Biochem

Purification and characterization of tyrosinase from gill tissue of Portabella mushrooms

Yan Fan, William H. Flurkey*

Chemistry Department, Science Building, Indiana State University, Terre Haute, IN 47809, USA

Received 22 August 2003; received in revised form 19 November 2003

Abstract

A group of tyrosinase isoforms with isoelectric points between 4.9 and 5.2 was isolated from gill tissue of Portabella mushrooms.

Use of protease inhibitors was not able to increase the amount of latent forms significantly in crude extracts or to preserve latent tyrosinase activity during purification. The tyrosinase in gill tissue extracts showed latent activity above pH 5.5 and suppressed or displayed no latent activity below pH 5.5 when assayed in the presence of SDS. The purified isoforms showed monophenolase activity toward 4-hydroxyanisole but practically no activity toward tyrosine or tyramine. The purified isoforms showed greater

activity toward catechol than either 4-methylcatechol, dopa, dopamine, chlorogenic acid, t-butylcatechol, or catechin. The Km for catechol was similar for the group of isolated isoforms (4.3 mM) compared to the isoforms in crude extracts (5.3 mM). Crude extracts showed several isoforms ranging from 50 to 230 kDa after partially denaturing SDS PAGE, while the purified isoforms

showed molecular weights of 70 kDa. # 2004 Elsevier Ltd. All rights reserved.

Keywords: Agaricus bisporus; Portabella mushrooms; Gill tissue; Tyrosinase; Purification

1. Introduction

The enzyme tyrosinase (E.C. 1.14.18.1) is a member of the tyrosinase related protein family and is found throughout the phylogenetic scale of living organisms (for a review of tyrosinase and tyrosinase related pro- teins, see del Marmol and Beermann, 1996; Garcia- Borron and Solano, 2002; Hearing and Tsukamoto, 1991; van Gelder et al., 1997). Tyrosinase performs a variety of functions in these organisms ranging from pigmentation to defense to sclerotization. The enzyme catalyzes at least two different reactions using phenolic compounds and molecular oxygen as substrates. These reactions include hydroxylation of monophenols to form ortho-diphenols (monophenolase or cresolase activity) and oxidation of diphenols to form ortho- diquinones (diphenolase or catechol oxidase activity). It has also been suggested that tyrosinase can oxidize 5,6- dihydroxyindole to the 5,6-dihydroxyquinone (Korner and Pawelek, 1982).

There are several oxidases that can use phenolic sub- strates present in mushrooms, but tyrosinase is prob- ably the principal enzyme involved in browning reactions in Agaricus bisporus (review by Jolivet et al., 1998). For example, tyrosinase, laccase, and peroxidase were detected in Portabella mushrooms, a brown strain of Agaricus bisporus, but tyrosinase was present in lar- ger amounts than either laccase or peroxidase (Zhang and Flurkey, 1997). These authors also found that more tyrosinase was present in the stalk than in the cap skin, gill tissue, or cap flesh tissue. However, it is difficult to accurately determine quantitative tyrosinase activities associated with different tissues because of latent and active enzyme forms, the difficulty of determining pro- tein content, and different isoform composition at dif- ferent developmental stages (Jolivet et al., 1998; Van Leeuwen and Wichers, 1999). Zhang and Flurkey (1999) also showed that crude extracts of different mushroom tissues showed a slightly different isoform composition after analysis by isoelectric focusing (IEF). Crude gill tissue extracts showed less of the low pI isoforms and more of the higher pI isoforms (Zhang and Flurkey, 1999; Zhang et al., 1999). Tyrosinase isolated from cap skin and cap flesh of Portabella mushrooms and from

0031-9422/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.phytochem.2004.01.008

Phytochemistry 65 (2004) 671–678

www.elsevier.com/locate/phytochem

* Corresponding author. Tel.: +812-237-2245; fax: +812-237-

2232.

E-mail address: [email protected] (W.H. Flurkey).

Agaricus bisporus has a subunit molecular weight of around 43–48 kDa (Zhang and Flurkey, 1999; Ylostalo et al., 2001; Gerritsen et al., 1994; Wichers et al., 1996), although latent enzyme can be isolated with a higher molecular weight using freshly picked/frozen Agaricus bisporus tissue (Espin and Wichers, 1999). Gill tissues contain abundant melanized spores in

mature mushrooms. g-glutaminyl-4-hydroxybenzene (GHB) is considered to be the main substrate for mela- nogenesis catalyzed by tyrosinase/phenolase in Agaricus bisporus (Rast, et al., 1981). Tyrosinase has been shown to hydroxylate GHB into g-glutaminyl-3,4-dihydroxy- benzene (GDHB) and then to oxidize GDHB into g- glutaminyl-3,4-benzoquinone during the melanization process of sporogenesis (Boekelheide et al. 1979, 1980; Rast et al., 1981). However, the fate and physiological role of GHB may be different in mycelium (Soulier et al., 1993; Jolivet et al., 1998). Tyrosinase has been loca- ted in ‘‘tyrosinase vesicles’’ associated with cell walls and perhaps in cell wall regions containing amorphous melanin as well (Rast et al., 2003). Since melanin deposition in fungi takes place in extracellular locations, tyrosinase may play a key role in the development of the fungal cell wall constituents. Perhaps pigmentation in the different extracellular cell wall locations and in spores relies on different tyrosinase isoforms or isoforms with different substrate specificities. Characterization of the enzyme located in the tissues, i.e. gills, involved in converting GHB into GDHB and further oxidation into melanin may provide further insight into the synthesis of spore melanins and melanization. This study is one of the first to examine some of the biochemical character- istics associated with tyrosinase isolated from gill tissue of Portabella mushrooms.

2. Results and discussion

Purification of tyrosinase from gill tissue is somewhat more difficult due to the smaller amount of tissue avail- able per mushroom and greater amount of pigmenta- tion in these tissues. Initially, we tried various adsorbents (polyvinylpyrrolidone, Polyclar AT, XAD nonionic adsorbents, AG anion exchangers, and celite) as well as precipitation methods (polyethyleneglycol, acetone, ammonium sulfate, and ethanol) to remove colored material or to concentrate tyrosinase. None of these methods were successful without causing a decrease in the recovery of enzyme activity or not being able to remove a substantial amount of colored mate- rial. Secondly, in order to isolate a group of closely related pI tyrosinase isoforms, selective pooling from column chromatography was also necessary. As a result, lower recovery and yields were obtained. Lastly, both latent and active enzymes were present in crude extracts. This is rather surprising to find latent enzyme

in these tissues given that store-bought mushrooms usually have little latent enzyme in them. However, even with the use of protease inhibitors, we were not able to prevent activation of latent enzyme during purification. Table 1 shows the effect of using protease inhibitors

on the extraction of latent tyrosinase from gill tissues. Approximately half of the enzyme was latent in extracts prepared in the absence of protease inhibitors. Prepara- tion of extracts in the presence of protease inhibitors that would inhibit serine proteases, metallo-proteases, and cysteine proteases using PMSF, EDTA, and E-64, respectively, did not increase the percentage of latent enzyme appreciably although the total activity increased. Use of pepstatin to inhibit acidic proteases had no appreciable effect on increasing latent activity. These results indicate that at least half of the enzyme had already been converted into an active form in the tissue samples irrespective of what type of inhibitor was used. During the purification of tyrosinase, we were not able to preserve latent enzyme, and most of the latent enzyme was converted into active enzyme (data not shown). We also noticed that the amount of latent enzyme present in the isolated fractions varied from preparation to preparation. Presumably this variation is a result of limited proteolysis or partial unfolding of the latent enzyme according to the model of the maturation and activation of latent Agaricus bisporus mushroom tyrosinase proposed by Espin and Wichers (1999) and Espin et al. (2000a). In contrast, Espin et al. (1999) were able to isolate a latent tyrosinase from freshly harvested and frozen Agaricus bisporus mushrooms without the use of protease inhibitors. This latent enzyme could be activated in vitro by trypsin, a serine protease. To date

Table 1

Effect of protease inhibitors during the extraction of gill tissue

tyrosinase

Protease inhibitor

SDS

Total

tyrosinase

activity

(units/ml)

Latent

tyrosinase

activity

(units/ml)

% Latent

activity

Control

+

15.7

8.6

55

�

7.1

PMSF (1 mM)

+

22.7

13.9

61

�

8.8

EDTA (1 mM)

+

20.8

12.9

62

�

7.9

E-64 (1 mg/ml)

+

20.0

12.3

62

�

7.7

pepstatin (1 mg/ml)

+

17.8

9.9

56

�

7.9

Crude extracts, with and without the presence of added inhibitors,

were prepared by blending 10 g of tissues with 100 ml of 10 mM

phosphate buffer (pH 6.5), centrifuging the homogenate, and assaying

the supernatant for active and latent tyrosinase activity as described in

the material and methods. Latent activity is the activity detected in the

presence of 0.1% SDS minus the activity detected in the absence of

0.1% SDS.

672 Y. Fan, W.H. Flurkey / Phytochemistry 65 (2004) 671–678

this is the only group of investigators able to isolate a fully latent tyrosinase from mushrooms. The latent tyrosinase activity measured in crude

extracts from gill tissues is dependent upon the pH of the assay medium. At pH values below 5 there was little to no latent activity detected when assayed in the pre- sence of SDS, although there was some activity detected in the absence of SDS (Fig. 1). Above pH 6.0 the amount of tyrosinase activity was significantly greater than that assayed in the absence of SDS, indicating a greater amount of detectable latent enzyme. At more acidic pHs the ability to be activated by SDS was sup- pressed. The pH optimum was approximately pH 7.0 when assayed in the presence and absence of SDS for total and active tyrosinase activity, respectively. Latent Agaricus bisporus tyrosinase isolated by Espin et al. (1999) did show a small peak of activity from pH 4–4.5 in the absence of SDS due to acid shocking. However, the latent activity in gill tyrosinase (this report) showed no such peak of activity at low pH in the absence of SDS. It is possible that the brown strain of mushrooms may show different activation/latency characteristics of tyrosinase at lower pH compared to the tyrosinase found in the white strain of A. bisporus. SDS activation profiles of latent tyrosinase/polyphenoloxidase versus pH were also reported in broad beans (Moore and Flurkey, 1990; Jimenez and Garcia-Carmona, 1996), apples (Marques et al., 1995), Prunus fruits (Fragnier et al., 1995), and persimmon (Nunez-Delicado et al., 2003). In general, they showed similar activation by SDS at higher pH, similar suppression of activation at lower pH, and, in some cases, a pH optimum at acidic pH values when assayed in the absence of SDS. This pH

Fig. 1. Effect of pH and SDS on gill tissue tyrosinase activity. Tyrosinase activity was measured using 10 mM catechol as the substrate, with or

without 0.1% SDS, at various pH as described in the experimental section. Open symbols refer to assays without SDS. Filled symbols refer to assays

with SDS. Square symbols refer to citrate buffers. Triangle symbols refer to acetate buffers, and circles refer to phosphate buffers.

Fig. 2. Isoelectric focusing of crude and purified gill tissue tyrosinase.

Isoeletric focusing was carried out in pH 3–7 gradients at 10 �C. Gels

were stained with 1 mM catechol and 2 mM MBTH. pI values were

determined by comparison to pI standards. 1 and 2 refer to two

different HA purified preparations of gill tissue tyrosinase. CE refers

to a crude extract of gill tissues. pI groupings are designated by

brackets.

Y. Fan, W.H. Flurkey / Phytochemistry 65 (2004) 671–678 673

dependency on latency, SDS, and acid shocking may represent different mechanisms for activation. Crude gill extracts contained several pI isoforms that

clustered around pH 4.9–5.2 and another group of iso- forms that clustered around pH 5.4–5.7 (Fig. 2). Only small amounts of the lower pI isoforms around pH 4.3– 4.5 were present in gill tissues. There were approxi- mately four heavily stained isoforms present in the 4.9– 5.2 pI range and four heavily stained isoforms in the 5.4-5.7 pI range. At least 16 different isoforms with varying intensity could be identified by IEF in crude extracts after prolonged staining. This number is larger than that reported by van Leeuwen and Wichers (1999) for tyrosinase in crude extracts of gill tissues from Agaricus bisporus and larger than that in an earlier report by Zhang and Flurkey (1999) using crude extracts of gill tissues from Portabella mushrooms. Two different tyrosinase preparations that were isolated fol-

lowing the purification procedure outlined above con- tained the same 3–4 isoforms that clustered between pI 4.9–5.2. Although we only examined active IEF iso- forms from these preparations, the distribution of indi- vidual IEF forms within the 4.9–5.2 pI cluster was slightly different. This suggests that there may be some conversion or loss of these isoforms during the pur- ification process that we could not prevent and illus- trates the difficulty in isolating specific tyrosinase isoforms. Chromatography on DEAE columns can partially

separate many of the isoforms with higher and lower pIs. Using DEAE columns equilibrated at pH 7, tyr- osinase was eluted in the order of high to low pI iso- forms with increasing salt concentrations. Further separation of the isoforms was achieved using hydro- xyapatite (HA) chromatography (Fig. 3). Depending on what fractions were pooled from the DEAE column,

Fig. 3. Chromatography of gill tyrosinase on hydroxyapatite. Selective fractions from tyrosinase subjected to chromatography on DEAE cellulose

were applied to a hydroxyapatite column equilibrated in 10 mM phosphate buffer (pH 7.0). Tyrosinase activity was eluted with a 10–250 mM

phosphate buffer gradient. Fractions containing the group of IEF isoforms from 4.9 to 5.2 (first peak) were pooled and used for further experiments.

Solid line denotes absorbance at 280 nm. Dashed line represents tyrosinase activity. Open circles designate conductivity of phosphate buffer gradient

in diluted samples from fractions.

674 Y. Fan, W.H. Flurkey / Phytochemistry 65 (2004) 671–678

HA chromatography resulted in two to three peaks of tyrosinase activity with middle to higher pI forms elut- ing later in the column fractions. Fig. 3 shows two major peaks of tyrosinase activity and a very minor peak at fraction 51 eluting during HA chromatography. The order of elution of tyrosinase isoforms using HA chromatography was from low to middle to high pI isoforms with increasing phosphate buffer concentra- tions. Isoelectric focusing of two different purification attempts showed that the middle group of isoforms, pI 4.9–5.2, was separated from lower and high pI isoforms (Fig. 2). However, there were still several isoforms (3–4) present with minor differences in pI. Although several isoforms were detected by IEF in the purified samples, these same samples showed a single, albeit smeary, band of tyrosinase activity after native PAGE (data not shown). This indicates that the isoforms have similar sizes and charges. At present, we have not been able to separate the members of this isoform group from one another using conventional chromatographic methods. In spite of the isoform separation, the material

obtained from HA chromatography was not completely homogenous when analyzed by SDS PAGE (Fig. 4 a). The purification scheme removed most of the major proteins with lower molecular weight found in the crude extracts (Fig. 4a HA vs CE). Two major stained bands of protein were present in the final sample along with a protein with less staining intensity. The estimated sizes of the major bands were 48 and 43 kDa, respectively. The minor protein band was estimated to have a size of 60 kDa. Several minor proteins smaller than 45 kDa were present but not readily visible in the scanned gels. We assume that the bands in the 43–48 kDa are the active proteolyzed forms of tyrosinase that many other investigators have observed (Wichers et al., 1996; Ger- ritsen et al., 1994; Zhang et al., 1999). The minor band at 60 kDa could be the latent tyrosinase that is known to have a higher molecular weight (Espin and Wichers, 1999; Espin et al., 2000a). Alternatively, this 60 kDa protein could be a protein contaminant still remaining in the preparation. Partial protein sequencing or Wes- tern blotting with specific antibodies would be needed to confirm this potential relationship. Recently, we have been able to remove some 50–70 kDa proteins from commercial tyrosinase preparations using hydrophobic interaction chromatography (data not shown). Since the commercial tyrosinase contains little latent enzyme, this suggests that the 60 kDa protein in the isolated gill tyr- osinase could be a contaminant rather than a latent enzyme form. Under partially denaturing SDS PAGE, samples not

boiled or treated with reducing agents, several isoforms of tyrosinase were detected in crude extracts of gill tis- sue (Fig. 4b). The size of these isoforms ranged from 50 to 230 kDa when stained for enzyme activity. A broad band at approximately 70 kDa showed heavy staining.

Fig. 4. (a) Denaturing SDS PAGE of crude and purified tyrosinase

from gill tissue. Samples from the crude extract (CE) and tyrosinase

purified through hydroxyapatite chromatography (HA) were subjected

to SDS PAGE using the Laemmli method. Protein standards (NOV)

were used to estimate molecular weights. (b) Partially denaturing SDS

PAGE of crude and HA purified tyrosinase from gill tissue. Samples

from CE and HA, not denatured, were subjected to SDS PAGE and

stained for tyrosinase activity with 2 mM dopa/2 mM MBTH in the

presence of 0.1% SDS. See-Blue (SB) pre-stained molecular weight

protein standards were from Invitrogen (formerly Novex).

Y. Fan, W.H. Flurkey / Phytochemistry 65 (2004) 671–678 675

The purified gill tyrosinase isoforms also showed a sin- gle band at this same position, suggesting that the pI 4.9–5.2 IEF isoforms have an apparent MW of 70 kDa under these conditions. The other isoforms observed after partially denaturing SDS PAGE were apparently removed during the purification procedure. Native PAGE showed three broad bands of staining activity in crude extracts and one broad band in the purified gill tyrosinase fractions that corresponded to the major stained band in crude extracts (data not shown). Even though the subunit size determined by denaturing SDS PAGE of purified gill tyrosinase was 43–48 kDa, par- tially denaturing SDS PAGE indicated a much larger size. This increase in size could be due to conforma- tional changes and/or aggregation of monomeric units. However, conformational changes would not likely account for those isoforms with apparent sizes larger than 70 kDa, especially if the subunit molecular weight is 43–48 kDa. The results from partially denaturing SDS PAGE, SDS PAGE, IEF, and native PAGE illustrate the difficulty in trying to determine tyrosinase isoform composition and characteristics of specific tyrosinase isoforms. Tyrosinase activity in crude extracts and in the par-

tially purified sample was examined with regard to its monophenolase and diphenol oxidase activity Table 2. Both the crude extracts and purified enzyme were able to hydroxylate monophenols and oxidize diphenols. 4- Hydroxyanisole was the substrate that showed the most activity based on a relative comparison of the three monophenols tested for monophenolase activity. How- ever, the monophenolase activity was very low in the purified samples, indicating a substantial loss of this

activity during the purification. We have observed that this activity was lowered further when assayed in the presence of SDS, suggesting that the monophenolase activity is more susceptible to conformational changes around the active site than the catechol oxidase activity (data not shown). The labile nature of the mono- phenolase activity of tyrosinase has been noted in many earlier reports. Of the diphenols tested, highest activity was shown

with catechol as the substrate using either crude extracts or purified enzyme. Except for 4-methyl catechol, a similar percent relative activity was observed with dopa, dopamine, chlorogenic acid, t-butylcatechol, or catechin as substrates using crude extracts compared to the pur- ified enzyme. This indicates that purification did not change the general substrate preferences of tyrosinase. The overall relative diphenol oxidase activity decreased approximately 67% when the enzyme was purified compared to the crude extract. This decrease in diphe- nol oxidase activity (5.6 units/ml vs 1.9 units/ml) was not nearly as great as the decrease observed in the monophenolase activity (16.1 units/ml vs 0.28 units/ml). In addition, much of the latent tyrosinase activity had disappeared in the purified preparations compared to crude extracts (data not shown). In general, these results are in agreement with those found for the tyrosinase IEF isoforms (pI 5.1–5.3) isolated from cap flesh of Portabella mushroom (Zhang et al., 1999) but not as similar for the tyrosinase IEF isoform (pI 4.3) isolated from cap skins of Portabella mushrooms (Ylostalo et al., 2001). Using catechol as the substrate, the Km was deter-

mined for active tyrosinase from crude extracts and from the purified tyrosinase preparations. In the absence of SDS in the assays, the tyrosinase in crude extracts and in purified preparations showed apparent hyperbolic curves in velocity versus substrate plots over a broad range of catechol concentrations (Fig. 5). In the absence of SDS, the apparent Km of catechol for tyr- osinase in crude extracts was determined to be 5.3 mM while a Km of 4.3 mM for catechol was obtained using tyrosinase in purified samples. The Km for crude tyr- osinase extracts assayed in the presence of SDS to detect total tyrosinase activity was 3.6 mM (data not shown). These Km values determined in the absence of SDS represent contributions from all active nonlatent tyr- osinase forms in each sample. The Km values deter- mined in the presence of SDS represent contributions from all active and activated forms of tyrosinase. Based on the above Km values, there may be a relationship between Km and the forms of tyrosinase that are acti- vated, but this requires further study to confirm this possibility. The Km values for Portabella gill tyrosinase in this study seem to be somewhat greater than those reported for tyrosinase from A. bisporus (Espin and Wichers, 1999; Espin et al., 2000a). Even though sub-

Table 2

Monophenolase and diphenol oxidase activity of crude and partially

purified gill tyrosinase

Relative activity (%)

Crude enzyme

Partially purified

enzyme

Monophenols

4-hydroxyanisole

100 (16.1)

100 (0.28)

tyrosine

<1

<1

tyramine

3

<1

Diphenols

catechol

100 (5.6)

100 (1.9)

4-methylcatechol

59

22

dl-dopa

23

21

dopamine

31

35

chlorogenic acid

39

36

t-butylcatechol

13

15

catechin

12

11

Tyrosinase activity was measured spectrophotometrically as described

in the experimental section. Relative activities were based on the sub-

strate with the greatest amount of activity as 100%. Numbers in par-

entheses refer to actual enzyme activity in units/ml.

676 Y. Fan, W.H. Flurkey / Phytochemistry 65 (2004) 671–678

unit molecular weights and IEF isoform composition of tyrosinase seem to be similar in the brown strain of A. bisporus, Portabella mushrooms, compared to the white strain of A. bisporus, there may be some underlying differences in their kinetic properties and substrates preferences.

3. Experimental

3.1. Materials

Fully expanded large caps (ca. 10 cm diameter) from Portabella mushrooms were obtained from local gro- ceries. The gill tissue was scraped from the caps, frozen in liquid nitrogen, and stored at �80 �C until processed. Protease inhibitors were obtained from Sigma Chemical Co., St. Louis, MO. Isoelectric focusing gels were obtained from Hypure Inc., Norton, OH, and from BioWhittaker Molecular Applications, Rockland, MA. Hydroxyapatite HTP was purchased from BioRad Laboratories, Hercules, CA.

3.2. Purification of tyrosinase from gill tissue

Gill tissue tyrosinase was purified using modifications of the methods for isolating tyrosinase from the cap skin and cap flesh tissue of Portabella mushrooms as reported by Ylostalo et al. (2001) and Zhang and Flurkey (1999). Briefly, crude extracts were prepared by grinding 50–100 g of tissue into a powder using liquid

nitrogen. The powder was homogenized in five volumes of 10 mM sodium phosphate buffer (pH 6.5) containing the following protease inhibitors: 1 mM PMSF, 1 mM EDTA, 1 mg/ml of pepstatin A, and 1 ug/ml of E-64. After centrifugation, the supernatant was applied onto a DEAE cellulose column (2.5 � 12 cm) equilibrated in 10 mM phosphate buffer (pH 7.0). Enzyme was eluted with a 0–500 mM NaCl gradient. After SDS PAGE and IEF analysis of column fractions, selective pooling of var- ious tyrosinase IEF group isoforms was carried out. The tyrosinase group of IEF isoforms in the range of pI 4.9– 5.2 was concentrated and applied onto a hydroxyapatite column (1 � 9 cm) equilibrated in 10 mM sodium phosphate buffer (7.0). Tyrosinase isoforms were eluted with a 10–250 mM phosphate buffer gradient. After IEF and SDS PAGE analysis, isoforms in the pI range of 4.9–5.2 were collected for further characterization. In some cases, it was necessary to repeat the hydroxy- apatite chromatography to remove other tyrosinase IEF isoforms present in minor amounts.

3.3. Enzyme assays

Tyrosinase activity was monitored spectro- photometrically using catechol as the substrate as reported earlier (Zhang et al., 1999). One unit of enzyme activity was defined as a change in one absorbance per min at 410 nm. Stock solutions of catechol were pre- pared in 0.5 mM H3PO4. Assays were conducted in the presence and absence of 0.1% SDS to detect latent enzyme activity. Latent activity is the amount of enzyme detected in the presence of SDS minus the amount detected in the absence of SDS. Monophenolase activity was determined using tyrosine (484 nm), tyramine (476 nm), and 4-hydroxyanisole (464 nm) as substrates along with MBTH in 50 mM phosphate buffer (pH 6.5) as described by Espin et al. (2000b) and Rodriquez-Lopez et al. (1994). The substrate concentrations were 0.5 mM and the MBTH concentration was 2 mM. Diphenol oxidase activity was determined using 4-methylcatechol (410 nm), catechol (410 nm), dl-dopa (475 nm), dopa- mine (480 nm), chlorogenic acid (400 nm), t-butylca- techol (400 nm), and catechin (390 nm) as substrates in 50 mM sodium phosphate buffer (pH 6.5) as described by Zhang et al. (1999). The final substrate concentra- tions were 5 mM. One unit of enzyme was defined as a change of one absorbance unit per min.

3.4. Kinetic constants

Kinetic constants were determined using catechol as the substrate. Catechol concentrations ranged from 0 to 55 mM and rates were determined from the linear por- tion of the absorbance versus time curves. Kinetic con- stants were evaluated using non-linear regression analysis by the method of Wilkinson in the Enzpack

Fig. 5. Velocity vs [catechol] plot of crude (dark colored squares) and

purified (light colored squares) gill tissue tyrosinase. Tyrosinase activ-

ity of the crude gill tissue extracts was measured in the presence of

different catechol concentrations ranging from 0.5 to 50 mM. Tyr-

osinase activity of the purified tyrosinase was measured in the presence

of catechol concentrations ranging from 0.5 to 35 mM. No SDS was

included in the assays. Km and Vmax values were determined from

nonlinear regression analysis using Enzpack software.

Y. Fan, W.H. Flurkey / Phytochemistry 65 (2004) 671–678 677

software (Biosoft, Cambridge, UK). Kinetic constants were determined using crude extracts and the purified enzyme in the presence of absence of 0.1% SDS in the assay mixtures.

3.5. Electrophoresis and isoelectric focusing

Native PAGE was carried out using the Laemmli SDS PAGE system without SDS (Chen and Flurkey, 2002) and under partially denaturing conditions (with SDS, no reducing agent, samples not boiled). SDS PAGE was carried out using the Laemmli system in which samples were denatured and treated with 50 mM dithioery- threitol (DTE, final concentration) before application (Zhang et al., 1999). Isoelectric focusing was carried out as described earlier by (Zhang et al., 1999) and more recently by Chen and Flurkey (2002). IEF gels were stained for tyrosinase activity using 1 mM catechol and 2 mM MBTH and destained in water.

3.6. pH optimum and pH effect on SDS activation

The pH optimum of tyrosinase was determined using catechol as in substrate. All buffer concentrations were 100 mM. Sodium citrate buffers were used to encompass a pH range from 3 to 4. Sodium acetate buffers were used for pH ranges from 4 to 6, and sodium phosphate buffers were used in pH ranges from 6 to 8. All assays were conducted in the presence and absence of 0.1% SDS.

References

Boekelheide, K., Graham, D.G., Mize, P.D., Anderson, C.W., Jeffs,

P.W., 1979. Synthesis of g-l-glutaminyl-[3,5-3H]4-hydroxybenzene

and the study of reactions catalyzed by the tyrosinase of Agaricus

bisporus. J. Biol. Chem. 254, 12185–12191.

Boekelheide, K., Graham, D.G., Mize, P.D., Jeffs, P.W., 1980. The

metabolic pathway catalyzed by the tyrosinase of Agaricus bisporus.

J. Biol. Chem. 255, 4766–4771.

Chen, L., Flurkey, W.H., 2002. Effect of protease inhibitors on the

extraction of Crimini mushroom tyrosinase isoforms. Current

Topics in Phytochem 5, 109–120.

del Marmol, V., Beermann, F., 1996. Tyrosinase and related proteins

in mammalian pigmentation. FEBS. Letts 381, 165–168.

Espin, J.C., Wichers, H.J., 1999. Activation of a latent mushroom

(Agaricus bisporus) tyrosinase isoform by sodium docecyl sulfate

(SDS). Kinetic properties of the SDS-activated isoform. J. Ag. Food

Chem. 47, 3518–3525.

Espin, J.C., van Leeuwen, J., Wichers, H.J., 1999. Kinetic study of the

activation process of a latent mushroom (Agaricus bisporus) tyr-

osinase by serine proteases. J. Ag. Food Chem. 47, 3509–3517.

Espin, J.C., Soler-Rivas, S., Wichers, H.J., 2000a. Maturation and

activation of latent tyrosinase from Agaricus bisporus. International.

Soc. Mushroom Science Proceed. May 15–19.

Espin, J.C., Varon, R., Fenoll, L.G., Gilabert, M.A., Garcia-Ruiz,

P.A., Tudela, J., Garcia-Canovas, F., 2000b. Kinetic characteriza-

tion of the substrate specificity and mechanism of mushroom

tyrosinase. Eur. J. Biochem. 267, 1270–1279.

Fraignier, M.P., Marques, L., Fleuriet, A., Macheix, J.J., 1995.

Biochemical and immunochemical characteristics of polyphenol

oxidases from different fruits of Prunus. J. Agric. Food Chem. 43,

2375–2380.

Garcia-Borron, J.C., Solano, F., 2002. Molecular anatomy of tyr-

osinase and its related proteins: beyond the histidine-bound metal

catalytic center. Pigment Cell Res. 15, 162–173.

Gerritsen, Y.A.M., Chapelon, C.G.J., Wichers, H.J., 1994. The low

isoelectric point tyrosinase of Agaricus bisporus may be a glycopro-

tein. Phytochem 35, 573–577.

Hearing, V.J., Tsukamoto, K., 1991. Enzymatic control of pigmenta-

tion in mammals. The FASEB J. 5, 2903–2910.

Jimenez, M., Garcia-Carmona, F., 1996. The effect of sodium dodecyl

sulfate on polyphenol oxidase. Phytochem 42, 1503–1509.

Jolivet, S., Arpin, N., Wichers, H.J., Pellon, G., 1998. Agaricus bis-

porus browning: a review. Mycol. Res. 102, 1459–1483.

Korner, A., Pawelek, J., 1982. Mammalian tyrosinase catalyzes three

reactions in the biosynthesis of melanin. Science 217, 1163–1165.

Marques, L., Fleuriet, A., Machiex, J.J., 1995. Characterization of

multiple forms of polyphenol-oxidase from apple fruit. Plant Phy-

siol. Biochem. 33, 193–200.

Moore, B.M., Flurkey, W.H., 1990. Sodium dodecyl sulfate activation

of a plant polyphenol-oxidase. J. Biol. Chem. 265, 4982–4988.

Nunez-Delicado, E., Sojo, M.M., Garcia-Carmona, F., Sanchez-

Ferrer, A., 2003. Partial purification of latent persimmon fruit

polyphenol oxidase. J. Agric. Food Chem. 51, 2058–2063.

Rast, D.M., Stussi, H., Hegnauer, H., Nyhlen, L.E., 1981. Melanins.

In: Turian, G., Hohl, H.R. (Eds.), The Fungal Spore: Morpho-

genetic Controls. Academic Press, London, pp. 507–531.

Rast, D.M., Baumgartner, D., Mayer, C., Hollenstein, G.O., 2003.

Cell wall-associated enzymes in fungi. Phytochem 64, 339–366.

Rodriquez-Lopez, J.N., Escribano, J., Garcia-Canovas, F., 1994. A

continuous spectrophotometric method for determination of mono-

phenolase activity of tyrosinase using 3-methyl-2-benzothiazolinone

hydrazone. Anal. Biochem. 216, 2005–2012.

Soulier, L., Foret, V., Arpin, N., 1993. Occurrence of agaritine and g- glutaminyl-4-hydroxybenzene (GHB) in the fructifying mycelium of

Agaricus bisporus. Mycol Res. 97, 529–532.

Van Gelder, C.W.G., Flurkey, W.H., Wichers, H.J., 1997. Sequence

and structural features of plant and fungal tyrosinases. Phytochem

45, 1309–1323.

Van Leeuwen, J., Wichers, H.J., 1999. Tyrosinase activity and isoform

composition in separate tissues during development of Agaricus

bisporus fruit bodies. Mycol. Res. 103, 413–418.

Wichers, H.J., Gerritsen, Y.A.M., Chapelon, C.G.J., 1996. Tyrosinase

isoforms from the fruit bodies of Agaricus bisporus. Phytochem 43,

333–337.

Ylostalo, J., Srivastava, K., Flurkey, W.H., 2001. Characterization of

a tyrosinase isoform from the cap skin of Portabella mushrooms. J.

Food Biochem. 25, 493–507.

Zhang, X., Flurkey, W.H., 1999. Purification and partial character-

ization of tyrosinase isoforms from cap flesh of Portabella mush-

rooms. J. Food Biochem. 23, 95–108.

Zhang, X., Flurkey, W.H., 1997. Phenoloxidases in Portabella mush-

rooms. J. Food Sci. 62, 97–100.

Zhang, X., van Leeuwen, J., Wichers, H.J., Flurkey, W.H., 1999.

Characterization of tyrosinase from the cap flesh of Portabella

mushrooms. J. Agric. Food Chem. 47, 374–378.

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