Michael E. Friedman and Harlow H. Daron Auburn University Auburn. Alabama 36830
Tyrosinase An introductory experiment with enzymes
Students frequently feel that enzymes are exotic materials ohtained from exotic sources. T h i s is partly because t h e enzyme they have worked with came from a tissue (or worse, a bottle) provided h y their teacher a n d which was relatively inaccessible to t h e student. W e feel that t h e student's initial contact with enzymes should emphasize their ubiquitous occurrence a n d availability from all living material, a n d suggest tyrosinase as-an enzyme that is easily extracted from sources t h a t s t u d e n t s are familiar with a n d can readily obtain from a grocery store. The entire cost of material in this experiment will h e less t h a n $1per student. Most students a r e aware t h a t when potatoes, apples, hananas, sweet potatoes, or mushrooms are injured they t u r n brown. This is d u e to the conversion of tyrosine to t h e pigment melanin, b y t h e sequence of reactions shown in Figure 1.T h e same process causes skin to t u r n hrown, following exposure to ultraviolet radiation. The enzymatic reactions are catalyzed b y tyrosinase (monophenol, dihydroxyphenylalanine: oxygen oxidoreductase; EC 1.14.18.1).' The enzyme is present in t h e interior of t h e plant material and, since t h e reaction requires molecular oxygen, the pigmentation does n o t occur until t h e interior is exposed.
Materials 0.1 M sodium phosphate buffer, p H 7.2 0.1 M sodium phosphate buffer, p H 6.0 20 mM D,L-dihydroxyphenylalanine(DOPA),prepared by adding 4 mg D,L DOPA11 mlO.l p H 6.0 buffer Source of enzyme: apple, banana, mushroom, potato, etc. Sand Cheesecloth Mortar and pestle Calorimeter or spectraphotometer
Experimental Enzyme Extraction
As carried out in our teaching laboratory, the experiment uses potatoes, apples, bananas, and mushrooms as sources of tyrosinase. Between 10 and 15 g of material is placed in a precooled mortar and one-half the amount, vollwt of 0.1 M phosphate buffer, p H 7.2, is added. With banana, an equal amount of buffer is added to get a filterable mixture. A few grams of acid-washed sand are added and the material is crushed thoroughly until it has a pasty texture. This mixture is filtered through a double layer of cheesecloth and centrifuged at full speed in a clinical centrifuge for approximately 5 min. The supernatant solution is decanted and placed in an ice bath. The extracts are characteristically dark brown and may darken upon standing. The extracts from mushroom and potato are diluted with 9 volumes of buffer for assay, and the extract from banana is diluted with 4 volumes of buffer. The extract from apples is assayed without dilution. Therefore, 0.5 ml of apple extract is equivalent to 1g af solid while 5.0 ml of the other extracts are equivalent to 1g of solid. Enzyme Assay
The enzyme is easily assayed wlorimetridy by measuring the rate of conversion of 3.4-dihydroxyphenylalanine (DOPA) to DOPA~ h r o m e An . ~ initial lag period complicates the assay when tyrosine is used as substrate. The assay is conducted by placing0.1-0.4 mlof suitably diluted extract in a eolorimeter tube and bringing the total '"Enzyme Nomenclature," International Union of Biochemistry, Elsevier Scientific Publishing Co., Inc., Amsterdam, 1973, p. 114. 2Horowitz, N. H., and Fling, M., Genetics, 38,360, (1953). 258 1 J m l of Chemical Education
dihydmryphenylalanine (DOPA)
a,- ~ m r l t m
HO
COOH leu- compovnd
DOPA
qvinone
Figure 1. Scheme of the formation of melanin from tyrosine as catalyzed by Wrosinase. volume to 4.0 ml with 0.1 M phosphate huffer,pH 6.0. The reaction is initiated by adding 1.0 ml of substrate solution. The absorbance at 420 nm is measured immediately after adding the substrate, and readings are made at room temperature every minute, for a period of 4-5 min, or at zero time and 5 min after the reaction has been initiated. Tubes are thorouehlv mixed before measurine the absorbance. The activity is computed by taking the difference between the initial and final readings and dividing by the time interval. The activity can also be determined from the slope of graph of absorbance versus time. I t will also be advisable to keep the temperature of the reaction constant by immersing the assay tubes in a water bath a t 30°C. An extra exoeriment can be oerformed bv heatine some of the extract for 5 10 min in a boiling u,ater bath and rhrn performlnn thp exprimens alter cmling thr solutim. ' h e denarm-d enqmp shc,uld yield little or no activity.
".
.~
Results An example of a typical series of data is given for the potato extract in Figure 2. It is observed from this figure that the absorbance is linear with time for about 5 min, after which theactivity decreases. This is due to the slow conversion of the DOPAchrome to other products which are precursors of melanin. The color of the solution will begin to turn from reddish-orange to reddish-gray after 5-10 min of standing. The computation of the data From Figure 2, as well as some additional data, for the activity of potato tyrosinase is given in Table 1. The data for apple, mushroom and banana are given in Table 2. The results from both Tables 1and 2 show that the substance with the highest specific activity of tyrosinase is the banana, with an activity of 0.89 AAlminlg of material. The potato tyrosinase with a specific activity of 0.41 AAlminlg and the mushroom enzyme with aspecifie activity of 0.39 AAIminlgare about the same, with avalue slightly less than half that of the banana. The lowest amount of tyrosinase was obtained from the apple which yielded avalue of 0.041. A 10%error was observed in all assays and this value appears consistent regardless of the source of enzyme.
Discussion The source of tyrasinase came from the pulp of each of these materials and not the skin. It might be interesting to compare the activity
Table 1. Activity of Potato Tyrosinare as a Function of Enzyme Concentration Volume
Astivitvlml Original
of Diluted
Extract (ml) 0.10 0.15 0.20 0.25 0.30 0.40
Activity ( A A l m i n ) 0.0075 0.0132 0.0176 0.0228 0.0253 0.0312
Extract
i 0.0005a
0.75 0.88 0.88 0.91 0.84 0.78
0.375 0.44 0.44 0.455 0.42 0.39 Average = 0.42 i 0.02
t 0.0009 0.0008 t 0.0011 t 0.0009 f
f
Recoverable Actiuitvlgrn Potato
0.0015
he original extract war diluted 1:10 with Duffer. The reaction mixture containea the volume of diiutea extract given in column one, buffer to bring the volume t o 4 ml, and 1 m l of rubrtrate rolu-
tion. a Average deviation of at least three tertr. Table 2.
Activity of Apple, Mushroom. and Banana Tyrosinas8 as a Function o f Enzyme Concentration
Activitvlml original extract Extract Ivolume) IAAlmin.ml original extract1
~ecoverable Activirvlgm tissue
Apple (undiluted)
0
v
1 2 3 4 5 TIME, MINUTES
6
7
8
Mushroom (diluted lr9)
Figure 2. Absorbance versus time measuremems of he tymsinaw, enzyme ham potato extracts using variws a m t s of exbact. The aliquots of @to exbact used in each of the above experiments are given as fallows: Solid line (--). 0.15 mi exbact: Broken line (- -1. 0.20 ml eman: Dashed line (-). 0.30 ml emact: and Bmken and dotted line (- - .).0.40 ml extract. The bracketed lines (I) represent the range of data f a several points on a given curve. At least three runs on each of two different samples were made.
.
0.10 0.15 0.25 0.30 0.40
--- ---
of the enzyme in each of these parts of thematerial, especially the banana. When the substrate concentration was 2-3 times greater than in the normal assay the activity increased only 1C-20%. so there seems to he no great advantage in increasing the substrate concentration. The method presented here is aquickand easy way toobtain, and to determine, tyrosinase activity in a variety of materials. The more inquisitive student may wish to further investigate the distribution of the enzyme in other tissues, the effect of substrate concentration
0.08 0.06 0.05 0.05 0.07 0.08
0.1 0.2 0.25 0.30 0.40 0.50
Average = 0.39
Banana (diluted 1:4) 0.10 0.15 0.20 0.25 0.30 0.50
0.82 f 0.06 0.795 f 0.07 0.96 i 0.05 0.07 0.82 0.895 t 0.06
* 0.82 0.795 0.96 0.82
+
0.895
Average = 0.89
+ 0.08
The assay conditions were the same as thore in t able 1.
a Average deviation of at least three tertr.
on reaction rate (determination of Michaelis constant), the effect of inhibitors (e.g. cyanide, a noncompetitive inhibitor), and the copper content in the enzvme.
Volume 54, Number 4, April 1977 / 257
