FACTORS WHICH GREATLY INCREASE THE ACTIVITY OF THE PHENOLIC HYDROXYL GROUP OF

I-TYROSINE*

BY DONALD E. BOWMAN

(From the Department of Biochemistry, School of Medicine, Western Reserve University, Cleveland)

(Received for publication, June 18, 1941)

That tyrosine constitutes an integral part of the structure of many enzymes, hormones, and proteins of immunity and plays an important r-ale in their physiological action is becoming evident. Thus, the fundamental relations between the phenolic hydroxyl of this amino acid and the physiological properties of pepsin (l-3), pepsinogen (4), insulin (5, G), and the chorionic gonado- tropic (7) and lactogenic hormones (8) have been demonstrated and it is quite possible that the contribution of this group to the essential nature of many other protein or peptide catalysts will also be observed.

Mirsky and Anson (9) have shown that ferricyanide is reduced not only by the sulfhydryl components of protein molecules but also by tyrosine and tryptophane. These authors found that as reductants tyrosine and tryptophane react very slowly and yet their reducing capacity is greater than that of the sulfhydryl groups. It was pointed out that, although the activity of the non-sulfhydryl reducing groups is enhanced by an increase in pH, rise in temperature, and denaturation, the reduction of ferri- cyanide by these groups ordinarily extends over a period of at least 5 hours.

In an earlier publication (10) we have presented evidence which indicates that the reducing action of the chorionic gonadotropic hormone is greatly intensified in the presence of phosphate and moderate heat. In the absence of these accelerating factors

* The material in this paper has been presented in part at the meeting of the American Society of Biological Chemists at Chicago, April, 1941.

877

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878 Reducing Action of Z-Tyrosine

oxidation of the hormone proceeds very slowly even in the pres- ence of strong oxidants. Since the physiological activity of the hormone, which decreases with oxidation (lo), is apparently de- pendent upon the presence of the phenolic hydroxyl group of tyrosine (7), it is not surprising to find that this group has reducing properties similar to those of the hormone. These observations have led to a more detailed study of crystalline tyrosine; and the object of the present paper is to present data which indicate that under certain conditions this amino acid is much more reactive as a reductant than has been previously supposed. It would appear that the normal physiological state should provide these conditions.

EXPERIMENTAL

The intensity of the reducing action of crystalline I-tyrosine (Eastman) was measured by observing the time required for the reduction of a given amount of an oxidant in a dilute solution. The influence of various accelerating or inhibiting agents upon this reduction time was determined. The 0.001 N iodine employed as an oxidant was prepared from potassium iodate, potassium iodide, and hydrochloric acid by combining, in order, 1 cc. of a stock iodate solution containing 3.567 gm. of KIOI per liter, 1 cc. of a stock iodide solution containing 13.835 gm. of KI per liter, 15 cc. of distilled water at O”, 1 cc. of 2 N HCl, and, after thorough mixing, sufficient distilled water at 0” to provide a total volume of 100 cc. This dilute solution was prepared each time just before it was used and was kept in an ice bath at 0”. A stock solution of iodine-potassium iodide which is more commonly used was not employed, since it is important to avoid an excess of potassium iodide. This will be discussed below. The dilute potassium permanganate was prepared by diluting a 0.1 N solution.

The oxidation-reduction dyes which may be easily obtained cannot be used as oxidants, since they apparently have E,J values below that of the phenolic hydroxyl of tyrosine. It may be pointed out that epinephrine and homogentisic acid also have E. values above those of the usual oxidation-reduction dyes.

The phosphate buffer mixtures were prepared according to SQrensen’s tables. Merck’s soluble starch, prepared according to Lintner, was employed as an indicator. In each case 1 cc. of

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D. E. Bowman 879

an exactly 0.2 per cent solution of starch prepared from a single source was used. The total volume of all constitutents of a given test was adjusted to 10 cc. unless otherwise indicated.

The phenol color values of solutions were obtained by a modi- fication of Herriott’s procedure (ll), with the phenol reagent of Folin. Before the colors were developed, the reagents and test solutions were cooled to 0”. After color development with the phenol reagent the solutions were allowed to stand 5 minutes at the same temperature. The phosphate precipitate was dissolved with a minimum of glacial acetic acid and the solution was quickly filtered. The color was matched against simultaneously pre- pared tyrosine standards within 8 minutes after color develop- ment. It was found that this procedure did not decrease the phenol value of unoxidized tyrosine; yet it minimized the reduc- tion of the phenol color reagent by reaction products such as hydriodic acid.

Results

Agents Which Accelerate the Reaction-The oxidant most fre- quently employed was iodine. Being a milder oxidant than potas- sium permanganate, it has certain advantages in following the kinetics of the reaction. Also the more intense color given by iodine, with starch, in the dilute solutions used is desirable. Ferricyanide was not used in order to avoid turbidity which results when the ferric indicator is added to a solution strongly buffered with phosphate. The use of phosphate buffer is particularly significant, since the addition of this salt to an aqueous solution of tyrosine or phenol greatly accelerates the rate at which these substances react with various oxidants. This is indicated by the data presented in Table I.

It will be seen that in the absence of the buffer a week or more may be required for 1 cc. of 0.001 M tyrosine or phenol completely to reduce 1 cc. of 0.001 N iodine at 25”. In the presence of phos- phate not more t’han 1 or 2 minutes is required. Similar relations were observed a,t 38”, although t,he reaction was somewhat more rapid.

It has been found that the substitution of some of the other buffers such as citrate or acetate in place of phosphate gives results similar to those just described. However, in some instances

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880 Reducing Action of I-Tyrosine

phosphates show a certain degree of specificity in addition to maintenance of pH. This is particularly true in the reduction of silver described below:

From Table I it is also apparent that the complete decoloration of potassium permanganate by tyrosine or phenol is accelerated by the phosphate in a similar manner, and it is of particular in- terest to note that phenylalanine does not possess this ability to reduce iodine or permanganate rapidly in the presence of phosphate.

In the presence of this buffer tyrosine readily reduces silver nitrate at room temperature when exposed to light. This can be

TABLE I

InfEuence of Phosphate Ion on Oxidation of Phenol, Tyrosine, and Phenylalanine

Phenol L< + phosphate*.

Tyrosine. ‘I + phosphate

Phenylalanine. “ + phos-

phate................. Phosphate alone.

- Time required for 1 cc. 0.001 M reductant to react with

1 cc. 0.001 i-i oxidant

25”

Iodine

1 wk. 15 sec. > 1 wk. 120 sec. > 1 wk.

>l L‘ >l “

KMnOa

24-72 hrs. 2.5 min. > 1 wk. 60 sec. > 1 wk.

>l ‘( >l “

38”

Iodine

> 24 hrs. 5 sec. 20 hrs. 3 sec. 20 hrs.

23 “ 1 wk.,

KMn04

> 24 hrs. 60 sec. 90 min. 45 sec. > 24 hrs.

2.5 “ >24 ‘l

* 0.5 cc. of 1 M Serensen’s phosphate buffer, pH 6.81.

demonstrated by combining 1 cc. of 0.001 M tyrosine, 0.5 cc. of 1 M phosphate buffer, pH 6.8, and an excess of silver nitrate such as 1 cc. of a 10 per cent solution. After standing 5 to 10 minutes in daylight (such as that equivalent to 300 foot-candles) the black metallic silver can be observed mixed with the yellow precipitate of silver phosphate. If sufficient ammonium hydroxide is added to dissolve the silver phosphate, the remaining reduced silver is very striking. In the absence of phosphate this reduction does not take place nor does it occur in the presence of phosphate if pure phenylalanine is substituted for tyrosine.

At pH 5.8 the reduction of silver by tyrosine readily takes place

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when the solution is buffered with phosphate but it was not ob- served when acetate, citrate, or phthalate buffers were employed at the same concentration and pH.

Since phenylalanine differs from tyrosine in that it does not react rapidly with iodine, permanganate, or silver nitrate even in the presence of phosphate, it would appear that the phenolic hydroxyl is the group primarily involved. The reaction of this group with permanganate or silver nitrate is undoubtedly one of simple oxidation-reduction; however, in the reaction with iodine substitution must also be considered. Yet increased ease in substitution would in itself suggest a more active phenolic hydroxyl group as the primary factor.

!&i % 2 0

B < 20

d 5 40 B

f 60 ti L.5 oc 80 I "

2 "'0

r-t I I 2 3 4 5 6 7 8

CUBIC CENTIMETERS OF 0001 M IODINE ADDED CUBIC CENTIMETERS OF 0001 M IODINE ADDED

FIG. 1. Decrease in phenol color value of 1 cc. of 0.001 M tyrosine after reaction with iodine.

In order to observe changes in this group which may accompany substitution of iodine in the benzene ring the phenolic color value of tyrosine was followed after the amino acid was allowed to react with various amounts of iodine. From Fig. 1 it will be seen that the phenol color value decreases progressively as the tyrosine reacts with increasing amounts of iodine, suggesting simultaneous oxidation and substitution. While the possibility that the de- crease in phenolic color value is due to the stabilizing influence of the substituted iodine has not been excluded, similar decreases in the phenol color value of tyrosine have been observed after the amino acid has been allowed to react with permanganate or silver nitrate in the presence of phosphate. It is of course neces-

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882 Reducing Action of I-Tyrosine

sary to avoid the interference caused by the reduced form of the oxidant in each case.

It was found that relatively large amounts of phosphate are required to accelerate to a maximum degree the reaction of tyro- sine with iodine. From the data presented in Table II it will be

TABLE II Influence of Phosphate Salts on Reaction of LTyrosine and Phenol with

Weight of salt added* T 0.1 mg. I-tyrosine 0.06 mg. phenol

w. min. min.

0 720 390 3.52 577 270 6.85 480 135

10.3 158 30 13.7 20.5 3.25 17.1 8.5 2.0 20.6 6.5 1.5 24.0 5.5 1.5 27.4 5.2 1.5 41.0 4.5 1.5 48.0 4.5 1.5

Iodine

Time required to react with 3 M proportions of iodine at 38”

* S@rensen’s phosphate buffer mixture, pH 5.9.

TABLE III InJluence of Temperature on Reaction of l-Tyrosine with Iodine

Temperature Time required for 0.1 &g. I-tyrosine to react with 3 M proportions of iodine at 38’*

“C. min.

20 265 30 28.5 40 2.75 50 Instantaneous

* In the presence of 68 mg. of &rensen’s phosphate salt mixture, pH 5.9.

seen that, at pH 5.9, about 41 mg. of the buffer salt are required for maximum rate of reaction of 1 mg. of tyrosine with 3 M pro- portions of iodine. Somewhat less phosphate is required at higher pH values.

That moderate heat also exerts a very striking influence on the rate of the reaction is indicated by the data given in Table III.

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D. E. Bowman

It will be seen that in the presence of phosphate, pH 5.9, the rate increases about 10 times for each 10” rise in temperature until the reaction becomes instantaneous at 50”. At slightly higher pH values it becomes instantaneous at lower temperatures.

The rate of reaction with iodine is also affected by a change in pH at a constant temperature. Thus from the data presented in Table IV it will be seen that the speed of reaction is approximately inversely proportional to the hydrogen ion concentration. Still further acceleration might be anticipated if the pH were increased to that of the normal physiological range.

The influence of pH and temperature upon the rate of reaction is less evident when permanganate is employed as the oxidant. Also considerably less phosphate is required to bring the reaction

TABLE IV

Injluence of Hydrogen Ion Concentration on Reaction of I-Tyrosine with Iodine

-~ _____

pH of phosphate buffer mixture* Time required for 0.08 mg. Z-tyrosine to react with 3.6 M proportions of iodine at 38”

- - min.

5.90 11.0 6.24 3.1 6.47 1.5 6.64 0.8 6.81 0.5 6.98 0.25

* 0.5 cc. of 1 M buffer solution was employed in each case.

to its maximum speed. This may be attributed to the stronger oxidizing power of permanganate, making it less dependent upon the favorable conditions which are necessary to support hhe rapid reaction with iodine.

By keeping all of the T-ariablcs ronstanf,, csrept the concentra- tion of the reductant), it may be observed that the time required to reduce a given amount, of iodine incrcascs exponontia.ll~ as the

concentrabion of tyrosine decreases. Agents Which Retard Reaction with Iodine--In the presence of

phosphate and at elevated temperatures pot,assium iodide is capable of greatly retarding the reaction of tyrosine with iodine. Therefore it’ is necessary carefully to st’andardize the potassium

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884 Reducing Action of I-Tyrosine

iodide content of the iodine solution which is to be employed. By preparing the iodine in very dilute solutions from iodate, iodide, and acid as described above it is possible to avoid the marked excess of iodide which is necessary to keep iodine in a more con- centrated solution. This is advantageous from the point of view of the present study.

The rate of reaction is also altered by the starch employed as an indicator. The majority of soluble starch preparations retard the reaction somewhat, but some allow it to proceed much more rapidly than others. Although the reason for this is not entirely clear, it should not interfere with the study of the influence of other

TABLE V

Reduction of Iodine by Casein, Egg Albumin, and Gelatin in Presence of Phosphate Ion

Weight of protein

mg.

1.0 1.25 1.50 1.75 3.0 4.0 5.0

Time required to reduce 5 co. 0.0005 N iodine at 38” *

Casein

min. min. 31 140 13.5 74 6.5 47 4.5 30

Egg albumin Gelatin

min.

1140 480 300

* In the presence of 68 mg. of buffer salt, pH 5.9.

factors as long as a uniform amount of starch obtained from a single preparation is employed throughout.

“Tyrosine Reaction” of Proteins-It is of interest to compare the reducing properties of various proteins which differ with respect to their tyrosine content. From the results represented in Table V it will be observed that casein, which contains more tyrosine but less cystine than dried commercial egg albumin1 reacts with more iodine in the presence of phosphate than does egg albumin. Gelatin containing very little tyrosine shows only

1 Casein contains 6.6 per cent tyrosine and 0.3 per cent cystine, while egg albumin contains 4.2 per cent tyrosine and 1.3 per cent cystine (from Schmidt (12)).

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a slight reaction. If these observations are repeated with the various proteins under the same conditions except that water is substituted for the phosphate buffer solution, only slight activity is apparent even after 96 hours at 38”. These data are apparently in accord with the belief that the reducing capacity of the non- sulfhydryl reducing groups of protein materials is greater than that of the sulfhydryl groups.

It has already been pointed out (9, 13) that the reducing activity of the phenolic groups of common proteins increa,ses as the pro- teins are denatured. Our experience is in accord with this ob- servation except in the case of some of the more uncommon labile proteins such as gonadotropin (9) which show a decrease rather than an increase in activity after being heated. Preliminary experiments also indicate that a labile component may be asso- ciated with serum globulin. For example, while heat denatura- tion of serum albumin causes about a 25-fold increase in the rate of oxidation of iodine in the presence of phosphate, similar dena- turation of the carefully prepared globulin fraction causes a moderate decrease in this activity. It would appear that,, in the globulin fraction, the liberation of addit.ional phenolic groups through denaturation is somewhat overshadowed by the oxidation of similar groups which is accelerated by the heat.

DISCUSSION

From the data presented it is apparent that while tyrosine is ordinarily oxidized in vitro at a very slow rate, even in the presence of relatively strong oxidants, under certain conditions the reaction takes place almost instantaneously. It would appear that the normal physiological environment should provide conditions neces- sary to support the increased activity of this group, at least in highly specialized proteins which though present in very small quantities have profound metabolic effects.

The fundamental relations between the phenolic hydroxyl group and the physiological activity of various enzymes and hormones have been pointed out by a number of investigators. The chem- ical activity of this group under physiological conditions is also of considerable interest in immunochemistry. It has been re- garded as playing a dominant role in determining the immuno- logical character of proteins. The specificity of proteins as an-

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886 Reducing Action of I-Tyrosine

tigens can be entirely changed by alterations in the tyrosine groups through nitration or halogenation ortho to the phenolic hydroxyl groups. Yet it has been stated that one outstanding difficulty is to account for the specific activities of antigens in the absence of evidence that the majority contain any specially reactive groups.

In view of the present findings it would s,puear that this group may not be as stable in vivo as was once thought, but it may be one of the active groups being sought. If this is the case, it is quite possible that proteins differ not only in the spatial distribution of these groups but also in the activity patterns which they present, the activity of each group reflecting its molecular environment.

SUMMARY

In a study of the reducing action of I-tyrosine it was found that the rate of reaction is far more rapid, under certain conditions, than was hitherto supposed. The results of the present study may be summarized as follows:

1. The rate at which tyrosine reacts with iodine, potassium permanganate, and silver nitrate is ordinarily quite slow; however, it may be greatly increased by the addition of phosphate buffer. Small increases in pH greatly intensify the reaction.

2. In the presence of phosphate further marked acceleration results from a moderate increase in temperature, until the reaction becomes instantaneous.

3. This reducing action of I-tyrosine may be att,ributed to the phenolic hydroxyl group. .

4. It would appear that the normal physiological state should provide the conditions necessary to support the increased activity of this group. This may further explain why this group is capable of playing such a dominant role in the physiological action of various protein catalysts.

BIBLIOGRAPHY

1. Herriott, R. M., and Northrop, J. H., J. Gen. Physiol., 18, 35 (1934-35). 2. Herriott, R. M., J. Gen. Physiol., 19, 283 (1935-36). 3. Philpot, J. St. L., and Small, P. A., Biochem. J., 32, 542 (1938). 4. Herriott, R. M., J. Gen. Physiol., 21, 501 (1937-38). 5. Stern, K. G., and White, A., J. Biol. Chem., 123,371 (1937-38).

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6. White, A., in Cold Spring Harbor symposia on quantitative biology, Cold Spring Harbor, 6, 262 (1938).

7. Li, C. H., Simpson, M. E., and Evans, H. M., J. Biol. Chem., 131, 259 (1939).

8. Li, C. H., Lyons, W. R., and Evans, H. M., J. Biol. Chem., 139, 43 (1941).

9. Mirsky, A. E., and Anson, M. L., J. Gen. Physiol., 19, 451 (1935-36). 10. Bowman, D. E., J. Biol. Chem., 137, 293 (1941). 11. Herriott, R. M., J. Gen. Physiol., 20, 335 (1937). 12. Schmidt, C. L. A., The chemistry of the amino acids and proteins,

Baltimore (1938). 13. Mirsky, A. E., in Cold Spring Harbor symposia on quantitative biology,

Cold Spring Harbor, 6, 150 (1938).

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Donald E. Bowman-TYROSINE

lPHENOLIC HYDROXYL GROUP OF INCREASE THE ACTIVITY OF THE

FACTORS WHICH GREATLY

1941, 141:877-887.J. Biol. Chem. 

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