THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 238, No. 6,June1963

Printed in U.S.A.

The Isolation and Properties of Crystalline

Tyrosinase from Neurospora*

MARGUERITE FLING, N. H. HOROWITZ, AND STEPHEN F. HEINEMANN~

From the Division of Biology, Calijornia Institute of Technology, Pasadena, California

(Received for publication, February 4, 1963)

Tyrosinase (EC 1.10.3.1) occurs in Neurospcra crassa in a number of forms which can be distinguished from one another by their thermostability or electrophoretic properties. Four such forms of the enzyme have been recognized, and these are in- herited as a series of Mendelian alternatives, or alleles (1, 2). In an earlier publication, we described a procedure for obtaining a 40- to 45-fold purification of the enzyme (1). Since the tyro- sinase content of the crude extracts was not over 0.5% of the soluble protein, the best preparations were less than 25% pure. Since then, it has been found that much higher quantities of tyrosinase-up to 5% of the extractable proteins-are produced by cultures which have been starved in phosphate buffer (3). These starved cultures may correspond to derepressed bacterial cells. This finding, combined with the use of columns of diato- maceous earth (Celite), whose special utility for the isolation of copper-containing proteins from Neurospora will be indicated be- low, has made it possible to isolate two of the four allelic forms of the enzyme in a crystalline state. A description of the fractiona- tion procedure and of some of the physical and chemical proper- ties of these enzymes follows. A preliminary account of some of the results has appeared (3).

EXPERIMENTAL PROCEDURE

Materials-All reagents were of analytical grade, unless other- wise indicated. Celite 535 (obtained from Johns-Manville) was washed several times by suspending it in distilled water and decanting. The slurry was poured into columns and equilibrated with 5 mM sodium phosphate buffer, pH 7.2, by running several hold-up volumes of buffer through each column.

Enzyme Assay-Tyrosinase was assayed by measuring the formation of 2-carboxy-2,3-dihydroindoled , 6-quinone (dopa- chrome) from 3,4-dihydroxy-nn-phenylalanine (nn-dopa) at pH 6 and 30”. The assay procedure has been described (4). The reaction was followed routinely with a Klett-Summerson photoelectric calorimeter equipped with a No. 42 filter. Under the conditions of the assay, the rate of dopachrome formation is linear with enzyme concentration between 0 and 0.14 pg of tyrosinase per ml (corresponding to readings of 0 to 150 colorime- ter units in the first 5 minutes of the reaction). When it was desired to monitor the reaction continuously, a Cary recording spectrophotometer set at 475 rnp was used.

* This work was supported by the National Science Foundation (Grant G-10718).

t Present address, the Biological Laboratories, Harvard Uni- versity, Cambridge, Massachusetts.

Tyrosinase Unit-In previous publications, we adopted as the unit of tyrosinase activity that amount of enzyme which produces an absorbancy increase, at 475 rnp, of 1.0 in the first 5 minutes after mixing the enzyme with IX-dopa in a final volume of 5 ml under the conditions described above. This is one of a variety of units, not all interconvertible, which have been employed by investigators of plant, animal, and fungal phenolases. Recently, the Enzyme Commission of the International Union of Bio- chemistry recommended that an enzyme unit be defined as the amount of enzyme which catalyzes the transformation of 1 pmole of substrate per minute under defined conditions (5). Since the adoption of a common unit would facilitate comparisons among different tyrosinases, we have derived a conversion factor by which our unit can be translated into the Enzyme Commission unit. The conversion is based on the following considerations.

1. Since our assay measures the rate of dopachrome appear- ance, rather than of dopa disappearance, it was necessary to establish the relation between these two quantities. It cannot be assumed that the appearance of a mole of dopachrome cor- responds to the disappearance of a mole of dopa, because dopa- chrome is not the immediate product of dopa oxidation (6). Investigation of this question showed, in fact, that 2 moles of dopa are oxidized for every mole of dopachrome produced (see “Molecular Activity” for details).

2. We use nn-dopa as substrate in routine assays because, in our experience, it is a more reliable product than commercial L-dopa. The two enantiomorphs of dopa are oxidized by Neuro- spora tyrosinase at different rates, however, so that the racemate constitutes a mixed substrate. In converting to the Enzyme Commission unit, a correction is therefore made to refer the data to L-dopa as substrate. For this purpose, the relative rates of oxidation of L-, DL-, and u-dopa by the Neurospora enzyme are taken as 1, 0.6, and 0.45, respectively.

3. The Neurospora enzyme shares with tyrosinases from other sources the property of becoming inactivated during the oxida- tion of dopa and other substrates (reaction-inactivation). Con- tinuous recordings made under the conditions of our assay show that the absorbancy at 475 rnp increases at a constant rate for about 2 minutes after mixing the reagents, after which the rate of increase gradually declines. The magnitude of the decline is such that the average rate of dopachrome formation per minute calculated from the first 5-minute reading is approximately 0.75 times the true initial rate. This correction is also incorporated into the conversion factor. There is an uncertainty of &lo% here, owing to the fact that crude preparations are less rapidly inactivated than purified ones. This uncertainty could be

2045

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2046 Crystalline Tyrosinase from Neurospora Vol. 238, n-o. 6

TABLE I Pwijication of tyrosinase

Values in the table are average yields per kg (wet weight) of N. crassa

Fraction

Crude extract.. MnS04 supernatant. First ammonium sulfatt

precipitate. Extract of acetone

precipitate. . . . Second ammonium sul-

fate precipitate. . . First Celite eluate Second Celite eluate

ml

3200

3200

200

100

50

150 100

Total activity

nits X IO-

10 9

8.6

7.5

7.2 3.9

2.36

-

4

Total protein

SpWitiC

activity

g rntdS/rn~ %

17 5.8

13.7 6.5 90

9.8 8.8 86

.- z

-

4.9 15.3 75

4.1 17.6 72 0.098 398 39 0.044 536 23.6

eliminated by taking readings at 1 or 2 minutes after mixing, instead of 5 minutes. The values recorded in this paper are based on 5-minute readings, however, unless otherwise indicated.

Given the molar extinction coefficient of dopachrome, ~~7~ ,,,,, = 3600 (7), we are led by the foregoing considerations to the result that a AA at 475 rnp of 0.81 per cm of light path in the initial 5 minutes of reaction under our assay conditions represents 1 Enzyme Commission unit. If L-dopa is employed as substrate, with other conditions the same, AA = 1.35 is 1 unit. The En- zyme Commission unit will be used henceforth in this paper.

Protein Determination-During the course of the isolation, protein concentrations were measured by the method of Lowry et al. (8), with crystalline bovine serum albumin as the standard. The protein content of pure preparations of the enzyme was determined by the optical density at 280 rnp, or by the biuret method (9), with a dried sample of the enzyme as the primary standard.

Copper Determination-Copper was determined by Stark and Dawson’s modification of the oxalyldihydrazide method (lo), with metallic copper as the standard. In our procedure, the final volume was reduced to 1.85 ml, and the absorbancy was read at 542 rnp in the Rotocell attachment of a Bausch and Lomb Spectronic-20 calorimeter.

Strains and Conditions of Culture-Selected high yielding strains of N. crassa producing either the S or the L form of tyro- sinase (1, 2) were maintained on slants of the complete medium described by Horowitz (11). Conidia from these cultures were used to inoculate 2.5-gallon carboys containing 8 liters of Vogel’s Medium N (12), containing 2% sucrose. The carboys were incubated in the dark at 25” with aeration, at the rate of approxi- mately 4 liters per minute, through a glass tube extending to the bottom of the carboy. After 3 days, the mycelium from each carboy was collected aseptically by gravity flow filtration through cheesecloth held in a large Buchner funnel. The mycelium was washed on the funnel with 2 to 3 liters of sterile distilled water and then was transferred to a second carboy containing 8 liters of sterile 0.05 M sodium phosphate buffer, pH 5.5. The filtration and transfer were carried out in a dust-free enclosure. The carboys containing mycelium in phosphate buffer were incubated for an additional 4 days at 25”, with aeration at the rate of 6 liters par minute. Production of tyrosinase occurs at this time. The mycelium was collected on cheesecloth, washed with water,

Yield

and the excess water squeezed out between paper towels with the aid of a rolling pin. The mycelial mats were weighed, a small sample of each was tested for tyrosinase activity, and they were then stored at -25” until needed. The yield of mycelium per carboy was 40 to 80 g, wet weight, and the tyrosinase content was 0.1 to 0.5 mg (approximately 50 to 250 units) per g, wet weight.

RESULTS

Isolation of Neurospora Tyrosinase

A summary of the isolation procedure, applicable to both forms of the enzyme, is shown in Table I. The procedure through the acetone precipitation is essentially the same as that described previously (1). All steps were carried out at 2-4”. Unless otherwise indicated, the buffer referred to below is 0.1 M sodium phosphate, pH 7.2.

E&action-The frozen mycelium was immersed in liquid nitro- gen and was then ground in a precooled corn mill. After thaw- ing, the ground mycelium was mixed with 2 volumes of cold buffer in a blendor and was allowed to stand for 3 days in the cold. Insoluble material was removed by centrifugation and was re- extracted three times, the blended suspension being allowed to stand overnight each time. The residue still contains extractable tyrosinase, but at too low a concentration to be useful.

Preliminary PuriJcation-The combined extracts were treated with 50 ml of 0.2 M manganous sulfate per liter. The pH was maintained at 7 to 7.2 with 0.1 N sodium hydroxide during the addition of the manganous sulfate. The precipitate was removed by centrifugation and was washed once with cold water. The supernatant + washing were transferred to dialyzing tubing. The filled tubing was coiled in a bucket or large beaker and was covered with the amount of solid ammonium sulfate calculated to give a 60% saturated solution. After 24 hours, the protein had precipitated within the dialysis tubing, and half or more of the liquid had diffused through the tubing and could be dis- carded. The precipitated protein, containing the tyrosinase, was collected by centrifugation at 14,000 x g for 30 minutes. The precipitate could be stored at -25” for several months at this point.

Acetone Precipitation-The precipitate was dissolved in the minimal amount of buffer and was maintained at -10” while 2 volumes of acetone were added slowly with stirring. The suspen- sion was centrifuged at -10’ and the supernatant discarded. The tyrosinase was removed from the copious precipitate by or four or five extractions with small quantities of buffer and was precipitated from the combined extracts by the addition of solid ammonium sulfate.

Celite Chromatography-The ammonium sulfate precipitate from the preceding step was dissolved in sufficient buffer to give a solution containing approximately 1250 units per ml. The solution was freed of ammonium sulfate by passage through Sephadex G-50 which had been equilibrated with 10 mM buffer. A column (4 X 20 cm) of Sephadex can be used to treat 50 ml of the solution. The ammonium-free eluate was diluted with 2 volumes of water and placed on a column (3 x 70 cm) of Celite 535 which had been equilibrated with 5 mM buffer. One such column can be used to chromatograph approximately 67,000 units of partially purified tyrosinase. Two hold-up volumes of 5 mM buffer were next passed through the column by gravity flow. About 20% of the tyrosinase, together with the bulk of the non-

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June 1963 144. Fling, N. H. Horowitz, and X. F. Heinemann 2047

tyrosinase proteins and pigmented materials, appeared in the front and was discarded. The tyrosinase remaining on the column was eluted with 50 mM buffer. From 50 to 70% of the activity originally placed on the column was recovered in the 50 mM phosphate buffer front. In early isolation experiments, the chromatography was carried out at pH 6. The behavior of such columns is the same as at pH 7.2, except that a higher salt con- centration is required for elution (Fig. 1).

The proteins were precipitated from the column eluate by dialysis against solid ammonium sulfate, as described above. To insure precipitation of all the activity, the enzyme concentra- tion in the eluate should be at least 160 units per ml. More dilute solutions can be effectively concentrated by dialysis against solid sucrose, followed by precipitation with ammonium sulfate.’

Removal of Blue Protein and Final Puti&ation-The tyro- sinase-containing eluate from the Celite column has a bluish tint, owing to the presence of a small amount (approximately 1%) of a blue, copper-containing protein contaminant. That the color is due to a contaminant, and not to tyrosinase, was first recognized in a free boundary electrophoresis experiment at pH 8.5, in which the blue component moved away rapidly from the major peak, toward the anode. The blue component has about the same copper content as tyrosinase, but is devoid of tyrosinase activity. It can be precipitated from the Celite eluate by adjust- ing the pH to 5.0 in the presence of 10% saturated ammonium sulfate, and this procedure was used for its routine removal.

After removal of the blue protein, the preparation was again chromatographed on Celite. Further chromatography after the second column did not increase the specific activity.

Crystallization-Crystallization was induced by adding satu- rated ammonium sulfate to a 1% solution of the enzyme until a faint turbidity just persisted. The solution was allowed to stand in the refrigerator for several weeks, at which time small needles had formed (Fig. 2). Use of these crystals as seed considerably hastened subsequent crystallizations. The crystalline product shows the same specific activity as the amorphous enzyme from the second Celite column. Concentrated solutions of the crystal- line enzyme are straw colored.

Physical and Chemical Properties of Enzymes

Electrophoretic Mobility-Electrophoretic measurements were carried out between pH 5 and 9 with a Perkin-Elmer model 38 Tiselius apparatus. The purified enzyme migrates as a single boundary throughout the pH range (Fig. 3). Electrophoretic mobility as a function of pH is plotted in Fig. 4. An electro- phoretic difference between the S and L tyrosinases was originally detected by paper electrophoresis (2), and free boundary electro- phoresis confirms this difference. It is interesting to note that both forms are nearly isoelectric between pH 6 and 8; this interval corresponds to the flat pH optimum of the enzyme (13).

Sedimentation Analysissedimentation velocity and sedi- mentation equilibrium studies were carried out with a Spinco model E ultracentrifuge equipped with a phase plate and Ray-

1 Solid sucrose or ammonium sulfate is superior, in our ex- perience, to solutions of polyvinylpyrrolidone or polyethylene glycol (Carbowax) for concentrating dilute protein solutions by dialysis. The latter compounds pass through some dialyzing membranes, and once they get into a protein solution they are very hard to get rid of.

0.05 M 0.1 M T

0.6 ,3

2 0.4 -

ELUATE, ml

FIG. 1. Elution of tyrosinase from diatomaceous earth. Ty- rosinase solution (0.5 ml; specific activity, 4.2 units per mg of pro- tein) was placed on a column (12 X 1 cm) of Celite 535 and eluted with successive 25-ml portions of phosphate buffer, pH 6, of the molarities shown. -, Enzyme activity; - - -, protein.

x

FIG. 2. Crystals of tyrosinasc S from ammonium sulfate. The scale mark is 10 p.

leigh interference optics.* Sucrose gradient centrifugation was performed with a Spinco model L ultracentrifuge according to the method of Martin and Ames (14), with crystalline egg white lysozyme as the reference standard.3

The purified enzyme shows a single schlieren boundary in the ultracentrifuge (Fig. 3). The sedimentation coefficient, cor- rected to water and 20”, was 4.3 f 0.1 S (average and standard error of the first three determinations shown in Table II). The sedimentation coefficient of the enzymes by the sucrose-gradient method was 3.6 S (Table II). No difference between the S and L forms was detected by either method.

Sedimentation equilibrium measurements were made with interference optics at 546 mp. Short (2 to 4 mm) columns were used to reduce the time required to attain equilibrium. Only tyrosinase S was examined. The results (Fig. 5) show that the enzyme is polydisperse under the conditions employed. The range of molecular weights indicated in Fig. 5 is from 35,000 to 120,000. Attempts to prevent aggregation of the enzyme by carrying out sedimentation equilibrium analyses in glycine-NaCl

2 These measurements were performed by Dr. Edward A. Ca- rusi and Prof. R. L. Sinsheimer. to whom we are areatlv indebted.

3 We wish to thank Mr. John Urey for carrying out the sucrose gradient centrifugation.

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2048 Crystalline Tyrosinase from Neurospora Vol. 238, n-0. fj

FIG. 3. Above, sedimentation of tyrosinase S, 0.370 in 0.1 M NaCl + 0.02 M sodium phosphate, pH 6. Rotor speed, 56,100 p.m.; temperature, 20”. Below, electrophoresis of tyrosinase S, 0.4% in 0.1 M acetate, pH 5.05, 125 minutes after start of the experi- ment; temperature, O-2’.

3- These results will be discussed below. Diffusion Coeficient-Diffusion measurements were made at

“0 2- - 20” in a Stokes glass diqphragm diffusion cell (15). The cell was

x - _ calibrated with KCI, and its reliability was checked by deter-

: ’ mining the diffusion coefficients of horse heart cytochrome c and

i - sucrose. Satisfactory agreement with values in the literature ob-

g o- tained by other methods was found (Table III).

I - The rate of diffusion of enzyme from the lower into the upper

chamber of the cell was measured by following the enzymatic -I - activity in the upper chamber. Several days were required for

I I I I I I I t I the attainment of a constant diffusion rate. 5 6 7 8 9

Since highly purified

PH tyrosinase is unstable in dilute solution, enzyme of approximately

FIG. 4. Electrophoretic mobility as a function of pH. Upper curve ( l ), tyrosinase L; lower curve (O), tyrosinase S. Buffers as

TABLE II

follows : Sedimentation coefkients

PH B&X Ionic strength Experiments 1, 2, and 3 were run at 20” in 0.1 M NaCl buffered

5.0 Acetate 0.033 with 20 mM phosphate. The rotor speed was 56,100 r.p.m. in 5.05 Acetate 0.067 Experiments 1 and 2 and 50,740 r.p.m. in Experiment 3. Experi- 5.5 Acetate 0.10 ments 4a, b, and c were run for 16 hours at 4” and 38,000 r.p.m.

6.0 Phosphate 0.12 The enzyme was layered on the sucrose gradient in 0.1 ml of 0.1 M 7.0 7.5

8.1 8.5

9.0 9.15

Cacodylate Phosphate

Tris Verona1

Glycine Glycine

0.10 0.10

0.10 0.09

0.20 0.10

phosphate buffer, PH 6. - ̂

Experiment

1 2

Method ‘yrosinase Concentration PH no,w

Schlieren Schlieren

Schlieren Sucrose gradient Sucrose gradient Sucrose gradient

buffer at pH 9.2, or in 0.001% sodium dodecyl sulfate, were unsuccessful. In fact, results opposite to that anticipated were

34a

obtained, since in both cases the minimal molecular weight was 4b

4c now found to be 65,000 to 70,000.

w/ml

S 1.8 S 3.0 L 2.6

S 0.024 S 0.024 L 0.035

7.2 6.0 7.2

6.0 6.0 6.0

s

4.4

4.1 4.3

3.6 3.6 3.6

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June 1963 M. Fling, N. H. Horowitz, and S. F. Heinemann 2049

25% purity was used for these measurements. The enzyme was further stabilized by dissolving it in phosphate buffer con- taining penicillin, streptomycin, and bovine serum albumin. The same solvent was used in the upper chamber. No loss of enzymatic activity was detected in this system during the course of a 5-day run with tyrosinase S. With tyrosinase L, a 15 y0 loss of activity was observed over the same period.

The results are shown in Table III. The difference between the two tyrosinases is not significant, but since the value obtained for the S form is the more reliable one, we shall use it for calcula- tions.

Molecular Weight-Examination of the sedimentation (schli- eren method) and diffusion coefficients obtained above shows that they cannot refer to the same molecular species. If we take the partial specific volume of the enzyme as 0.73 (based on the amino acid composition, see bdow), the molecular weight of the equivalent anhydrous sphere calculated from Dz~,W = 10.7 x lo-’ cm2 set-l is 27,600 (95 % confidence limits: 25,000 to 31,000). On the other hand, the molecular weight of the equivalent anhydrous sphere calculated from ~20,~ = 4.3 S is 40,500 (95 ‘$& confidence limits: 35,000 to 46,000). Since the former is an upper limit on the molecular weight and the latter a lower limit, an inconsistency is evident. The value of s obtained by the sucrose gradient method, however, is more nearly compatible with the observed diffusion coefficient; the molecular weight of the equivalent sphere in this case is 31,100.

These inconsistencies can be explained by assuming that the enzyme undergoes rapid association-dissociation reactions, such as have been observed for a number of other proteins in solution. Such solutions contain equilibrium mixtures of monomers and polymers, the proportions depending on the protein concentra- tion; a single, nearly symmetrical boundary is observed during sedimentation, but s increases with concentration in dilute solu- tions (19). In the case of tyrosinase, the diffusion measurements and the sedimentation measurements made by the sucrose gra- dient technique were done at low enzyme concentrations, where the monomer would be expected to predominate. Sedi- mentation measurements by the schlieren technique, however, involved much higher enzyme concentrations, and, as predicted by the above interpretation, significantly higher s values were obtained. This interpretation is also supported by the results of the sedimentation equilibrium experiments, described above.

Further information on the molecular weight of the enzyme can be derived from its composition. The equivalent weight per atom of copper (see below) is 32,600. The molecular weight

calculated from the amino acid analyses given below is 31,500. The difference is not significant. Examination of tryptic hy- drolysates of the enzyme by the fingerprint method (20) con- firms that the molecular weight of the monomer lies in the range indicated by these values. By this method, the number of dif- ferent peptides formed on tryptic hydrolysis can be counted and, ideally, equals one plus the number of different arginine + lysine residues in the protein. Fingerprints of S and L tyrosinase pre- pared by the procedure of Katz, Dreyer, and Anfinsen (21) show 26 to 28 peptides, in agreement with the number expected on the basis of a molecular weight of about 32,000.

All of the evidence is thus consistent with a monomeric mo- lecular weight of 33,000 f 2,000.

Molecular Activity (Turnover Number)-If we take the specific activity of the pure enzyme as 536 (Table I) and the molecular

- 0.750 - Ii .

E

5 I=

2 0.650 -

5

Y

E

:: -I 0.550 -

J 0.450 ’ 0 1 I I I I I

47 48 49 50 51

x2 (cm*)

FIG. 5. Sedimentation equilibrium of tyrosinase S in 0.1 M N&l + 0.02 M sodium phosphate, pH 6. Rotor speed, 7,928 r.p.m.; temperature, 20”; time to reach equilibrium, 24 hours. The tan- gents correspond to molecular weights of approximately 35,000 and 120,090, respectively.

TaBLE 111

L)iffksion coejicients determined with the Stokes cell

Cell constant was 0.404; stirring rate, 50 to 80 r.p.m. Solution A: 0.1 al sodium phosphate buffer, pH 6, containing 50 pg per ml of bovine serum albumin + 30 rg per ml each of penicillin and streptomycin. Viscosities were measured with an Ostwald viscometer at 2o”. Numbers in parentheses are references.

Substance

Sucrose ..................... Cytochromec ............... Tyrosinase S ................ Tyrosinase L ................

Concentration

0.5 M

0.4% 35 units/ml 35 units/ml

Solvent Dzo.w f standard error

Found I

Values in literature

mz2 se0 x 107

Water 1 40.9 f 3.2 41 (16) 0.05 M Tris, pH 8 1.020 11.1 f 0.4 10.1 (17), 12 (18) Solution A 1.042 10.7 f 0.2 Solution A 1.042 11.9 ZJZ 0.8

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2050 Crystalline Tyrosinase from Neurospora Vol. 238, Tu’o. 6

TABLE IV Relative rates of dopaquinone and dopachrome formation

All reactants were dissolved in 0.1 in sodium phosphate, pH 6, in a final volume of 3 ml. Ascorbate, where used, was 70 FM. All cuvettes contained 15 PM ethylenediaminetetraacetate. The en- zyme was tyrosinase S purified through the first Celite column. Continuous readings were taken with a Cary recording spectro- photometer for the first 2 minutes after mixing: l 66 mp, ascorbate = 15,360; cd’16 m,,, dopachrome = 3600.

ml mM

0.2 0.267

0.4 0.267 0.8 0.267 0.2 0.133

0.4 0.133 0.8 0.133 0.4 0.067

0.8 0.067

L-Dopa

0.58 1.30

2.58 0.375 0.80

1.60 0.44 0.90

lAmmr /min AAscorbate min-1

ADopacbrome min-1

0.075 1.82

0.15 2.03 0.30 2.02

0.044 2.03 0.085 2.21 0.17 2.21

0.055 1.89 0.09 2.35

2.07 f 0.06*

* Mean f standard error.

weight as 32,000, the molecular activity is found to be 17,150. This slightly underestimates the true value, owing to the fact, mentioned earlier, that in converting to the Enzyme Commission unit of activity, the reaction inactivation of the purified enzyme is underestimated by some 10%. When this is corrected for, the molecular activity is found to be about 19,000 moles of L- dopa transformed per minute per mole of enzyme during the initial, linear phase of the reaction. No difference in activity was found between the S and L forms.

It is interesting to note that under steady state conditions, dopaquinone (phenylalanine 3 ,Cquinone) is formed twice as fast as dopachrome, so that if the latter is used to measure tyrosinase activity, the molecular activity will be underestimated by a factor of two. The relation between the rates of dopaquinone and dopachrome formation is shown in Table IV. Dopaquinone for- mation was measured by the ascorbate method of El-Bayoumi and Frieden (22)

Dopa + 3 0~. + dopaquinone + HtO

Dopaquinone + ascorbate --f dopa + dehydroascorbate

The procedure used was described by Sussman (13).4 At the same time, the formation of dopachrome from dopa was followed in parallel cuvettes without ascorbate.

This unexpected finding is interpretable on the basis of the mechanism proposed by Evans and Raper (6) to explain the ac- cumulation of dopa during the enzymatic oxidation of tyrosine

2 Dopa + 02 -+ 2 dopaquinone + 2 HtO (1)

Dopaquinone --) leukodopachrome (2)

Leukodopachrome + dopaquinone -+ dopachrome + dopa Sum: Dopa + 02 --) dopachrome + 2 Hz0 (3)

According to this scheme, 1 molecule of dopachrome appears for every 2 molecules of dopaquinone formed under steady state con-

* The final concentration of ascorbate was 7 X 10m5 M, not 7 X 10-G M as stated in (13).

ditions, which is what we observe. We also find, as did Evans and Raper, that dopa accumulates during the oxidation of tyro- sine.

Thermostability-Previous studies with crude and partially purified preparations showed a marked thermostability difference between the S and L forms of the enzyme (1). It was of interest to repeat these measurements with the crystalline enzymes. In agreement with the earlier results, it was found that in 0.1 M sodium phosphate at pH 6, both forms are inactivated in a first order reaction at 59”, with half-lives of 65 minutes and 5 minutes for the S and L forms, respectively.

Absorption Spectrum-The pure enzyme shows a typical pro- tein absorption spectrum in the ultraviolet region, with a maxi- mum at 280 rnp and a shoulder at 290 rnp. There is a suggestion of a broad shoulder in the region around 340 mp. No specific absorption is seen in the visible. The absorbancy, at 280 mp, of a 1% solution at pH 6 is 15.6 to 19.0 per cm of light path, depending on the amount of correction that is made for extra- neous absorption. The slight shoulder at 340 rnp makes the usual method of obtaining the extraneous absorption (by extrap- olating from the absorbancies at 340 and 370 rnp (23)) somewhat ambiguous.

Copper, Content and Oxidation State-Like other phenol oxi- dases, Neurospora tyrosinase contains copper. The average copper content of five different preparations of the enzyme (two of the S form, three of L) was 0.195 + 0.005%. The copper atom is not removable by dialysis against cyanide, in contrast to what has been reported for phenol oxidases from potato (24) and mouse melanoma (25). That the copper of Neurospora tyrosinase is essential for enzymatic activity is shown, however, by the marked sensitivity of the enzyme to diethyldithiocarbam- ate, azide, cyanide, phenylthiourea, and cysteine (13).

The absence of a blue color, or of any specific absorption in the visible region in concentrated solutions of the crystalline enzyme, suggested that the copper is in the cuprous state (3). This was confirmed by electron spin resonance measurements carried out on a sample of pure tyrosinase S.5 No cupric copper was de- tected, and it is estimated that not over 1 y. of the copper in the sample was in the cupric state.

Amino Acid Composition-Amino acids were determined in acid hydrolysates of the lyophilized enzyme. The conditions of hydrolysis were: 6 N HCl in nitrogen-filled or evacuated sealed tubes at 110’ for 22 or 70 hours. Analyses were performed with a Beckman/Spinco amino acid analyzer, model 120 (26).6

The results are shown in Table V. The values for serine and threonine were corrected for destruction during hydrolysis by extrapolation to zero time. The tyrosine values were variable, probably owing to oxidation during hydrolysis in some cases. In addition, two enzyme samples showed a pink color after lyo- philization, suggesting formation of an oxidation product of ty- rosine, possibly catalyzed by the enzyme itself. In both cases, the tyrosine values were low, and a number of small, unidentified ninhydrin-positive peaks appeared in the eluate. For these rea- sons, the tyrosine values were not averaged, but the highest ones found are recorded. These values are 10 to 25% higher than

5 Electron spin resonance measurements were performed by Dr. A. S. Kwiram and Prof. Harden McConnell, to whom we ex- press our appreciation.

6 We wish to thank Dr. R. T. Jones, Dr. H. J. Burkhardt, Miss Nancy Martin, and Miss Joyce Bullock for assistance with these analyses.

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June 1963 M. Fling, N. H. Horowitz, and S. F. Heinemann

TABLE V rlmino acid composition of Neurospora tyrosinase

Each value is an average (A mean deviation) of a total of five analyses, carried out on three different preparations.

2051

Amino acid

Aspartic acid. . Threonine* . Serine*. . Proline. . . . . . . Glutamic acid. . Glycine. . Alanine. Cystine*. Valine. Methionine. Isoleucine Leucine Tyrosine* Phenylalanine Lysine. Histidine. Arginine Tryptophan*.

- I Molar ratios of recovered amino acids based on

alanine = 1.000

T

--

-

L S AWXlge

1.336 f 0.036 1.325 f 0.011 30.6 f 0.7

0.672 f 0.008 0.685 f 0.022 15.4 f 0.2

1.217 zt 0.043 1.353 f 0.026 27.9 f 1.0 0.969 f 0.022 0.966 f 0.031 22.2 f 0.5

0.985 f 0.089 0.913 f 0.961 22.6 f 2.0

0.804 f 0.017 0.802 f 0.014 18.4 f 0.4

1.999 f 0.010 1.999 f 0.999 22.9 f 0.2

0.712 f 0.031 0.076 f 0.022 0.395 f 0.023 1.010 f 0.016 0.519 0.690 f 0.012 0.522 f 0.017 0.262 f 0.015

0.566 f 0.018

0.714 f 0.030 0.082 f 0.029 0.396 f 0.012

0.995 f 0.027

0.509 0.718 f 0.026 0.507 f 0.020

0.253 f 0.006

0.593 f 0.013

16.3 i 0.7

1.7 f 0.5

9.1 f 0.5 23.1 f 0.3 11.9 15.8 f 0.3 12.0 f 0.4

6.0 f 0.3

13.0 f 0.4

8.1

* See the text for details.

the estimates obtained by the spectrophotometric method of chromatographed on paper with butanol-acetic acid-water (4: Goodwin and Morton (23), and these in turn are 10% higher than 1:4). Both ninhydrin and the iodoplatinic acid reagent (31) the values given by the Bencze and Schmid procedure (27). The were used to locate S-carboxymethylcysteine. S-Carboxymeth- cause of the discrepancies is unknown, although they suggest ylcysteine controls reacted with both reagents and showed a that some of the phenolic hydroxyl groups of the enzyme are not Rp value of 0.15 to 0.17. All six unknowns showed faintly nin- available for ionization. Tryptophan was estimated both spec- hydrin-positive spots at RF 0.14, but in no case did they react trophotometrically (23) and calorimetrically with dimethylbenz- with the iodoplatinic acid reagent. Since this reagent is some- aldehyde (28). The spectrophotometric estimates were, in this what more sensitive than ninhydrin, it was concluded that none case, 5 to 10% higher than the calorimetric ones. of the fractions contained S-carboxymethylcysteine.

Total amino acid recoveries, based on dry weight, varied from 70 to 90 ‘%, with alanine showing the least variation. In Columns 2 and 3 of Table V, molar ratios of recovered amino acids relative to alanine are shown. The calculation of residue frequencies (Columns 4 to 7) is based on histidine, rather than methionine, the least frequent amino acid, because the latter undergoes a certain amount of oxidation during hydrolysis.

3. Titration with p-mercuribenzoate (32). The enzyme re- acted sluggishly when titrated at pH 5 in the presence of sodium dodecyl sulfate. Thus, in two experiments, the uptake of p- mercuribenzoate corresponded to approximately 0.25 and 0.5 mole per 32,000 molecular weight after the addition of, respec- tively, a 4000- and a 7000-fold excess of the mercurial.

No cystine was detected in hydrolysates of the untreated pro- teins. The following additional analyses were therefore carried out.

1. Performic acid oxidation (29). The hydrolysate of a per- formic acid-oxidized sample of tyrosinase S (10 mg) was chro- matographed on Amberlite IR-120. An amount of ninhydrin- reacting material corresponding to only 0.3 residue per 32,000 molecular weight was obtained in the fraction that normally contains cysteic acid. The identity of the material was not in- vestigated further.

In inhibition experiments, the activity of the crude enzyme was not affected by 0.5 rnM p-mercuribenzoate, whereas pure preparations were inhibited 25 to 50% by the same concentra- tion. Enzymes with essential sulfhydryl groups are much more sensitive than this (33, 34). Furthermore, benzoic acid itself inhibits the enzyme in this concentration range (13), a fact which throws further doubt on the interpretation of the p-mercuriben- zoate inhibition as a sulfhydryl-blocking effect.

4. Titration with 5,5’-dithiobis(2-nitrobenzoic acid) (35). The results with this reagent were negative with both native and denatured (8 M urea) enzyme.

2. S-Carboxymethylation (30). A 15-mg sample of enzyme was alkylated with iodoacetic acid and chromatographed on Am- berlite IR-120. All fractions between the solvent front and that containing aspartic acid (the region expected to contain S-car- boxymethylcysteine) were analyzed with ninhydrin. Six small peaks were obtained. Each was concentrated, desalted, and

DISCUSSION

This study shows that the tyrosinase of Neurospora is a pro- tein of moderately low molecular weight which can be isolated in good yields. The usefulness of the enzyme for the investiga-

Amino acid ratios based on histidine = 6

-7

L

Nearest integet

31 15

28 22

23 18

23 0

16 2

9 23 12 16

12 6

13

8

S

Average

31.4 f 0.3

16.2 f 0.5

32.1 f 0.6

22.9 f 0.7 21.6 f 1.4

19.0 f 0.3 23.7 f 0.2

16.9 f 0.7 1.9 f 0.7 9.4 f 0.3

23.6 f 0.6 12.1

17.0 f 0.6

12.0 f 0.5 6.0 f 0.1

14.1 f 0.3 8.4

- karest integer

31 16

32 23 22

19 24

0 17

2

9 24 12

17 12

6 14

8

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Crystalline Tgrosinase from Neurospora Vol. 238, No. 6

tion of a variety of biochemical and genetic problems should same authors obtained only a tyrosinase of molecular weight thereby be enhanced. 128,000 (40).

The extraordinary selectivity which Celite displays for ty- rosinase and the blue protein suggests that this material may be useful for the isolation of other copper proteins. A clue to the selectivity of the columns is possibly to be found in the ob- servation of Quinot and Claeys (36) that potato tyrosinase is adsorbed by ground quartz. The adsorption did not take place if the copper was removed from the enzyme, but did so when the copper was replaced. The authors propose that the enzyme is held to the quartz through the copper atom.

SUMMARY

Among the features of the enzyme that deserve comment are the apparent absence of cysteine from the molecule and the high serine content. The elimination of cysteine as a possible binding site for the copper atom makes histidine the most likely site of attachment. The formation of copper (I)-imidazole complexes has been studied in detail (37). Involvement of 2 of the 6 his- tidine residues in copper binding would help to explain the flat shape of the pH-mobility curve in the neighborhood of pH 6, where the imidazole groups of histidine are normally titrated.

Two allelic tyrosinases, S and L, of Neurospora crassa have been isolated in a homogeneous, crystalline state by a procedure involving chromatography on diatomaceous earth. The molec- ular weight of both enzymes is 33,000 f 2,000, and both appear to aggregate reversibly in solution. There is 1 atom of cuprous copper per molecule. The molecular activity (turnover number) is approximately 19,000 moles per minute, with L-dopa as sub- strate. Both enzymes are nearly isoelectric between pH 6 and 8, with the L form showing a slightly greater mobility over the pH range studied. The L form is considerably more thermolabile than the S form. Amino acid analyses suggest that the S form contains more serine than the L form, but both enzymes are un- usually rich in this amino acid. Cyst(e)ine could not be detected in the enzymes. The results are discussed in relation to problems of tyrosinase chemistry in Neurospora and the mushroom.

In regard to serine, it should be noted that, besides being present in an unusually high concentration, it is the only amino acid in respect to which the data indicate a significant difference between the S and L forms. In view of the inherent uncertainty of the serine determination, this difference should be regarded with reserve. This and other possible structural differences be- tween the enzymes are being investigated further.

Acknowledgment-The invaluable assistance of Miss Helen Macleod is gratefully acknowledged. We also wish to thank Dr. Edward A. Carusi for many fruitful discussions.

REFERENCES

It is of some interest to compare our enzyme with the classical fungal tyrosinase, that of mushrooms. An important step in our understanding of mushroom tyrosinase was the recent finding by Smith and Krueger (38) that it consists of a mixture of several enzymes which can be differentiated on the basis of their chro- matographic behavior and substrate specificities. This hetero- geneity suggests the possibility of genetic heterogeneity in the starting material. The Neurospora enzyme is also highly varia- ble, with at least four forms, differing in therrnostability and/or electrophoretic mobility, established in nature (2). This varia- bility has a genetic basis. Genetically pure cultures make one form of the enzyme, but heterocaryons (cultures in which genet- ically dissimilar nuclei are mixed in a common cytoplasm) make corresponding mixtures of tyrosinases (39). In the case of the mushroom also, it is possible that genetically uncontrolled cul- tures produce mixtures of genetically diverse tyrosinases. Such variability in the starting material could explain many of the difficulties which are encountered in this field. Some workers have, in fact, stressed the importance of the source of the enzyme in discussing the variability of mushroom tyrosinase (38, 40). What has perhaps not been realized is that considerable genetic heterogeneity can exist even within a single culture if steps have not been taken to safeguard against it.

1. HOROWITZ, N. H., AND FLING, M., Genetics, 38, 360 (1953). 2. HOROWITZ, N. H., FLING, M., MACLEOD, H., AND SUEOKA, N.,

Genetics, 46, 1015 (1961). 3. HOROWITZ, N. H., FLING, M., MACLEOD, H., AND WATANABE,

Y., Cold Spring Harbor symposia on quantitative biology, VoZ. 26, Long Island Biological Association, Cold Spring Harbor, Long Island, 1961, p. 233.

4. HOROWITZ, N. H., FLING, M., MACLEOD, H., AND SUEOKA, N., J. Molecular Biol., 2,96 (1960).

5. Report on the Commission on Enzymes of the International Union of Biochemistry, Pergamon Press, New York, 1961, p. 8.

6. EVANS, W. C., AND RAPER, H. S., Biochem. J., 31, 2162 (1937). 7. MASON, H. S., J. Biol. Chem., 172,83 (1948). 8. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND

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The Neurospora enzyme differs from the classically described mushroom tyrosinase in its apparent inability to oxidize catechol (41). Sussman (13) has shown that this is due to the rapid re- action-inactivation of the enzyme on this substrate. It is in- teresting to note that fraction Bi of Smith and Krueger resembles the Neurospora enzyme in its substrate specificity, including inactivation by catechol. In molecular weight and copper (I) content our enzyme is similar to that isolated from mushrooms by Kertesz and Zito (42, 43). The substrate specificity of this enzyme was not stated. In later preparations, however, the

NICHOLS, J. B., AND BA~IZEY, E.’ D.,‘i’n A. WEISSBERGER (Editor), Physical methods of organic chemistry, Vol. 2, In- terscience Publishers. Inc.. New York. 1949. D. 704.

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June 1963 M. Fling, N. H. Horowitz, and S. F. Heinemann

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(1949). 29. HIRS, C. H. W., J. Biol. Chem., 219,611 (1956). 30. COLE, R. D., STEIN, W. H.. AND MOORE. S., J. Biol. Chem.. 233.

1359 (1958). ’ , ,

31. WINEGARD, H. M., AND TOENNIES, G., Science, 108,506 (1948). 32. BOYER, P. D., J. Am. Chem. Sot., 76.4331 (1954). 33. VELI&, S. F:, in W. D. MCELROY AND B: GL&S (Editors),

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2053

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Marguerite Fling, N. H. Horowitz and Stephen F. HeinemannThe Isolation and Properties of Crystalline Tyrosinase from Neurospora

1963, 238:2045-2053.J. Biol. Chem. 

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