Polyphenol Oxidase Activity and Enzymatic Browning in Mushrooms

Chapter 4

Polyphenol Oxidase Activity and Enzymatic Browning in Mushrooms William H. Flurkey and Janis Ingebrigtsen

Downloaded by UCSF LIB CKM RSCS MGMT on November 26, 2014 | http://pubs.acs.org Publication Date: September 7, 1989 | doi: 10.1021/bk-1989-0405.ch004

Chemistry Department, Indiana State University, Terre Haute, IN 47809

Tyrosinase activities and isoenzyme forms were examined in developing mushrooms. In extracts prepared in the absence of phenolic adsorbents, no trend was apparent in catechol oxidase activity, but dopa oxidase and tyrosine hydroxylase activity decreased from small pins to the mature mushroom. Latent enzyme activity, detected by including SDS in the assay, was associated with dopa oxidase activity more than catechol oxidase activity or tyrosine hydroxylase activity. The decrease in dopa oxidase and tyrosine hydroxylase was observed in two different strains of mushrooms irrespective of whether fresh, frozen, or freeze dried mushrooms were used as samples. Dopa oxidase activity also decreased during development in samples from each break harvested from a single compost innoculum. Concomitant with the decrease in dopa oxidase activity was an increase in latent enzyme activity. Histochemical staining of mushroom tissues blotted into nitrocellulose showed that the enzyme was localized in the skin, cap flesh, g i l l s , and stalk regions of developing mushrooms. These results suggest that inactive, active, and latent enzyme forms exist at several stages of development and that potential browning problems in mushrooms may be more complicated than once believed.

Polyphenoloxidases have been the subject of several recent reviews (1-8). The enzyme i s ubiquitous and found in a variety of vertebrate, invertebrate, plant, and fungal organisms. Numerous 0097-6156/89/0405-0044$06.00/0 © 1989 American Chemical Society

In Quality Factors of Fruits and Vegetables; Jen, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


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Polyphenol Oxidase Activity

45

reports have s p e c i f i c a l l y examined the mushroom enzyme, tyrosinase, with regard t o i t s enzymatic, physical and molecular c h a r a c t e r i s t i c s (1,4-7). In recent years, many investigations have used the crude enzyme obtained from commercial sources rather than enzyme obtained d i r e c t l y from mushroom t i s s u e s . Tyrosinases catalyze the hydroxylation of monophenols, such as tyrosine, t o ortho-diphenols and the conversion of ortho-diphenols to ortho-diquinones. The mushroom enzyme can u t i l i z e either tyrosine, catechol, or dopa as substrates and therefore e x h i b i t s tyrosine hydroxylase, catechol oxidase, or dopa oxidase a c t i v i t y respectively. Boekelheide e t a l . (10-11) and others (12-13) have proposed that mushroom tyrosinase catalyzes the conversion of gamma L-glutamyl-4-hydroxybenzene t o the corresponding dihydroxyl and 3,4-dibenzoquinone d e r i v a t i v e s during spore formation. More recently, Sugamaran (14) reported that commercial mushroom tyrosinase preparations catalyzed the unusual oxidative decarboxylation of 3,4-dihydroxymandelate t o 3,4dihydroxybenzaldehyde through a quinone methide intermediate. Sugamaran e t a l . (15) have also reported that mushroom tyrosinase can catalyze the oxidative dimerization of 1,2-dehydro-Nacetyldopamine t o a benzodioxane d e r i v a t i v e . While the i n vivo substrates of mushroom tyrosinase have not been i d e n t i f i e d , N-( -Lglutamyl)-4-hydroxyaniline, tyrosine and dihydroxyphenylalanine (dopa) are a v a i l a b l e as substrate for browning reactions (16). Many physical c h a r a c t e r i s t i c s have been determined f o r the mushroom tyrosinases. These include d i f f u s i o n c o e f f i c i e n t s , sedimentation c o e f f i c i e n t s , f r i c t i o n a l r a t i o s , Stoke s r a d i i , i s o e l e c t r i c points, and apparent molecular weights (17-20). Recent evidence suggests that tyrosinase i s composed of two heavy chain components (H) of approximately 43-45 kd each and two l i g h t chain components (L) of approximately 13 kd each (18-20) . The heavy chains contain the c a t a l y t i c s i t e s . Podila and Flurkey (21) have suggested that the heavy chains were synthesized as smaller molecular weight precursors t o the native enzyme. D i f f e r e n t isoenzyme forms of tyrosinase apparently contain d i f f e r e n t H chains (7,17). No r o l e f o r the L chain subunits have been reported. In s p i t e of the wealth of information a v a i l a b l e on mushroom tyrosinase with regard t o i t s enzymatic and physical c h a r a c t e r i s t i c s , l i t t l e i s known about the enzyme i n developing mushrooms. Yamaguchi e t a l . (22) reported that only a small portion of the t o t a l enzyme a c t i v i t y (latent + active) was i n the active form during development from the t i g h t button stage t o the f l a t p i l e u s stage. Some of t h i s enzyme existed i n an inactive or latent state and could be released from latency by exposing the enzyme t o sodium dodecylsulfate (SDS). This latent a c t i v i t y i s c h a r a c t e r i s t i c of many polyphenoloxidases (1-7). Yamaguchi e t a l . (22) also reported that the latent tyrosinase a c t i v i t y i n the cap f l e s h and stalk increased with development. In contrast, Burton (23) examined the tyrosinase i n pre-harvest developing mushrooms and found that skin t i s s u e had more non-latent or a c t i v e tyrosinase than f l e s h t i s s u e . The tyrosinase i n the skin was activated more 1


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QUALITY FACTORS OF FRUITS AND

VEGETABLES

by t r y p s i n treatment than by SDS but the tyrosinase i n the f l e s h t i s s u e was activated by SDS to greater extents. Burton also reported that nonlatent and active enzyme i n the f l e s h showed no consistent trendsin a c t i v i t y with mushroom development. On the other hand, f u l l y activated tyrosinase i n the skin declined during development while nonlatent tyrosinase a c t i v i t y showed l i t t l e change. E a r l i e r , Burton (24) showed that the skin t i s s u e contained three times more tyrosinase than the f l e s h t i s s u e when compared on a fresh weight b a s i s . Both Yamaguchi et a l . (22) and Burton (23-24) u t i l i z e d substrates which could be acted upon by other oxidative enzymes and thus may not be a true r e f l e c t i o n of tyrosinase a c t i v i t y or l e v e l s of tyrosinase present i n the samples.


r

Materials and Methods Commercial mushroom tyrosinase was obtained from Sigma Chemical Company (St. L o u i s MO). Fresh, frozen, or freeze d r i e d mushrooms (Agaricus bisporus) were supplied by the Campbell I n s t i t u t e for Research and Technology (Napoleon, OH). Mushroom tyrosinase was extracted as described by Ingebrigtsen and Flurkey ( J ^ Food S c i . , i n press). Tyrosinase a c t i v i t y was monitored using e i t h e r catechol, dopa or tyrosine as the substrates. A l l assays were c a r r i e d out i n the presence and absence of 0.1% SDS (w/v) to detect active and latent enzyme a c t i v i t i e s . The catechol oxidase a c t i v i t y of tyrosinase was assayed i n 50 mM phosphate (pH 6.0) containing 10 mM catechol and the absorbance monitored at 410 nm (25-26) . The dopa oxidase a c t i v i t y of tyrosinase was assayed i n 50 mM phosphate (pH 6.0) containing 5 mM L-dopa and the absorbance monitored at 475 nm. The tyrosine hydroxylase a c t i v i t y of tyrosinase was assayed i n 33 mM phosphate (pH 6.0) containing 0.33 mM L-tyrosine and the absorbance monitored at 280 nm. Protein content was determined by the method of Lowry et a l . (26). Native and p a r t i a l l y denaturing electrophoresis was c a r r i e d out using the method described by Angleton and Flurkey (27). Denaturing electrophoresis was followed according to the method of Laemmli (28). Equivalent amounts of protein were used for each sample a p p l i c a t i o n . Gels were stained for dopa oxidase a c t i v i t y i n 0.1 M phosphate (pH 6.0) containing 1 mM L-dopa (25,27). Western b l o t t i n g procedures were conducted as described previously using anti-tyrosinase antibodies and goat a n t i - r a b b i t conjugated a l k a l i n e phosphatase antibodies (26). Tyrosinase i n whole t i s s u e sections was l o c a l i z e d by pressing t i s s u e sections onto n i t r o c e l l u l o s e membranes using a procedure s i m i l a r to that described by Spruce et a l . (29). Histochemical l o c a l i z a t i o n was determined by incubating b l o t s i n dopa as described by Moore et a l . (30,31). Immunochemical l o c a l i z a t i o n of tyrosinase at the whole t i s s u e l e v e l was c a r r i e d out using the method of Moore et a l . (30). f


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FLURKEY &INGEBRIGTSEN


47


Results and Discussion Investigators have described novel reactions (14,15), prepared antibodies (32), or described unusual a c t i v i t i e s associated with the enzyme (33) using commercial mushroom tyrosinase. Much of the recent information on mushroom tyrosinase has r e l i e d e x c l u s i v e l y upon the use of commercial enzyme. This i s somewhat of a problem considering less than 10% of the material i n commercial preparations has been estimated to be the enzyme (7)· No estimates have been made concerning the amount of i n a c t i v e (denatured) or latent enzyme present i n the same samples. This problem i s further compounded because commercial enzymes may be derived from more than a s i n g l e Agaricus s t r a i n (M. Wach, personal communication). For example, F i g . 1 shows the SDS PAGE pattern of two l o t s of commercial mushroom tyrosinase preparations. Lot A was derived from a s i n g l e s t r a i n of Agaricus while l o t Β was derived from a mixture of two s t r a i n s . I t i s r e a d i l y apparent that (1) both l o t s contain prominent protein bands at 43-45, 24-26, and 13-14 kd, (2) many other proteins are present i n each sample, and (3) l o t A and Β d i f f e r s i g n i f i c a n t l y i n t h e i r protein patterns at the 40-48 kd region. Presumably, the proteins i n t h i s 40-48 kd region correspond to the c a t a l y t i c heavy chain subunits of tyrosinases. While the e f f e c t s of d i f f e r e n t H subunit d i s t r i b u t i o n on the enzymatic c h a r a c t e r i s t i c s have not been examined thoroughly, mixtures of d i f f e r e n t H chains would give r i s e t o d i f f e r e n t isoenzymes and would necessitate réévaluation of e x i s t i n g k i n e t i c data. In addition, the isoenzyme composition of tyrosinase may change with development. Therefore, we examined the tyrosinase a c t i v i t i e s and isoenzymes i n developing mushrooms. Tyrosinase a c t i v i t y was monitored i n four stages of mushroom development. Small pins (0-.5 cm), large pins (.5-1 cm), immature and mature mushrooms were c l a s s i f i e d by cap s i z e , g i l l development, and v e i l covering (K. Dahlberg, personal communication). These samples were assayed f o r a c t i v e and latent tyrosinase a c t i v i t y after extraction i n the absence of phenolic adsorbents (Fig. 2). No apparent trend i n enzyme a c t i v i t y was noted during development using catechol as the substrate. Latent enzyme a c t i v i t y was also present i n a l l stages. In contrast, dopa oxidase a c t i v i t y appeared to decrease with development concommitant with an increase i n latent tyrosinase a c t i v i t y . Tyrosine hydroxylase a c t i v i t y also decreased with development using tyrosine as the substrate, but no latent a c t i v i t y was noted using t h i s substrate. Yamaguchi e t a l . (22) found a somewhat s i m i l a r r e s u l t using a d i f f e r e n t substrate (catechol/proline) and assay method for monitoring a c t i v e and latent tyrosinase i n mushrooms. They observed that active tyrosinase was r e l a t i v e l y constant from t i g h t buttons to the f l a t p i l e u s stage; however, latent a c t i v i t y increased with development. Using catechol as the substrate, Burton (23) found that nonlatent or active tyrosinase a c t i v i t y was r e l a t i v e l y constant during preharvest development. Burton also observed that latent a c t i v i t y d i d not change s i g n i f i c a n t l y during pre-harvest development.

American Chemical Society Library 1155 16th St., N.W. In Quality Factors of Fruits and Vegetables; Jen, J.; Washington, D.C. Society: 20036Washington, DC, 1989. ACS Symposium Series; American Chemical

48

QUALITY FACTORS OF FRUITS AND VEGETABLES


SDS Page of Tyrosinase

Figure 1. Letters A and Β refer to two different lots of commercial mushroom tyrosinase. Numbers refer to molecular weight markers in kd.

CATECHOL

DOPA

TYROSINE

Figure 2. Clear bars represent active tyrosinase in four different developmental stages of mushrooms, while the hatched bars represent latent and active enzyme. (Reproduced with permission from ref. 35. Copyright 1989/. Food ScL)



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49

Measurements of t o t a l tyrosinase a c t i v i t y do not r e f l e c t i t s isoenzyme composition. Therefore, we examined the isoenzyme forms of tyrosinase i n small pins, large pins, immature and mature mushrooms by native electrophoresis followed by staining f o r dopa oxidase a c t i v i t y . Using dopa oxidase as an indicator of tyrosinase, one dominant isoenzyme form (I) was present i n a l l development stages (Fig. 3). Two faster migrating forms (IIIa,b) were also apparent as well as a f a i n t intermediate migrating form (II). No new isoenzyme forms were observed during development. A l l forms appeared to decrease during development with the III a and b forms decreasing more r a p i d l y . I f these same samples were extracted i n the presence of phenolic adsorbents (+), the intermediate and faster migrating forms were inactivated by or completely adsorbed on t o the phenolic adsorbents. Identical patterns were obtained i r r e s p e c t i v e of whether electrophoresis was c a r r i e d out i n the presence or absence of SDS, indicating that no new latent isoenzyme forms were made which were s p e c i f i c a l l y a c t i v i a t e d by SDS. Because tyrosinase can be activated from a latent state, neither enzyme assays or electrophoresis can d i s t i n g u i s h the mass amount of tyrosinase present on a protein b a s i s . To determine the amount of tyrosinase present on a protein b a s i s , we subjected the same samples as i n F i g . 3 A to denaturing electrophoresis followed by Western b l o t t i n g . Anti-mushroom tyrosinase antibodies were used to locate the enzyme i n each of the samples. As seen i n F i g . 3B, a broad band of immunostaining was observed i n each extract. This band appeared t o remain r e l a t i v e l y constant during development. A minor band at 24-26 kd appeared to decrease with devleopment. This band was e n t i r e l y absent i n samples treated with phenolic adsorbents and suggests adsorption to the resins. In addition, t h i s band cannot account f o r the decrease i n enzyme a c t i v i t y , because only the H subunits are believed t o contain the c a t a l y t i c s i t e . In general, the mass amount of tyrosinase detected immunologically d i d not appear to c o r r e l a t e well with the decrease i n a c t i v i t y observed by using enzyme assays or by electrophoresis. This suggests that the decrease i n enzyme a c t i v i t y during development may not be related t o decreased enzyme synthesis but may be related to the presence of an i n h i b i t o r or some other mechanism of i n a c t i v a t i o n . Commercial mushroom growers harvest three t o four cycles of mature mushrooms from a s i n g l e compost innoculum. Each c y c l e i s c a l l e d a break or f l u s h . To determine i f tyrosinase a c t i v i t y and content changed with the breaks harvested, freeze d r i e d samples were obtained from small pins, large pins, immature and mature stages i n the f i r s t three breaks from the same mushroom innoculum and assayed f o r a c t i v e and latent tyrosinase a c t i v i t y (Fig. 4). Using dopa oxidase as an indicator f o r tyrosinase enzyme a c t i v i t y decreased i n a l l three breaks as the mushrooms matured. Tyrosinase a c t i v i t y was somewhat greater i n e a r l i e r developmental stages i n break 1 than i n breaks 2 or 3. This general pattern of a c t i v i t y was s i m i l a r to that obtained using fresh or frozen samples and latent a c t i v i t y appeared t o be more pronounced i n l a t e r



50


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+ Figure 3. Extracts prepared in the absence (-) and presence (+) of phenolic adsorbents were subjected to native electrophoresis and stained for tyrosinase using dopa (A). Western blot of mushroom tyrosinase in four developmental stages after SDS PAGE (B). Samples were the same as in A. (Top photograph reproduced with permission from ref. 35. Copyright 1989/. Food Set)



4.



51

developmental stages than i n e a r l i e r stages. In general, the trend in decreased tyrosinase a c t i v i t y and increased l a t e n t / a c t i v e r a t i o of enzyme appeared t o be s i m i l a r i n a l l three breaks. The above samples i n each break were analyzed f o r tyrosinase isoenzymes by native electrophoresis to determine i f any changes i n isoenzyme forms occurred from break to break (Fig. 5). A l l samples applied to electrophoresis contained s i m i l a r amounts of protein (15 ug). One dominant slower form (I) was present i n a l l three breaks. The i n t e n s i t y of t h i s form appeared to decrease with development i n each break. Two faster migrating forms (III a,b) were also observed i n each break and these two forms also decreased i n i n t e n s i t y with development. No apparent trend was noted i n the amount of the intermediate migrating forms (II). These p r o f i l e s were q u a l i t a t i v e l y s i m i l a r to fresh mushroom extract p r o f i l e s at d i f f e r e n t developmental stages and once again suggest that no new isoenzyme forms were made during development or i n d i f f e r e n t breaks. Using a method of histochemical l o c a l i z a t i o n developed by Spruce et a l . (29), tyrosinase a c t i v i t y was v i s u a l i z e d at the whole tissue l e v e l . After b l o t t i n g mushroom s l i c e s onto n i t r o c e l l u l o s e , the membranes were subsequently stained f o r dopa oxidase a c t i v i t y (31-32). As seen i n Fig. 6, tyrosinase was d i s t r i b u t e d throughout the e n t i r e t i s s u e section. However, darker areas of histochemical staining i n t e n s i t y were noted i n the epidermis, g i l l , and stalk tissues compared to the cap f l e s h . These r e s u l t s are i n general agreement with Burton (23-24) and Yamaguchi et a l . (22) but give no indication of the amount of latent a c t i v i t y present i n each t i s s u e . To examine the amount of active and latent a c t i v i t y i n d i f f e r e n t parts of the mushroom, cap skin, cap f l e s h , g i l l and stalk tissues were extracted and assayed for dopa oxidase a c t i v i t y in the presence and absence of SDS. Based upon s p e c i f i c a c t i v i t i e s , greater amounts of tyrosinase were present i n f l e s h and stalk tissues compared to skin or g i l l t i s s u e (Table I ) . These r e s u l t s are q u a l i t a t i v e l y s i m i l a r to those reported by Yamaguchi et a l . (22) and Boret et a U (34). In f a c t , Boret et a l ^ (34) appeared to f i n d a concentration gradient of tyrosinase a c t i v i t y i n the stem from bottom to top. However, they also found considerable v a r i a t i o n i n enzyme a c t i v i t y i n d i f f e r e n t t i s s u e sections from mushroom to mushroom. We also have found such v a r i a t i o n s between i n d i v i d u a l mushrooms and between d i f f e r e n t batches of mushrooms. When t i s s u e samples were measured f o r tyrosinase a c t i v i t y i n the presence of SDS, a l l tissues showed latent a c t i v i t y . This latent a c t i v i t y appeared to be s l i g h t l y greater i n the skin t i s s u e than i n the cap f l e s h , g i l l , or stalk t i s s u e s . Yamaguchi et a l . (22) observed that the s t i p e t i s s u e contained more a c t i v e and latent enzyme and Burton (23-24) reported that skin tissues contained more tyrosinase than the cap f l e s h . The differences between t h e i r data and ours may be related to the d i f f e r e n t substrates used to monitor tyrosinase a c t i v i t y .


52



Tyrosinase Activity in Freeze Dried Mushrooms

1st Break

Break

3rd Break

Figure 4. Clear bars represent active tyrosinase in four developmental stages in each of three different breaks. Hatched bars represent active and latent enzyme using dopa as the substrate. (Reproduced with permission from ref. 35. Copyright 1989/. Food Set)

Figure 5. Tyrosinase isoenzyme forms identified in three different breaks (1-3) after native electrophoresis and staining for dopa oxidase activity. A-D represent small pins, large pins, immature, and mature mushroom samples, respectively. (Reproduced with permission from ref. 35. Copyright 1989/. Food Set)

Figure 6. Histochemical localization of mushroom tyrosinase on nitrocellulose blots. Letters refer to epidermis (e), gills (g), and stalk (s). (Reproduced with permission from ref. 31. Copyright 1989 Histochemistry.)


4.

FLURKEY & INGEBRIGTSEN Table I.


Active and Latent Tyrosinase i n D i f f e r e n t Tissues of Mature Mushrooms

Tyrosinase A c t i v i t y


Tissue cap skin cap f l e s h gill stalk

-SDS .35 .69 .34 1.13

(Units/mg) +SDS 5.9 5.13 3.53 7.71

Fold Increase 17 7 10 7

Each tissue contains d i f f e r e n t amounts of tyrosinase a c t i v i t y and may have a d i f f e r e n t isoenzyme composition. Samples from the skin, g i l l , cap f l e s h , and the stalk were subjected to native electrophoresis followed by staining with dopa f o r dopa oxidase a c t i v i t y . In contrast to whole mushroom homogenates, each t i s s u e appeared to contain a s i n g l e dominant isoenzyme form (data not shown) but each t i s s u e also showed a d i f f e r e n t isoenzyme pattern. This suggests that when a l l tissues are homogenized together some interconversion or transformation process takes place, perhaps by p r o t e o l y s i s , which generates the patterns we observed i n d i f f e r e n t developmental stages. This a l s o suggests the need to monitor d i s c r e t e isoenzymes i n i n d i v i d u a l t i s s u e sections with development. In conclusion, the r e s u l t s presented i n t h i s report and by others tend to indicate that tyrosinase a c t i v i t y and the isoenzyme content i n mushrooms (1) are very complicated, (2) are d i f f e r e n t in pre-harvest development, (3) vary from tissue to t i s s u e , (4) are composed of latent and a c t i v e enzyme forms; and that no new isoenzyme forms are synthesized during development. The applications of these findings may be of interest to the mushroom industry but also indicate the need for further study of the enzyme during pre-harvest and post-harvest development and storage. Acknowledgments We wish to thank the Campbell Soup Company and the Campbell I n s t i t u t e f o r Research and Technology f o r t h e i r support of t h i s research p r o j e c t . We also appreciate the s e c r e t a r i a l services of P. Archer and K. Divan. We also thank M. Wach and K. Dahlberg for t h e i r comments on t h i s manuscript.

L i t e r a t u r e Cited 1. 2. 3. 4. 5. 6.

Mayer, A. M.; Harel, E. Phytochemistry 1979, 18, 193-215. Mayer, A. M. Phytochemistry 1987, 26, 11-20. Vaughn, K. C.; Duke, S. O. Physiol. Plant. 1984, 60, 106-112. Vaughn, K. C.; Lax, R. L.; Duke, S. O. Physiol. Plant. 1988, 72, 659-665. Butt, V. S. In The Biochemistry of Plants, Stumpf, P. K.; Conn Ε. Ε., Eds. Academic Press: New York, 1980, v o l . 2, p. 81. Vamos-Vigyazo, L. In CRC C r i t i c a l Reviews i n Food Science and N u t r i t i o n , CRC Press: Boca Raton, FL, 1981, p. 49.


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Robb, D. A. In Copper Proteins and Copper Enzymes; Lontie, R. Ed.; CRC Press: Boca Raton. 1984, Chapter 7, p. 207. 8. Hearing, V. J.; Jimenez, M. Eur.J.Biochem. 1987, 12, 11411147. 9. Korner, Α.; Pawlek, J. Science 1982, 217, 1163. 10. Boekelheide, K.; Graham, D. G.; Mize, P. D.; Anderson, C. W.; J e f f s , P. W. J. B i o l . Chem. 1979, 254, 12185-12191. 11. Boekelheide, K.; Graham, D. G.; Mize, P. D.; J e f f s , P. W. J. B i o l . Chem. 1980, 255, 4766-4771. 12. Rast, D. M.; Stussi, H.; Hegnauer, H.; Nyhlen, L. E. In The Fungal Spore: Morphogenetic Controls; Turian G.; Hohl, R. R. Eds.; Academic Press: London, 1981; p. 507. 13. Stussi, H.; Rast, D. M. Phytochemistry 1981, 20, 2347-2352. 14. Sugumaran. M. Biochemistry 1986, 25, 4489-4492. 15. Sugumaran, M.; D a l i , H.; Semensi, V.; Hennigan, B. J. B i o l . Chem. 1987, 262, 20546-20549. 16. Oka, Y.; Tsuji, H.; Ogawa, T.; Sasaoka, K. J. Nutr. S c i . Vitaminol. 1981, 27, 253-262. 17. Gutteridge, S.; Mason H. S. In Biochemical and C l i n i c a l Aspects of Oxygen; Caughy, W. S. Ed.; Academic Press: New York, 1979; p.589. 18. Robb, D. Α.; Gutteridge, S. Phytochemistry 1981, 20, 14811485. 19. Sharma, R. C.;Ali,R. Phytochemistry 1981, 20, 399-401. 20. Strothkamp, K. G.; Jolley, R. L.; Mason, H. S. Biochem. Biophys. Res. Commun. 1976, 70, 519-524. 21. Podila, G. K.; Flurkey, W. H. Biochem. Biophys. Res. Commun. 1986, 141, 697-703. 22. Yamaguchi, M.; Hwang, P. M.; Campbell, J. D. Can.J.Biochem. 1970, 48, 198-202. 23. Burton, K. S. J. Hort. S c i . 1988, 63, 255-260. 24. Burton, K. S. The Mushroom Journal 1986, 158, 68-70. 25. Lanker, T.; King, T. G.; Arnold, S. W.; Flurkey, W. H. Physiol. Plant. 1987, 69, 323-329. 26. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. B i o l . Chem. 1951, 193, 265-275. 27. Angleton, Α.; Flurkey, W. H. Phytochemistry 1984, 23, 27232725. 28. Laemmli, U. K. Nature 1970, 227, 680-685. 29. Spruce, J.; Mayer, A. M.; Osborne, D. J. Phytochemistry 1987, 26, 2901-2903. 30. Moore, Β. M.; Kang, B.; Flurkey, W. H. Phytochemistry 1988, 27, 3735-3737. 31. Moore, Β. M.; Kang, B. Flurkey, W. H. Histochemistry 1989, 90, 379-381. 32. Golan-Goldhirsh, Α.; Whitaker, J. R. In Nutritional and Toxicological Aspects of Food Safety; Friedman, M. Ed.; Ad vances in Experimental Medicine and Biology Vol 177. Plenum Press: New York, 1984; p. 437-456. 33. Boyer, R. F.; Mascotti, D. P.; Schori, Β. Ε. Phytochemistry 1986, 25, 1281-1283. 34. Boiret, M.; Marty, Α.; Deumie, M. Biochemical Education 1985, 13, 82-84. 35. Ingebrigtsen, J.; Flurkey, W. H. J. Food Sci. 1989, ms 88-253-2. RECEIVED February 7, 1989


Polyphenol Oxidase Activity and Enzymatic Browning in Mushrooms

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