S. glaucescens polyphenol oxidase (3) and 2:62 104 M and 4:62 104 M for L-tyrosine methyl ester for the - and isozymes of mushroom polyphenol oxidase (30). When the Km and kcat values of the monophenol and o-diphenol substrates are similar, the observed rates of the reactions of benzoquinone formation will be contributed by both rate constants (v0 k2 E0 S0 = k2 k3 =k2 k3 S0 . See discussion below for methodology to use.

Table 7 Ratios of kcat =Km and kcat =kcat , in Parentheses, for Some Monophenol/o-Diphenol Substrate Pairs Substrate pairs Monophenol L-Tyrosine p-Cresol p-Hydroxyphenylpropionic acid p-Hydroxyphenylacetic acid L-Tyrosine methyl ester L-Tyrosine methyl ester ()c L-Tyrosine methyl ester ( )c
a b

Diphenol 3,4-Dihydroxy-L-phenylalanine Homocatechol 3,4-Dihydroxyphenylpropionic acid 3,4-Dihydroxyphenylacetic acid 3,4-Dihydroxy-L-phenylalanine 3,4-Dihydroxyl-L-phenylalanine ()a 3,4-Dihydroxy-L-phenylalanine ( )a

(1)a 7.81 25.7 6.31 2.89 1.77 3.44 3.41

(2)b (110) (222) (13.6) (2.89) (6.17) (2.62) (5.20)

(kcat =Km ) diphenol/(kcat =Km ) monophenol. kcat , diphenol/kcat , monophenol. c Based on kinetic constants for - and -isozymes of mushroom polyphenol oxidase, using a molecular weight of 32,400. Source: Refs. 2830.

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B.

Measurement of o-Diphenol Oxidase Activity

Reports of Km values for S. glaucescens enzyme activity on o-diphenols range from 3:72 104 M for caffeic acid to 1:50 102 M for 3,4-dihydroxy-D-phenylalanine (3). For mushroom isozymes  and , Km values are 3:75 104 M and 4:00 104 M, respectively (30). Other reports of Km include those of Wong et al. (4) where Km values for catechol and peach polyphenol oxidase were 6.6, 4.2, 7.0, and 36.0 mM for isozymes A, B, C, and D, respectively, and of Rivas and Whitaker (5) with pear isozymes A and B, where Kms were 20.9 and 33.9 mM for pyrocatechol, 8.0 and 5.8 mM for 4-methyl catechol, 16.1 and 11.9 mM for chlorogenic acid, and 3.1 and 1.6 mM for dcatechin, respectively. These Km values permit one to use an appropriate concentration of substrate for measuring activities. 1. Spectrophotometric Method

In the authors laboratory, o-diphenol oxidase activity is generally determined using catechol or 4-methyl catechol (prepared fresh daily) by mixing 4.00 mL of 0.1 M sodium phosphate buffer, pH 7.0, 0.50 mL of 5:00 102 M 4-methylcatechol (nal concentration 5:00 103 M) and adding 0.50 mL of polyphenol oxidase of a concentration that gives a change in absorbance of $ 0:100/min. The wavelength used is 395 nm; the m for 4-methylbenzo-2,3-quinone is 1:350 103 M1 cm1 at 395 nm. Absorbance should be measured with a recording spectrophotometer so that v0 can be determined precisely at the very early stage of the reaction for reasons discussed above. The temperature and pH should also be controlled. 2. Polarographic Method

Figure 7 Relationship between enzyme concentration and L O2 consumed by oxidation of 4-methyl catechol by apple catechol oxidase, using an O2 electrode. The reaction was performed in phosphate-citrate buffer, pH 5.1 with 5 103 M 4-methyl catechol and stock solution of 50 g enzyme protein/mL. (From Ref. 47.)

The polarographic method, also called the O2 -electrode method, determines the rate of O2 uptake. As shown in Figures 7 and 8 and Table 6, the O2 electrode and spectrophotometric methods give linear relationships over a wide range of enzyme concentrations. The chronometric method measures the lag time before browning occurs or ascorbic acid is exhausted, and the manometric method (O2 uptake) does not give linear relationships between enzyme concentration and O2 uptake. The concentration of enzyme, substrate, buffers, and pH used may be the same as described for the spectrophotometric method (above). Specic conditions are also given in the legends of Figures 7 and 8 (47).

Figure 8 Relationship between enzyme concentration and L O2 consumed by oxidation of 4-methyl catechol by apple catechol oxidase using spectrophotometric, chronometric, and Warburg techniques. The reaction was performed under the experimental conditions given in Figure 7. (From Ref. 47.)

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VI.

PURIFICATION OF POLYPHENOL OXIDASES

Purication of polyphenol oxidase must be done without any development of browning. Otherwise, the polyphenol oxidase will be modied by reaction of the -amino group of lysyl residues with the o-benzoquinone. Modication leads articially to several bands with activity as shown by gel electrophoresis or by column chromatography (E. Ram rez and J.R. Whitaker, unpublished data). Several methods have been used by the authors to prevent browning. The tissue was frozen in dry ice or Freon (4, 5), lyophilized, and the powder extracted repeatedly with cold (5 to 10 C) acetone to remove the phenolic compounds. This method of removing phenols with acetone, while used widely in the 1950s to 1970s, has been shown to modify proteins under certain conditions. We have found Freon to be a good method for rapidly freezing the tissue (5). More recently, we have used 0.03 M ascorbic acid and insoluble polyvinyl polypyrrolidone with frozen whole grapes to prevent browning by binding the phenols. This gave an extract that did not brown. We have

also successfully used ascorbic acid and Sephadex G-25 to bind the phenols. This is done at 0 C. The following method (E. Ram rez and J.R. Whitaker, unpublished data) was used to purify grape polyphenol oxidase to homogeneity for crystallization for x-ray structure determinations. Acceptable extraction yields were obtained only when a maximum of 250 g of grapes per batch were used as starting material. Attempts to extract PPO from larger batches were unsuccessful, owing to insufcient mechanical agitation during the second extraction step because of high viscosity of the solution. All steps were done at 4 C. In a typical extraction, 100200 g of frozen whole Grenache grapes were blended in a Waring blender for 5 min at low speed with 2 volumes (w/v) of buffer A (100 mM sodium phosphate, 0.03 M ascorbic acid, pH 6.5) and 10% PVPP. Pulp was separated by centrifugation (23,000 g, 30 min) and the supernatant discarded. It contained no PPO activity. The precipitate was transferred to a beaker with 1 volume of buffer B (100 mM sodium phosphate, 0.1% Triton X-100, pH 6.5) and 2.5% PVPP. This suspension was mechanically stirred for 30 min. After centrifugation (23,000 g, 30 min) the precipitate was discarded and

Figure 9 Mono Q [R-CH2 -N CH3 3 HR 5/5 column chromatography of grape polyphenol oxidase. Protein was eluted initially with a sodium chloride linear gradient (00.3 M), held for 2 mL at 0.3 M sodium chloride, and then stepped up to 1 M (dashed line). Activity of polyphenol oxidase is indicated by closed circles (and shaded area). Protein is indicated by open squares. The small peak of activity at Fraction No. 30 is that designated as Mono Q II in the text.

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Table 8 Purication of Grenache Grape Leaf Polyphenol Oxidase Activity (units/mL) 103 3.64 13.8 90.2 164 33.3 Protein (g/mL) 53.1 26.9 272 260 105 Spec. activity (units/mg) 103 68.5 513 332 631 317 Volume (mL) 2267 480 22 3 14 Total protein (mg) 120 12.9 5.98 0.78 1.47 Total activity (units 103 ) 8250 6620 1980 492 466 Yield (%) 100 80.0 24.0 6.75 5.64 Purication (fold) 1.00 7.47 4.82 9.19 4.63

Sample Extraction 40% AS (sum) 95% AS (sum) Mono QI Mono QII

the supernatant was concentrated by ultraltration (Amicon microconcentrator, Diao PM10 membrane). The concentrated solution was brought to 40% saturation with ammonium sulfate, let stand for 30 min, centrifuged, and the precipitate discarded. The supernatant was further brought to 95% saturation with ammonium sulfate, let stand for 30 min, centrifuged, and the supernatant separated. The supernatant still contained 50% of total activity; owing to very low protein concentration, some PPO remained in solution. The precipitate was stored at 4 C (or frozen) until all extractions were done. The precipitates from all extractions were combined, dissolved in a minimal amount of buffer (5 mM sodium phosphate buffer, pH 8.0), and dialyzed with 200 volumes of the same buffer. The sample was applied to a Mono Q [(R-CH2 -N CH3 3 HR 5/5 column linked to an FPLC system and the polyphenol oxidase was eluted with 0.3 M NaCl in the 5 mM sodium phosphate buffer, pH 8.0, as shown in Figure 9 (Mono Q I peak; at Fraction No. 26). A second, smaller peak (Mono Q II) with polyphenol oxidase activity and brown color was eluted with 1 M NaCl at Fraction No. 31. Native PAGE of Mono Q I peak showed only one protein band (Coomassie Blue R-250 stain) and one concomitant activity band (stained with 8 mM catechol, 0.06% 3-methyl-2-benzothiazolinone hydrazone [MBTH]). Native PAGE of the Mono Q II peak gave ve protein bands, all of them active. Most likely, these are modied PPO owing to reaction with PPO-produced benzoquinones. The purication of Grenache PPO is summarized in Table 8. We used the rst peak (Mono Q I) for crystallization, which corresponded to unmodied polyphenol oxidase as indicated by no brown color. Protein was determined using the 1976 Bradford assay (48) or by following the absorbance at 214 nm for column chromatography. Activity was determined by a modication of the colorimetric method described by Ponting and Joslyn (49) using 5 mM catechol as

substrate and measuring the initial change in absorbance at 420 mM (pH 6.5, 25 C). One unit of activity is dened as an increase in absorbance of 0.001/min. The Mr of the mature PPO was determined on gel ltration columns. Mr was 41:2 2 kDa (replications 4) on Sephadex G-75 (ne), 34.6 kDa on Superose 12 column (50), 28.0 kDa on TSKgel G3000 SW, and 22.9 kDa on TSKgel 2000 SW. Superose 12 columns are known to give Mr values that are too low (51). Note that the Mr values on TSK gels are even lower than those on Superose 12. All standard proteins were the same (bovine serum albumin, ovalbumin, -lactoglobulin [dimer], chymotrypsinogen, and ribonuclease A). Based on our use of Sephadex columns for Mr determination for 37 years, we suggest the correct Mr is near 41.2 kDa. REFERENCES
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