THE ACTION OF TYROSINASE ON PROTEINS

BY IRWIN W. SIZER*

(From the Department of Biology, Massachusetts Institute of Technology, Cambridge)

(Received for publication, January 15, 1946)

Tyrosinase catalyzes the oxidation by oxygen of a large variety of mono- and polyphenols (1,2) as well as many phenolic derivatives such as adren- alin, certain sex hormones, tyrosine, and the poison ivy irritants (3, 4). Monophenols areoxidized to diphenols; these are in turn converted to unsta- ble quinone derivatives which polymerize to highly colored compounds of unknown structure. A typical example is the aerobic oxidation of tyrosine by tyrosinase to 3,4-dihydroxyphenylalanine (dopa), which is converted by way of unstable intermediaries to the pigment melanin (5, 6). This oxidation of tyrosine is believed to be involved in the production of pig- mentation in many different organisms.

As far as the author is aware the only enzymes which are known to attack simple intact proteins in vitro are proteolytic ones, although chemical reagents can readily bring about modifications in such constituents of native proteins as amino, carboxyl, and phenolic groups. The situation in vivo appears to be quite different, however, for tracer studies by Schoen- heimer (7) and others have clearly shown that proteins in the body are quite labile and that substitutions of amino acids appear to occur and that reactions take place which involve active available groups on the protein molecule. A consideration of the discrepancy between the reactivity in vivo and in vitro of proteins to non-proteolytic enzymes led to this study of the action of tyrosinase on proteins. In view of the low specificity of tyrosinase it was felt that if the tyrosine residue were available at the sur- face of the protein molecule its phenolic hydroxyl group might be subject to attack by mushroom tyrosinase.

Methods and Results

Manometric Experiment-The Barcroft differential manometer was used, at 37“ f 0.02” and at a shaking rate of about 120 oscillations per minute, to study any effect of tyrosinase in bringing about an oxygen consumption by the protein solutions. The reaction flasks had a volume of 15 ml. and possessed a single side arm in which the enzyme was placed. The ex- perimental flask contained 25 mg. of protein, 4.5 ml. of M/15 phosphate buffer, pH 7.3, 2 drops of toluene (or 0.001 per cent merthiolate), and 0.5 ml. of tyrosinase solution in the side arm. The control flask contained

* With the technical assistance of Miss Janette Robinson.

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146 TYROSINASE AND PROTEINS

exactly the same components as the experimental, except that boiled enzyme was placed in the side arm. In some experiments, however, distilled water was used in the control instead of the inactivated tyrosinase. After 5 minutes adaptation to temperature, the reactions were initiated by tipping the enzyme into the protein solution. In most experiments Upjohn mushroom tyrosinase was used, which contained 1100 Adams and Nelson catechol-hydroquinone units, 150 cresolase units, and 2750 chrono- metric catecholase units per ml. A few experiments with very similar results were also performed with Nelson’s highly purified mushroom

I 2

TIME3 IN tG”RS

5 6

FIG. 1. Oxygen consumption of proteins in the presence of tyrosinase. The solu- tion contained 25 mg. of protein, 4.5 ml. of ~/15 phosphate buffer, pH 7.3, and 0.5 ml. of Upjohn tyrosinase, plus 2 drops of toluene. The tyrosine solution contained only 2.5 mg. of tyrosine, 4.95 ml. of buffer, and 0.05 ml. of tyrosinase.

tyrosinase,’ which contained 4200 Miller and Dawson catecholase units and 70 Adams and Nelson p-cresolase units per ml. (1).

Results of typical experiments are presented in Fig. 1, from which it appears that the action of tyrosinase in the oxidation of certain proteins is clearly demonstrated by the oxygen consumption of protein solutions in- duced by the addition of tyrosinase. The reaction follows a smooth cur- vilinear course characteristic of the particular protein substrate employed, but the rate is much less than for the action of 0.05 ml. of tyrosinase on 2.5 mg. of t-tyrosine. Results for all the proteins studied are summarized

1 Kindly furnished by Dr. J. F. Nelson of Columbia University.

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TABLE I

Action of Tyrosinasc on Proteins with Reference to Oxygen Consumption, Pigment Production, and Reaction to Millon’s Reagent

Substrate

I-Tyrosinet Trypsinl Pepsin1 Chymotrypsint: Peptone (Difco) Caseins Zinc insulin (Merck) Hemogbbin (Difco) Gelatin (Difco) Protamine (Lilly) Tobacco mosaic virus/] Egg albumin (Merck) Serum albumin, human?

“ “ “ + 1 mg. trypsin “ “ bovine** I‘ “ “ + 1 mg. trypsin “ r-globulin, human7 <‘ “ “ + 1 mg. trypsin “ &globulin, bovine** “ “ “ + 1 mg. trypsin

Fibrinogen, bovine** “ “ + 1 mg. trypsin

Gramicidinsg CL + 1 mg. trypsin

:icro2iters 0 per hr.

250 163

90 43 58 11

9 2 0 0 0 0 0 6 0 0.5 0 1 0 2 0.1

13 0 0

x.orption ir zdue-violet

+ + + + + + + + - - - - -

+ -

+ -

+ -

+ f + - -

Millon’s test’

- - - -

- - - -

Not measured

+ + -

Not measured -

+ -

Not measured -

Not measured - - -

* If the production of a pink solution or precipitate by the protein treated with act,ive tyrosinase is less than t.he color of the protein treated with inactive tyrosinase, the reaction is called negative; if the same, it is called positive.

t Digest contained only 2.5 mg. of tyrosine and 0.05 ml. of tyrosinase. $ Obtained in crystalline form from the Plaut Research Laboratory. Each

enzyme preparation contains about 40 per cent MgS04. 8 Prepared according t,o Koch (8). 11 A saturated solution was used. It was kindly furnished by Dr. I. Fankuchen,

the Polytechnic Institute of Brooklyn. 7 The products of plasma fractionation employed in this work were developed

by the Department of Physical Chemistry, Harvard Medical School, Boston, Massa- chusetts, under a cont,ract recommended by the Committee on Medical Research, between the Office of Scientific Research and Development and Harvard University. Both the albumin and the r-globulin are about 98 per cent pure.

** Kindly furnished by the Armour Laboratories, These proteins were prepared under a contract recommended by the Committee on Medical Research, between the Office of Scientific Research and Development and Armour and Company. The al- bumin is 100 per cent pure; the B-globulin is 98 per cent pure (contains 2 per cent al- bumin) ; the fibrinogen is 77 per cent pure (contains 18 per cent globulin plas 5 per cent albumin) and is mixed with 40 per cent sodium citrate.

$8 Kindly furnished by Dr. R. Dubos, The Rockefeller Institute for Medical Re- search.

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148 TYROSINASE AND PROTEINS

in Table I. As would be expected, the substrates which contain no ty- rosine, such as gelatin, protamine, and gramicidin, show no evidence of oxidation by tyrosinase.

It is also apparent from Table I that, although many proteins are ox- idized, there remain a large number which contain appreciable amounts of tyrosine, but nevertheless are not oxidized under the influence of tyrosinase. In these proteins it is possible that the tyrosine residues are not accessible at the surface of the protein molecule, or the phenolic groups may be so combined as to be unavailable for attack by tyrosinase. With these pos- sibilities in mind it seemed likely that tyrosinase action on these resistant proteins could be demonstrated if the protein molecule were partially

TIME IN HOURS

FIG. 2. Oxygen consumption catalyzed by tyrosinase of trypsin-treated prot’eins. The solution contained 25 mg. of protein, 4.5 ml. of buffer, 1 mg. of cryastlline trypsin, 0.5 ml. of Upjohn tyrosinase, plus 2 drops of toluene.

digested with trypsin before or concurrently with the addit#ion of tyrosinase. That this is possible is shown for several different proteins in Fig. 2, from which it appears that the addition of 1 mg. of crystalline trypsin to the digest rendered the protein oxidizable by tyrosinase. Results of this study are summarized in Table I, which also shows that a substrate such as gramicidin which contains no tyrosine is not rendered oxidizable by ty- rosinase after preliminary digestion with crypsul.

E$ect of Tyrosinase on Biological Activity of Typical Proteins-Since tyrosinase most readily oxidizes pepsin, trypsin, and chymotrypsin (see Fig. l), a study was made of the effect of tyrosinase on the enzymatic activity of each of these crystalline enzymes. The technique employed in

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I. W. SIZER 149

studying the prot.eolytic activities of these enzymes was the same as that described previously (9). It involved the suspension in the proteolytic enzyme solution at 37” of a filament of reprecipitat,ed purified collagen to which was attached a 2 gm. paraffin-coated lead weight. The time re- quired for the filament to break and the weight to fall was measured with an automatic timing device (10). In the first series of tests the enzyme solutions were studied for proteolytic activity at the completion of the manometer experiments in which oxidation of the protein catalyzed by tyrosinase was measured. No change in pH of these solutions was neces- sary in order to study the activity of trypsin and chymotrypsin, but the measurement of peptic activity required the change in pH from 7.3 (neces- sary for tyrosinase action) to pH 2.0 by the addition of a few drops of 1 N

HCI to the pepsin solution containing boiled or native tyrosinase. In the second series of experiments the procedure was the same except that, in-

TABLE II

Bction of Tyrosinase on Activity of Certain Enzymes As Measured by Time Required to

Protease

Chymotrypsin “

Trypsin

Pepsin

Digest Collagen Filament

Tyrosinase

Boiled A7ative Roiled ljative Boiled Sative

Digestion time

hrs.

2.16 2.10 1.32 1.54 38 Approximately 38 “

stead of the manometric technique, the solutions were incubated with na- tive or boiled tyrosinase for 18 hours at 37” before the proteolytic activity was measured. As would be expected, results mere very similar by both methods. Typical results are presented in Table II, from which it is apparent that tyrosinase has no effect upon the activity of pepsin, trypsin, or chymotrypsin despite the fact that it catalyzes the oxidation of tyrosine in all three enzymes. These results are surprising in view of the fact that chemical reagents which combine with or oxidize the tyrosine residues of pepsin (11) and chymotrypsin (9) produce inactivation of these proteases. It seems likely that the enzymatic oxidation of tyrosine in proteins does not bring about as drastic a change in the tyrosine moiety as does the chemical oxidation.

Chemical Studies of Effect of Tyrosinase on Proteins-Upon completion of each manometric experiment the digests containing either native or boiled tyrosinase were saved for chemical and spectroscopic examination to de-

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150 TYROSINASE AND PROTEINS

termine whether or not the oxidation of the protein molecule induced by tyrosinase could be demonstrated by techniques other than the ma.no- metric one.

Studies on Native Protein

Phenol Test for Tyrosine Plus Tryptophane-The phenol reagent was pre- pared according to Folin and Ciocalteau (12), and a standardization curve was made for tyrosine measuring the absorption at 420 rnp with a Coleman spectrophotometer. The test on the tyrosinase-treated protein solution was made by adding 10 ml. of 0.5 N NaOH plus 3 ml. of phenol reagent, to 5 ml. of protein solution. After the solution had stood for 5 minutes, the absorption at 420 rnp was measured. In each experiment the absorption of the protein solution with native tyrosinase was compared with the absorp- tion of the solution containing boiled tyrosinasc. No significant difference in the amount of tyrosine plus tryptophane could be detected by this technique, which is not applicable to the study of byrosine of intact protein molecules before hydrolysis.

Qualitafice Millon’s Test for Tyrosine-This test was performed by adding to 1 ml. of tyrosinase-treated protein 3 ml. of water plus 3 drops of Millon’s reagent (13). The solution was t’hen brought to a boil and allowed to cool. Results of these studies were what might have been predict’ed (see Table I), In all cases in which oxidation of the protein by tyrosinase was indicated manometrically by oxygen consumption, a much fainter pink color was obtained than for the control solution containing inactive tyrosinase. On the other hand, for those sulustrates not oxidized under the influence of tyrosinase there was no detectable qualitative difference in color with Millon’s reagent between proteins treated with active or inactive tyrosinase. Of course gelatin, protamine, and gramicidin, which contain no tyrosine, gave no color at all with Millon’s reagent, regardless of the presence or absence of active tyrosinase. Those proteins resistant to tyrosinase, but rendered labile by digestion with 1 mg. of trypsin, did not react as intensely with Millon’s reagent as did the controls treated \vit,h inactive tyrosinase. From the results of these studies with Millon’s reagent it appears that tyrosinase so modifies the phenol groups of certain labile proteins that they no longer produce an intense pink color in this t’est.

Quantitative Determination of Tyrosine in Protein Hydrolysates

Bernhart’s modification of the Millon-Weiss method for tyrosine deter- mination (14) was used, according to the directions given by Block and Bolling (15). In place of the 10 mg. of protein used in the test, 2 ml. of tyrosinase-treated protein containing 5 mg. of protein per ml. were used. The final reddish colored solution which was obtained showed a maximum

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I. W. SIZER 151

absorption at 420 rnp and so the calibration curve for tyrosine was made at this wave-length. All subsequent measurements on the protein hydroly- sates were also made at 320 rnp. Results of the tyrosine analysis by this test, are present,ed in Table III, from which it appears that for tyrosinase- labile proteins the a&ion of tyrosinase results in all cases in a decrease

Effect of Tyrosimse on Tyrosine Content of Proteins As Measured by Modijied Jlillon- Weiss Test ,for Tyrosinc in Protein Hydrolysates

Protein Tyrosinase r

- I -

ryrosine in 10 mg. protein

Serum albumin, bovine “ “ “ “ “ human “ ‘< “

“ L‘ “ + trypsin “ “ “ + (‘

Casein “

Chymotrypsin “

Fibrinogen + trypsin ‘I + “

&Globulin, bcvine, + trypsin “ ‘< + id

T-Globulin, human, + “ “ “

+ iL Gramicidin + trypsin

“ + “ Hemoglobin

“

Insulin “

Pepsin “

Trypsin “

Tyrosine (0.1 mg.) “ (0.1 I1 )

Tyrosinase

Inactive Active Inactive Active Inactive Active Inactive i\ctive Inactive Act,ive Inactive Active Inactive Active Inactive Active Inactive Active Inactive Active Inactive Active Inactive Active Inactive Active Inactive Active

mg.

0.55 0.59 0.53 0.61 0.80 0.71 0.71 0.63 0.35 0.23 0.57 0.52 0.76 0.70 0.72 0.51 0.08 0.08 0.36 0.18 1.04 0.77 0.53 0.33 0.49 0.34 0.08 0.02 0.02

(as compared with the control which was treated with inactive tyrosinase) in the amount of tyrosine present in an alkaline hydrolysate of the protein. The results of the chemical tests confirm the manometric studies in re- flecting the oxidation of phenolic groups in native proteins catalyzed by tyrosinase.

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152 TYROSINASE AND PROTEINS

Effect of Tyrosinase on Color of Proteins

In view of the fact that tyrosinase, in acting on tyrosine, mono- and polyphenols and their derivatives, produces pigmented oxidation products, it is not surprising that proteins oxidized catalytically by tyrosinase are converted to colored end-products. The protein solutions studied mano- metrically for oxygen consumption gradually darkened to a reddish brown color in the presence of active tyrosinase, but not in the presence of boiled tyrosinase. This was true only of those proteins which were oxidized by tyrosinase; no change in color was observed, if the protein was not oxidized. The results of a typical experiment are shown in Fig. 3 for chymotrypsin.

I 400 450 500 550 600 650 700 750

WAVE-LENGTH,w FIG. 3. Effect of tyrosinase on the visible absorption spectrum of crystalline

chymotrypsin. The solution contained 25 mg. of chymotrypsin, 4.5 ml. of phosphate buffer, pH 7.3,2 drops of toluene, and 0.5 ml. of tyrosinase added at zero time. The chymotrypsin is oxidized to a brownish color which absorbs strongly in the blue- violet region.

The upper curve is the transmission curve immediately after adding 0.5 ml. of Upjohn tyrosinase to 25 mg. of crystalline chymytrypsin (in 4.5 ml. of phosphate buffer at pH 7.3). The transmission after 22 hours at 37” is shown by the lower curve. Although with many protein substrates there is some increase at other wave-lengths in absorption produced by tyrosinase, the maximum effect with all proteins is in the violet region at 410 rnp. Similarly the action of tyrosinase on tyrosine results in a maximum absorp- tion in the violet region.

In view of the fact that tyrosinase produces a color change of certain labile proteins, it is possible to use this production of pigment as a means of

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I. W. SIZER 153

studying the kinetics of oxidation of proteins under the influence of ty- rosinase (see Fig. 4). A comparison of Figs. 1 and 4 indicates that the ki- netics of protein oxidation are similar when followed manometrically or calorimetrically.

E$ect of Tyrosinase on Ultraviolet Absorption Spectrum of Proteins

Since the absorption peak characteristic of most proteins at about 276 rnp can be chiefly ascribed to the presence of tyrosine and tryptophane (16, 17), it seemed likely that any effect of tyrosinase on t.he tyrosine of proteins

I 2 3 4 5

TIME IN HOURS FIG. 4. Kinetics of the increase in color at 410 mp associated qTit,h the oxidation of

chymotrypsin catalyzed by tyrosinase. The composition of the solution is the same as for Fig. 3.

would be reflected in a change in the ultraviolet absorption spectrum, especially in the region of 276 rnp.

The absorption spectra were taken in the Spectroscopy Laboratory of the Massachusetts Institute of Technology with a Hilger quartz spectrograph equipped with a Spekker photometer. Detailed studies were made with untreated pepsin, chymotrypsin, and trypsin, and these same proteins after treatment with tyrosinase for several days. These solutions had been studied manometrically before their ultraviolet absorption was measured. As usual, the protein concentration was 5 mg. per ml. in phosphate buffer at pH 7.3. The control solutions contained 0.1 ml. of water per ml., while the experimental solution contained 0.1 ml. of Upjohn tyrosinase. The

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154 TYROSINA4SE AND PROTEINS

absorption of the tyrosinase solution was studied independently Tvith 0.1 ml. in 0.9 ml. of buffer. In order to measure the absorption it was necessary to adjust the concentration of protein in solution by dilution with water to different extents, depending on the absorption of the specific protein used. Before plotting the results the log extinction coefficients mere multiplied by the dilution factors to put the data for all solutions on a comparable basis.2

Results with pepsin are shown in Fig. 5. The control curve for pepsin in buffer agrees closely with that reported by Gates (18, 19) with a maximum absorption at 275 rnp attributable to tyrosine and tryptophane.

PEPSIN-TYR0SlNAS.E

250 280 310 340 370 4oc

WAVE-LENGTH,mp

FIG. 5. The ultraviolet absorption spectrum of certain protein solutions. Upper curve, the solution contains 25 mg. of crystalline pepsin, 4.5 ml. of buffer, 0.5 ml. of tyrosinase, 0.001 mg. of merthiolate. Middle curve, the same, but 0.5 ml. of water instead of tyrosinase. Lower curve, 4.5 ml. of buffer, 0.5 ml. of tyrosinase, 0.001 mg. of merthiolate. The oxidation of pepsin by tyrosinase has increased the absorption in the ultraviolet with the elimination of the maximum in absorption at 275 mp attrib- utable to tyroske.

The tyrosinase shows a typical protein absorption spectrum with a slight peak (more apparent when the data are plotted on a larger scale) at 268 rnp, agreeing fairly well with the absorption band reported by Dalton and Nelson (20). The oxidation of pepsin catalyzed by tyrosinase increases the absorption throughout the ultraviolet, but the specific peak at 275 rnp attributable to tyrosine has been completely eliminated, suggesting an

2 This correction involves the assumpt.ion that Beer’s law applies to the absorption of these solutions in the ult,raviolet. Even if it did not strictly apply, the error would not be great, since the dilution factors did not differ appreciably for the different solutions.

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I. W. SIZER 155

oxidation of the tyrosine of pepsin by tyrosinase. Results with chymotryp- sin are very similar to those for pepsin. The control solution is character- ized by a sharp peak at 272 rnp, a minimum at 253 rnp, and “end-absorp- tion” below 240 mp. The oxidation of chymotrypsin by tyrosinase gives rise to an increase in absorption over most of the ultraviolet, but the sharp peak in absorption at 272 rnp is eliminated. Tyrosinase also produced an increase in absorption of trypsin over most of the ultraviolet spectrum, but the solutions were not satisfactory for comparison at 280 rnp, at which point trypsin shows a sharp peak in absorption (21,22).

These studies on light absorption clearly demonstrate that oxidation of typical proteins catalyzed by tyrosinase results in an increased absorption by the protein in the blue-violet and ultraviolet, and that the specific absorption of the protein attributable to tyrosine is greatly decreased.

DISCUSSION

The oxidation by tyrosinase of tyrosine in certain labile proteins or in proteins rendered labile by treatment with trypsin is clearly indicated by studies of oxygen consumption, production of pigment, decrease in color produced wit.h Millon’s reagent, decrease in tyrosine in the protein hy- drolysate as measured quantitatively by the modified Millon-Weiss method, and increased absorption in the ultraviolet accompanied by the elimination of the peak at 276 rnp attributable to tyrosine. The rate of oxidation of proteins by tyrosinase is very much less than the corresponding rate of oxidation of tyrosine. Although in each case the oxidation is believed to proceed through the formation of dopa (3,4-dihydroxyphenylalanine), the further oxidation of tyrosine is not as extensive for the tyrosyl moiety of proteins as it is for free tyrosine which is converted to melanin (1).

The striking difference in susceptibility of different proteins to attack by tyrosinase suggests that in some proteins the tyrosyl group is available at the surface of the molecule, while in others the stereochemical configuration of the protein is such as to render the tyrosine residue inaccessible to ox- idation by tyrosinase. Even in those proteins labile to tyrosinase only a fraction (about 10 to 20 per cent) of the total tyrosine was oxidized by tprosinase; a large part, of the tyrosine appears inaccessible to tyrosinase. On the other hand a very large fraction of the tyrosyl residues of proteins reacts with certain chemical reagents (9, 11, 23, 24). The dif- ference in extent of reaction with tyrosyl groups of proteins by tyrosinase and chemical reagents is also illustrated by the fact that the former does not destroy the biological activity of the proteins, while the latter inactivate the proteins.

In view of the fact that t.he oxidative enzyme tyrosinase has been sh0Tv-n to act on certain proteins in vitro, it seems possible that other non-proteo-

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156 TYROSINASE AND PROTEINS

lytic enzymes will also attack native proteins. In particular the action on proteins of such enzymes as deaminases, decarboxylases, dehydrogenases, and certain oxidases deserves careful study. From such investigations may come at least a partial interpretation of the great lability of proteins in living systems.

SUMMARY

Both crude and highly purified mushroom tyrosinases have been demon- strated to oxidize the tyrosyl groups 01 such proteins as trypsin, pepsin, chymotrypsin, casein, peptone, insulin, and hemoglobin. Tyrosinase did not oxidize gelatin, protamine, and gramicidin, which are devoid of tyrosine. Certain proteins containing tyrosine were resistant to tyrosinase; these included egg albumin, human and bovine serum albumin, tobacco mosaic virus, human y-globulin, bovine fl-globulin, and bovine fibrinogen. All members of this group which were studied mere oxidized by tyrosine aft.er a preliminary treatment with crystalline trypsin.

Several techniques were employed in studying the action of tyrosinase on proteins, all of which methods yielded consistent data. These procedures included (I) the measurement of oxidation manometricallp from oxygen. consumption; (2) the measurement of phenolic groups with Millon’s test on intact proteins; (3) the quantitative determination of tyrosine in pro- tein hydrolysates; (4) the quantitative measurement of pigment production in the blue-violet caused by the action of tyrosinase on proteins; (5) the measurement of change in the ultraviolet absorption spectrum especially at 275 rnp, at which wave-length the protein absorption is due to tyrosine and tryptophane.

In contrast to most chemical reagents which react with the tyrosyl group to destroy the biological activity of the protein, tyrosinase has no effect on the enzymatic act.ivity of pepsin, trypsin, and chymotrypsin. This dif- ference may be related to the fact that only a small fraction of the total tyrosine of the protein is oxidized by tyrosinase, and that each tyrosyl group does not appear to undergo a very extensive oxidation.

BIBLIOGRAPHY

1. Selson, J. M., and Damson, C. R., in Sord, F. F., and Werkman, C. H., Advances in enzymology and related subjects, New York, 4,99 (1944).

2. Sumner, J. B., and Somers, G. F., Chemistry and methods of enzymes, New York (1943).

3. Sizer, I. W., and Prokesch, C. E., Science, 101,517 (1945). 4. Mason, II. S., Schwartz, L., and Peterson, D. C., J. Am. Chem. Sm., 67, 1233

(1945). 5. Beadle, G. W., Chem. Rev., 37, 15 (1945). 6. Wright,, S., Annual review of physiology, Stanford University, 7,75 (1945). 7. Schoenheimer, R., The dynamic state of body constituents, Cambridge (1942).

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I. W. SIZER 157

8. Koch, F. C., Practical methods in biochemistry, 3rd edition, Baltimore (1941). 9. Sizer, I. W., J. Biol. Cheln., 160, 547 (1945).

10. Lion, I<. S., and Sizer, I. W., Arch. Surg., 48, 120 (1944). 11. Herriott, R. AI., J. Gen. Physiol., 19, 283 (1935). 12. Folin, O., and Ciocalteu, V., J. Biol. Chem., 73, 627 (1927). 13. Harrow, B., Laboratory manual of biochemistry, Philadelphia (1940). 14. Bernhart, F. W., Proc. /l,n. Sot. Biol. Chem., J. Biol. Chem., 123, p. x (1938). 15. Block, R. J., and Bolling, D., The amino acid composition of proteins and foods,

Springfield (1945). 16. Sizer, I. W.: Proc. Sot. Exp. Biol. and Med., 49, 700 (1942). 17. Loofbourow, J. R., Rev. Modern Phus., 12,267 (1940). 18. Gates, F. L., J. Gen. Physiol., 18, 265 (1934). 19. Gates, F. L., J. Exp. Med., 60, 179 (1934). 20. Dalton, H. R., and Selsog, J. hf., J. Am. Chenl. Sot., 61, 2946 (1939). 21. Uber, F. &I., and McLaren, A. D., J. Biol. Chem., 141,231 (1941). 22. Verbrugge, F., J. Biol. Chem., 149,405 (1943). 23. Christensen, H. K., J. Biol. Chem., 160, 75 (1945). 24. Weill, C. E., and Caldyell, %I. L., J. Anz. Chem. Sot., 67,212,214 (1945).

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Irwin W. SizerPROTEINS

THE ACTION OF TYROSINASE ON

1946, 163:145-157.J. Biol. Chem. 

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