- Email: [email protected]
Activity, isoenzymes and purity of mushroom tyrosinase in commercial preparations
Phytochemistry,
Vol. 30. No. 12. pp. 3899 3902, 1991
Printedin Grrat Britain.
ACTIVITY,
0
ISOENZYMES AND PURITY OF MUSHROOM COMMERCIAL PREPARATIONS
0031 -9422/91 $3.00 + 0.00 1991Pcrgamon Press plc
TYROSINASE
IN
MANISH KUMAR and WILLIAM H.FLURKEY* Department of Chemistry, Indiana State University, Terre Haute, IN 47809, U.S.A. (Received
Key Word Index--Agarcius
bispow;
mushroom;
19 April 1991)
tyrosinase;
isoenzymes;
purity;
commercial
preparations
Abstract-Several lots of commercial tyrosinase preparations were examined with regard to their enzyme activity, isoenzyme composition and purity. Enzyme activity toward catechol, t-dopa and tyrosine showed significant variations from lot to lot and activation by SDS. Distribution of isoenzyme forms also varied from lot to lot. Comparisons of electrophoretic and isoelectric focusing protein profiles showed considerable differences and distributions of the proteins in each sample. Tyrosinase appeared to be a minor component in each preparation when compared to a partially purified enzyme. Investigators using commercial tyrosinase should exercise caution in interpreting data due to the presence of different isoenzyme forms, their distribution in various lots, and the presence of numerous other proteins.
INTRODUCTION Tyrosinase (monophenol, dihydroxyphenylalanine, oxygen oxidoreductase, EC 1.14.18. I) is found throughout plant and fungal species [l-3]. The enzyme from mushrooms has received considerable attention within the last 10 years. Most of this attention and subsequent investigations have relied on commercial sources for their studies. Representative topics in these studies have included examination of inhibitor effects using methimazole [4], mimosine [SJ, tropolone [63, ascorbic acid and its derivatives [7, 81, cysteine [9] and 3-amino t_-tyrosine [lo]. Other studies have used commercial tyrosinase to explore new substrates such as phosphonic analogue of tyrosine and dopa [ll], l-naphthol [12], oxidation of 3,4-dihydroxymandelic acid [ 133, oxidation of dopaquinone addition products [14], and novel substrates [15]. Other investigators have used the commercial enzyme to examine the effects of various compounds on the enzyme or kinetic characteristics of tyrosinase [16]. Three unusual enzyme activities have also been recently attributed to the mushroom enzyme. These include the oxidative dimerization of 1,2-dehydro-n-acetyldopamine [ 173, oxidative decarboxylation of 3,4-dihydroxymandelate [ 181 and ferroxidase activity [19]. Each of the above reports used commercial preparations although Boyer et al. [ 191 used a partially purified tyrosinase from commercial sources. The commercial enzyme has also been used for studies involving enzymatic reactions in organic media [20], affinity chromatography [21], development of new assays for the enzyme [22] and control of browning in processed mushrooms [23]. With few exceptions, most of the above investigations have not reported or examined the isoenzyme content of tyrosinase in these preparations, nor have they examined the purity of the enzyme preparations. These factors are important
*Author
to whom correspondence
should
be addressed.
because different isoenzyme forms could display specific inhibitor and kinetic characteristics. In addition, impure preparations could contain other enzymes capable of interfering with or catalyzing ancillary reactions utilizing potential substrates or inhibitors of tyrosinase. In this study we set out to examine the isoenzyme forms in commercial mushroom tyrosinase by looking at differences in specific activities and variations in isoenzyme forms by electrophoresis. Purity and comparisons between different lots were examined, by SDS-PAGE and IEF. RESL!LTSANDDlSCUS!3ON
There was considerable variation in the specific activity of mushroom tyrosinase from one preparation to another (Table 1). These variations occurred irrespective of the substrate used to monoitor the enzyme activity (i.e. catechol, dopa or tyrosine). Dopa oxidase and tyrosine hydroxylation activity appeared to follow a consistent pattern from lot to lot, but we did not observe a consistent trend with regard to catechol oxidase activity. We did not find any large differences in the K, for dopa (0.174.26 mM) or the V,,, for dopa (0.17-0.31 units min - 1 ml-‘) among the four lots examined. Using o-diphenol substrates, some preparations appeared to be activated by SDS (lot 39F) while others did not appear to be so responsive and/or were inhibited to a slight degree (26F, 127F, 99F). This suggests the presence of variable amounts of latent enzyme in each preparation. The ratio of activation by SDS showed no discernible pattern from lot to lot using catechol or dopa as substrates. On the other hand, SDS severely inhibited hydroxylation of tyrosine in all lots examined. The amount of total latent enzyme in each preparation was not determined, but the presence of latent enzyme could have serious implications in reports investigating enzyme kinetics, substrate or inhibitor analogs, and compounds that influence tyrosinase activity.
M. KUMAR and W. H. FLURKF.Y
:B
A
i
1
ycc:
2 Fig. 1. Native, partially denaturing, denaturing electrophoresis, and isoelectric focusing of commercial preparations. (A). Native electrophoresis. Lane I. lot 26F. Arrows denote presence of a faint staining area. Lane 2, lot 127F. Lane 3, lot 99F. Lane 4, lot 39F. Arrows in lanes 3 and 4 denote doublet of closely migrating bonds. Dopa oxidase activity was located as described in the Experimental. (B). Partially denaturing electrophoresis. Lane I. lot 26F. Arrows denote areas of weak staining in lanes I and 2. Lane 2, lot 127F. Lane 3, lot 99F. Arrows denote location of two bands of nearly equal staining intensity in lanes 3 and 4. Lane 4, lot 39F. Dopaoxidase activity was located as described above. Molecular weight marker positions are noted on the right.(C). Denaturing SDS PAGE. Lane I, lot 26F. Lane 2, lot 127 F. Lane 3, lot 99F. Lane 4. lot 39F. Approximately 50 /(g ofcrude solid was loaded each lane I- 4. Lane 5, partially purified mushroom (ca 3 pg) tyrosinase from lot 26F-9515. Molecular weight markers arc designated at the right. Proteins were located by staining wtth Coommassie Blue R 250. (D). Isoelectric focusing. Each lot (ca 35 pg solid) was loaded on a horizontal precast 3-7 IEF gel. Lane 1, lot 26F. Lane 2. lot 127F. Lane 3, lot 99F. Lane 4. lot 39F. Lane 5, partially purified mushroom tyrosinose. Proteins were located by staining the gel with Coommassie Blue R 250. Arrow indicates region exhibiting dopa oxidase activity. Isoelectric focusing was carried out as described in the Experimental.
3901
Mushroom tyrosinase Table 1. Enzyme activities associated with commercial mushroom tyrosinase
Lot
-SDS
26F 127F 99F 39F
15.0 16.0 9.2 10.2
Catechol (units mg- ‘) +SDS + 15.2 17.8 8.8 18.9
1.01 1.12 0.95 1.81
-SDS 18.8 23.1 13.3 28.0
Dopa (units mg-1) +SDS + 19.5 18.6 21.1 38.9
1.04 0.80 1.59 1.39
-SDS 1.82 1.96 1.51 4.60
Tyrosine (units mg- ‘) +SDS + 0.61 0.63 0.56 3.24
0.33 0.32 0.37 0.70
+ Indicates the ratio of activity measured in the presence of SDS to that measured in the absence
of SDS. Isoenzyme composition was determined by native and partially denaturing PAGE. Most lots of tyrosinase contained the same number of isoenzyme forms, although the distribution of the forms varied from lot to lot. For example, using native PAGE lot 26F showed a dominant slow moving form with very small amounts of two fast moving forms (Fig. 1A). On the other hand, lots 99F and 39F showed a similar distribution but contained much larger amounts of the faster moving forms. Each faster moving form was composed of a closely migrating doublet. Under partially denaturing conditions (+ SDS, but not boiled), a similar pattern was observed but the distribution of isoenzyme forms was much more clear (Fig. 1B). Lots 26F and 127F also contained minor amounts of intermediate and faster migrating forms. Crude mushrooms extracts contained isoenzyme patterns similar to lot 99F and 39F although the distribution of staining was different [24, 251. These results appear to indicate that some preparations may lack or contain inactive isoforms that are often found in crude mushroom extracts. Thus, commercial sources of tyrosinase may not be representative of the enzyme found in live mushrooms. Purity of the commercial mushroom tyrosinase was monitored by native IEF and denaturing SDS PAGE. Each tyrosinase preparation showed numerous proteins after SDS PAGE (Fig. 1C). The distribution of proteins in each sample varied from lot to lot although a similar protein profile was observed in all lots. Mushroom tyrosinase is thought to be composed of a heavy subunit (H, 43 000) and a light subunit (L, 13 000 [26, 271. Analysis of the proteins in this M, region identified three to four proteins which may be the H subunit and two or three proteins which may be the L subunit. In comparison to a partially purified enzyme from lot 26F, it would appear that the 43 C@O protein found in all preparations is the H subunit. However, the amount of this protein varies substantially from lot to lot. We have not been able to make a tentative identification of the L subunit because of two proteins in the 10000-15000 range in our partially purified samples. These results showed that the commercial tyrosinase preparations were not pure and that the tyrosinase enzyme is not a major protein component. We do not know how many of the remaining proteins are not related to tyrosinase, nor do we know if these other proteins represent degradation products or aggregation states of the enzyme. Native IEF showed that multiple proteins were present in each lot (Fig. 1D). Some lots showed similar distribution of proteins (26F and 127F) while other showed a much different distribution (39F and 99F). Enzyme activity staining with Ldopa indicated a broad band of staining in all samples and at a similar p1 (Fig. lD, arrow).
However, this band was so diffuse that we could not correlate it with a specific protein in this region. In any case, IEF showed that many other proteins were present and unrelated to active mushroom tyrosinase. The results of this investigation show that mushroom tyrosinase preparations contain several isoenzymes. These forms may or may not have similar substrate or inhibitor specificities. It is also apparent that each commercial preparation contains numerous proteins besides tyrosinase and that each preparation has its own characteristic protein profile. Speculatively, these undefined proteins could affect tyrosinase activity or account for other enzymatic activities associated with the enzyme. Until the enzyme can be obtained in a highly purified form from commercial preparations, many of these aforementioned observations remain uncertain. The variable protein content, composition, and tyrosinase distribution in each lot probably accounts for the observed differences in specific activities. We have nbt investigated how much mass of the enzyme preparation may contain carbohydrate, phenolic or other nonprotein material. This also may affect tyrosinase activity. Taken together, our results indicate that caution should be observed in interpreting data using commercial enzyme sources due to varied isoenzyme composition and contaminating proteins. EXPERIMENTAL
Materids. Mushroom tyrosinase (lots 26F-9515, 127F-9670, 99F-9535,39F-9515) were obtained from Sigma. Powder (10 mg) from each lot was resuspended in 1 ml deionized H,O. Samples were stored frozen at -20” in small aliquots. Enzyme assays. Enzyme activity was determined in the presence and absence of 0.1% SDS (w/v) using either catechol, ~-dopa, or tyrosine as substrates [24, 251. One unit of activity was defined as change of 1 absorbance unit per min at 25”. Electrophoresis. Native, partially denaturing SDS-PAGE, and denaturing SDS-PAGE were carried out as described earlier [28]. Gels were stained for dopa oxidase using 2 mM L-dopa in 0.1 M Pi (pH 6.0). Isoelectric focusing was carried out at 10” for 1 hr using precast agarose 3-7 IEF gels (FMC Corp., Rockland, ME) in a horizontal IEF unit with Peltier cooling. IEF gels were stained for protein using Serva Blue W and or Coommassie Blue R-250. Acknowledgements-We thank C. L.eClerc and P. Archer for help in typing this manuscript. REFERENCES
1. Mayer, A. M. and Harel, E. (1979) Phytochemislry 18, 193. 2. Mayer, A. M. (1987) Phytochemistry 26, 11.
3902
M. KUMAR and W. H. FLURKEY
3. Vamos-Vigyazo. L. (1981) CRC Crir. Rec. Food Sci. Nutrit. 15, 49. 4. Andrawis, A. and Kahn, V. (1986) Biochem. J. 235, 91. 5. Cabanes, J.. Garcia-Canovas, F., Tudela, J.. Lozano, J. A. and Garcia-Carmona, F. (1987) Phytochemistry 26, 917. 6. Kahn, V. and Andrawis, A. (1985) Phytochemistry 24, 905. 7. Golan-Goldhirsh, A. and Whitakcr, J. R. (1984) J. Agric. f’ood. Chem. 32, 1003. 8. Hsu. A. F., Shieh. J. J. and White. K. (1988) J. Food Sci. 53, 765. 9. Dudley, E. D. and Hotchkiss, J. H. (1989) J. Food Biochem. 13. 65. 10. Maddaluno, J. F. and Faull. K. F. (1988) Experientia 44,885. 11. Lejczak, B., Kafarski, P. and Makowiecka, E. (1987) Biothem. J. 242. 81. 12. Doherty, M. D., Cohen, G. M., Gant, T. W., Naish, S. and Riley, P. A. (1985) Biochem. Pharmacol. 34, 3167. 13. Ortiz, F. M. Serrano, J. T., Lopez, J. N. R., Castellanos, R. V., Teruel, J. A. L. and Garcia-Canovas, F. (1988) Biochim. Biophys. 14. Rorsman,
C.. Rosengren,
E. and Agrup,
(198 1) Pigment CelI 6. 67. M. E. and Cohen, G. (1983) Biochemistry 16. Garcia-Carmona, F., Valero, E. and Cabanes, Phyrochemistry 27, 196 1.
IS. Morrison,
M., Dali, H. and Hcnningan,
G.
22.5465.
J. (1988)
B. (I 987) J. BioL
Chem. 262, 10546.
18. Sugumaran, M. ( 1986) Biochcmisrr.v 25, 4489. 19. Boyer. R. F., Mascotti, D. P. and Schori. B. E. (1986) Phyrochemistry 25. 12X 1. 20. Zaks, A. and Khbanov. A. M. (19X8) J. Biol. Chem. 263.8017. 21. lngebrigtsen, J. and Flurkey, W. H. (1988) Phyfochemisrry 27, 1593. 22. Rzepecki.
L. M. and Waite. H. (1989) Awl.
Eiochem.
179,
375. 23. McCord,
J. D. and Kilara. A. (1983) J. Food Sci. 48, 1479. 24. Ingebrigtsen. J., Kang. B. and Flurkey. W. H. (1989) J. Food sci. 54, 128. 25. Flurkey, W. H. and Ingebrigtsen, J. (1989) in Quulify focrors of Fruirs and Veyetahles, ACS Symposium Series 405, (Jen, J. J., ed.), pp. 4454, American Chemical Society. Washington. DC. 26. Strothkamp, K. G., Jollcy. R. L. and Mason. H. S. (1976) Biochem.
Biophys.
27. Gutteridge,
Acta 957. 158.
H. Hannson,
17. Sugumaran,
and
Cfrn~c~I
Res. Commun. 70. 519.
S. and Aspecrs
Mason,
H. S. (1979) rn Biochemical (Caughey. W. S.. ed.), Press. New York.
c$ Oxygen
pp. 589602. Academic 28. Flurkey, W. H. (I 990) Phyrochemisrr>,
29. 387.
Vol. 30. No. 12. pp. 3899 3902, 1991
Printedin Grrat Britain.
ACTIVITY,
0
ISOENZYMES AND PURITY OF MUSHROOM COMMERCIAL PREPARATIONS
0031 -9422/91 $3.00 + 0.00 1991Pcrgamon Press plc
TYROSINASE
IN
MANISH KUMAR and WILLIAM H.FLURKEY* Department of Chemistry, Indiana State University, Terre Haute, IN 47809, U.S.A. (Received
Key Word Index--Agarcius
bispow;
mushroom;
19 April 1991)
tyrosinase;
isoenzymes;
purity;
commercial
preparations
Abstract-Several lots of commercial tyrosinase preparations were examined with regard to their enzyme activity, isoenzyme composition and purity. Enzyme activity toward catechol, t-dopa and tyrosine showed significant variations from lot to lot and activation by SDS. Distribution of isoenzyme forms also varied from lot to lot. Comparisons of electrophoretic and isoelectric focusing protein profiles showed considerable differences and distributions of the proteins in each sample. Tyrosinase appeared to be a minor component in each preparation when compared to a partially purified enzyme. Investigators using commercial tyrosinase should exercise caution in interpreting data due to the presence of different isoenzyme forms, their distribution in various lots, and the presence of numerous other proteins.
INTRODUCTION Tyrosinase (monophenol, dihydroxyphenylalanine, oxygen oxidoreductase, EC 1.14.18. I) is found throughout plant and fungal species [l-3]. The enzyme from mushrooms has received considerable attention within the last 10 years. Most of this attention and subsequent investigations have relied on commercial sources for their studies. Representative topics in these studies have included examination of inhibitor effects using methimazole [4], mimosine [SJ, tropolone [63, ascorbic acid and its derivatives [7, 81, cysteine [9] and 3-amino t_-tyrosine [lo]. Other studies have used commercial tyrosinase to explore new substrates such as phosphonic analogue of tyrosine and dopa [ll], l-naphthol [12], oxidation of 3,4-dihydroxymandelic acid [ 133, oxidation of dopaquinone addition products [14], and novel substrates [15]. Other investigators have used the commercial enzyme to examine the effects of various compounds on the enzyme or kinetic characteristics of tyrosinase [16]. Three unusual enzyme activities have also been recently attributed to the mushroom enzyme. These include the oxidative dimerization of 1,2-dehydro-n-acetyldopamine [ 173, oxidative decarboxylation of 3,4-dihydroxymandelate [ 181 and ferroxidase activity [19]. Each of the above reports used commercial preparations although Boyer et al. [ 191 used a partially purified tyrosinase from commercial sources. The commercial enzyme has also been used for studies involving enzymatic reactions in organic media [20], affinity chromatography [21], development of new assays for the enzyme [22] and control of browning in processed mushrooms [23]. With few exceptions, most of the above investigations have not reported or examined the isoenzyme content of tyrosinase in these preparations, nor have they examined the purity of the enzyme preparations. These factors are important
*Author
to whom correspondence
should
be addressed.
because different isoenzyme forms could display specific inhibitor and kinetic characteristics. In addition, impure preparations could contain other enzymes capable of interfering with or catalyzing ancillary reactions utilizing potential substrates or inhibitors of tyrosinase. In this study we set out to examine the isoenzyme forms in commercial mushroom tyrosinase by looking at differences in specific activities and variations in isoenzyme forms by electrophoresis. Purity and comparisons between different lots were examined, by SDS-PAGE and IEF. RESL!LTSANDDlSCUS!3ON
There was considerable variation in the specific activity of mushroom tyrosinase from one preparation to another (Table 1). These variations occurred irrespective of the substrate used to monoitor the enzyme activity (i.e. catechol, dopa or tyrosine). Dopa oxidase and tyrosine hydroxylation activity appeared to follow a consistent pattern from lot to lot, but we did not observe a consistent trend with regard to catechol oxidase activity. We did not find any large differences in the K, for dopa (0.174.26 mM) or the V,,, for dopa (0.17-0.31 units min - 1 ml-‘) among the four lots examined. Using o-diphenol substrates, some preparations appeared to be activated by SDS (lot 39F) while others did not appear to be so responsive and/or were inhibited to a slight degree (26F, 127F, 99F). This suggests the presence of variable amounts of latent enzyme in each preparation. The ratio of activation by SDS showed no discernible pattern from lot to lot using catechol or dopa as substrates. On the other hand, SDS severely inhibited hydroxylation of tyrosine in all lots examined. The amount of total latent enzyme in each preparation was not determined, but the presence of latent enzyme could have serious implications in reports investigating enzyme kinetics, substrate or inhibitor analogs, and compounds that influence tyrosinase activity.
M. KUMAR and W. H. FLURKF.Y
:B
A
i
1
ycc:
2 Fig. 1. Native, partially denaturing, denaturing electrophoresis, and isoelectric focusing of commercial preparations. (A). Native electrophoresis. Lane I. lot 26F. Arrows denote presence of a faint staining area. Lane 2, lot 127F. Lane 3, lot 99F. Lane 4, lot 39F. Arrows in lanes 3 and 4 denote doublet of closely migrating bonds. Dopa oxidase activity was located as described in the Experimental. (B). Partially denaturing electrophoresis. Lane I. lot 26F. Arrows denote areas of weak staining in lanes I and 2. Lane 2, lot 127F. Lane 3, lot 99F. Arrows denote location of two bands of nearly equal staining intensity in lanes 3 and 4. Lane 4, lot 39F. Dopaoxidase activity was located as described above. Molecular weight marker positions are noted on the right.(C). Denaturing SDS PAGE. Lane I, lot 26F. Lane 2, lot 127 F. Lane 3, lot 99F. Lane 4. lot 39F. Approximately 50 /(g ofcrude solid was loaded each lane I- 4. Lane 5, partially purified mushroom (ca 3 pg) tyrosinase from lot 26F-9515. Molecular weight markers arc designated at the right. Proteins were located by staining wtth Coommassie Blue R 250. (D). Isoelectric focusing. Each lot (ca 35 pg solid) was loaded on a horizontal precast 3-7 IEF gel. Lane 1, lot 26F. Lane 2. lot 127F. Lane 3, lot 99F. Lane 4. lot 39F. Lane 5, partially purified mushroom tyrosinose. Proteins were located by staining the gel with Coommassie Blue R 250. Arrow indicates region exhibiting dopa oxidase activity. Isoelectric focusing was carried out as described in the Experimental.
3901
Mushroom tyrosinase Table 1. Enzyme activities associated with commercial mushroom tyrosinase
Lot
-SDS
26F 127F 99F 39F
15.0 16.0 9.2 10.2
Catechol (units mg- ‘) +SDS + 15.2 17.8 8.8 18.9
1.01 1.12 0.95 1.81
-SDS 18.8 23.1 13.3 28.0
Dopa (units mg-1) +SDS + 19.5 18.6 21.1 38.9
1.04 0.80 1.59 1.39
-SDS 1.82 1.96 1.51 4.60
Tyrosine (units mg- ‘) +SDS + 0.61 0.63 0.56 3.24
0.33 0.32 0.37 0.70
+ Indicates the ratio of activity measured in the presence of SDS to that measured in the absence
of SDS. Isoenzyme composition was determined by native and partially denaturing PAGE. Most lots of tyrosinase contained the same number of isoenzyme forms, although the distribution of the forms varied from lot to lot. For example, using native PAGE lot 26F showed a dominant slow moving form with very small amounts of two fast moving forms (Fig. 1A). On the other hand, lots 99F and 39F showed a similar distribution but contained much larger amounts of the faster moving forms. Each faster moving form was composed of a closely migrating doublet. Under partially denaturing conditions (+ SDS, but not boiled), a similar pattern was observed but the distribution of isoenzyme forms was much more clear (Fig. 1B). Lots 26F and 127F also contained minor amounts of intermediate and faster migrating forms. Crude mushrooms extracts contained isoenzyme patterns similar to lot 99F and 39F although the distribution of staining was different [24, 251. These results appear to indicate that some preparations may lack or contain inactive isoforms that are often found in crude mushroom extracts. Thus, commercial sources of tyrosinase may not be representative of the enzyme found in live mushrooms. Purity of the commercial mushroom tyrosinase was monitored by native IEF and denaturing SDS PAGE. Each tyrosinase preparation showed numerous proteins after SDS PAGE (Fig. 1C). The distribution of proteins in each sample varied from lot to lot although a similar protein profile was observed in all lots. Mushroom tyrosinase is thought to be composed of a heavy subunit (H, 43 000) and a light subunit (L, 13 000 [26, 271. Analysis of the proteins in this M, region identified three to four proteins which may be the H subunit and two or three proteins which may be the L subunit. In comparison to a partially purified enzyme from lot 26F, it would appear that the 43 C@O protein found in all preparations is the H subunit. However, the amount of this protein varies substantially from lot to lot. We have not been able to make a tentative identification of the L subunit because of two proteins in the 10000-15000 range in our partially purified samples. These results showed that the commercial tyrosinase preparations were not pure and that the tyrosinase enzyme is not a major protein component. We do not know how many of the remaining proteins are not related to tyrosinase, nor do we know if these other proteins represent degradation products or aggregation states of the enzyme. Native IEF showed that multiple proteins were present in each lot (Fig. 1D). Some lots showed similar distribution of proteins (26F and 127F) while other showed a much different distribution (39F and 99F). Enzyme activity staining with Ldopa indicated a broad band of staining in all samples and at a similar p1 (Fig. lD, arrow).
However, this band was so diffuse that we could not correlate it with a specific protein in this region. In any case, IEF showed that many other proteins were present and unrelated to active mushroom tyrosinase. The results of this investigation show that mushroom tyrosinase preparations contain several isoenzymes. These forms may or may not have similar substrate or inhibitor specificities. It is also apparent that each commercial preparation contains numerous proteins besides tyrosinase and that each preparation has its own characteristic protein profile. Speculatively, these undefined proteins could affect tyrosinase activity or account for other enzymatic activities associated with the enzyme. Until the enzyme can be obtained in a highly purified form from commercial preparations, many of these aforementioned observations remain uncertain. The variable protein content, composition, and tyrosinase distribution in each lot probably accounts for the observed differences in specific activities. We have nbt investigated how much mass of the enzyme preparation may contain carbohydrate, phenolic or other nonprotein material. This also may affect tyrosinase activity. Taken together, our results indicate that caution should be observed in interpreting data using commercial enzyme sources due to varied isoenzyme composition and contaminating proteins. EXPERIMENTAL
Materids. Mushroom tyrosinase (lots 26F-9515, 127F-9670, 99F-9535,39F-9515) were obtained from Sigma. Powder (10 mg) from each lot was resuspended in 1 ml deionized H,O. Samples were stored frozen at -20” in small aliquots. Enzyme assays. Enzyme activity was determined in the presence and absence of 0.1% SDS (w/v) using either catechol, ~-dopa, or tyrosine as substrates [24, 251. One unit of activity was defined as change of 1 absorbance unit per min at 25”. Electrophoresis. Native, partially denaturing SDS-PAGE, and denaturing SDS-PAGE were carried out as described earlier [28]. Gels were stained for dopa oxidase using 2 mM L-dopa in 0.1 M Pi (pH 6.0). Isoelectric focusing was carried out at 10” for 1 hr using precast agarose 3-7 IEF gels (FMC Corp., Rockland, ME) in a horizontal IEF unit with Peltier cooling. IEF gels were stained for protein using Serva Blue W and or Coommassie Blue R-250. Acknowledgements-We thank C. L.eClerc and P. Archer for help in typing this manuscript. REFERENCES
1. Mayer, A. M. and Harel, E. (1979) Phytochemislry 18, 193. 2. Mayer, A. M. (1987) Phytochemistry 26, 11.
3902
M. KUMAR and W. H. FLURKEY
3. Vamos-Vigyazo. L. (1981) CRC Crir. Rec. Food Sci. Nutrit. 15, 49. 4. Andrawis, A. and Kahn, V. (1986) Biochem. J. 235, 91. 5. Cabanes, J.. Garcia-Canovas, F., Tudela, J.. Lozano, J. A. and Garcia-Carmona, F. (1987) Phytochemistry 26, 917. 6. Kahn, V. and Andrawis, A. (1985) Phytochemistry 24, 905. 7. Golan-Goldhirsh, A. and Whitakcr, J. R. (1984) J. Agric. f’ood. Chem. 32, 1003. 8. Hsu. A. F., Shieh. J. J. and White. K. (1988) J. Food Sci. 53, 765. 9. Dudley, E. D. and Hotchkiss, J. H. (1989) J. Food Biochem. 13. 65. 10. Maddaluno, J. F. and Faull. K. F. (1988) Experientia 44,885. 11. Lejczak, B., Kafarski, P. and Makowiecka, E. (1987) Biothem. J. 242. 81. 12. Doherty, M. D., Cohen, G. M., Gant, T. W., Naish, S. and Riley, P. A. (1985) Biochem. Pharmacol. 34, 3167. 13. Ortiz, F. M. Serrano, J. T., Lopez, J. N. R., Castellanos, R. V., Teruel, J. A. L. and Garcia-Canovas, F. (1988) Biochim. Biophys. 14. Rorsman,
C.. Rosengren,
E. and Agrup,
(198 1) Pigment CelI 6. 67. M. E. and Cohen, G. (1983) Biochemistry 16. Garcia-Carmona, F., Valero, E. and Cabanes, Phyrochemistry 27, 196 1.
IS. Morrison,
M., Dali, H. and Hcnningan,
G.
22.5465.
J. (1988)
B. (I 987) J. BioL
Chem. 262, 10546.
18. Sugumaran, M. ( 1986) Biochcmisrr.v 25, 4489. 19. Boyer. R. F., Mascotti, D. P. and Schori. B. E. (1986) Phyrochemistry 25. 12X 1. 20. Zaks, A. and Khbanov. A. M. (19X8) J. Biol. Chem. 263.8017. 21. lngebrigtsen, J. and Flurkey, W. H. (1988) Phyfochemisrry 27, 1593. 22. Rzepecki.
L. M. and Waite. H. (1989) Awl.
Eiochem.
179,
375. 23. McCord,
J. D. and Kilara. A. (1983) J. Food Sci. 48, 1479. 24. Ingebrigtsen. J., Kang. B. and Flurkey. W. H. (1989) J. Food sci. 54, 128. 25. Flurkey, W. H. and Ingebrigtsen, J. (1989) in Quulify focrors of Fruirs and Veyetahles, ACS Symposium Series 405, (Jen, J. J., ed.), pp. 4454, American Chemical Society. Washington. DC. 26. Strothkamp, K. G., Jollcy. R. L. and Mason. H. S. (1976) Biochem.
Biophys.
27. Gutteridge,
Acta 957. 158.
H. Hannson,
17. Sugumaran,
and
Cfrn~c~I
Res. Commun. 70. 519.
S. and Aspecrs
Mason,
H. S. (1979) rn Biochemical (Caughey. W. S.. ed.), Press. New York.
c$ Oxygen
pp. 589602. Academic 28. Flurkey, W. H. (I 990) Phyrochemisrr>,
29. 387.