TYROSINE AND TRYPTOPHANE DETERMINATIONS IN ONE-TENTH GRAM OF PROTEIN.

BY OTTO FOLIN AND A. D. MARENZI.*

(From the Biochemical Laboratory 0.f Harvard Medical School, Boston.)

(Received for publication, May 7, 1929.)

I. Preliminary Discussion.

In the calorimetric methods for the determination of tyrosine and tryptophane, described by Folin and Ciocalteu (1) in 1927, 1 or 2 gm. of protein material are taken for the preliminary hydrolysis. It has seemed desirable to try to adapt these methods to the use of smaller amounts of material (0.1 gm.) in order to broaden the applicability of the methods. While this work was in progress, there appeared a lengthy paper, by Hanke (2), on colori- metric tyrosine determinations, and it seems suitable first to con- sider that paper before taking up the constructive work.

Hanke’s method for tyrosine was published in 1922 and, during the intervening 7 years, Hanke has repeatedly used that method as a basis for criticism of other methods, particularly those published from this laboratory. He now finds that one essential step in his method, namely the removal of histidine with silver oxide, also removes tyrosine and, on the basis of that error alone, the tyrosine content of casein moves from 4.5 per cent (his earlier figures) to about 5.5 per cent. As the silver oxide treatment was a necessary prerequisite step in his method, he now definitely abandons that meth0d.l It is to be noted further that there is now no continuity between his tyrosine work of the past 7 years and the main part of the work reported in his present paper. What he now presents is just a modification of the Folin-Ciocalteu method, and on that

* Fellow of the Division of Medical Education, Rockefeller Foundation. l As late as 1927, Hanke insisted that the tyrosine in the silver oxide pre-

cipitates is not tyrosine, but an“ X factor,” the presence of which proved the superiority of hismethod. (Hanke, M. T., J. Biol. Chem., 74, p. x (1927).)

89

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90 Tyrosine and Tryptophane of Protein

basis he is taking a fresh start in the field of tyrosine determina- tions.

There is one slight link of continuity between Hanke’s past and his present work on tyrosine determination. He still main- tains that the tyrosine must be precipitated from the hydroly- sates and he uses for that precipitation the same treatment with mercuric acetate and sodium chloride as was used in his own method. It is well that some one should continue to take that

TABLE I.

Precipitation of Tyrosine from Pure Solutions by Means of Mercuric Acetate and Sodium Chloride.

Tyrosine taken. Tyrosine recovered. Difference. LOSS.

?w. mg. m!J. per cent

2 1.316 0.684 34.2

3 1.67 1.33 44.3 3 2.01 0.99 33.0 4 3.49 0.51 12.75 4 2.79 1.21 30.25 5 4.21 0.79 15.84 5 3.84 1.16 23.20

5 3.51 1.49 29.8 6 4.47 1.53 25.5 8 7.43 0.57 7.13

10 8.62 1.38 13.8 10 8.62 1.38 13.8 10 9.30 0.70 7.0 20 19.04 0.96 4.8

50 47.6 2.40 4.8 50 48.96 1.04 2.1

view, because it may ultimately lead to some quantitative method for the isolation of tyrosine. But after his unhappy experience withone of his precipitants (silver oxide) one would think that Hanke would not immediately set out on a new venture with the other (mercuric acetate and sodium chloride) without first positively proving that it does quantitatively precipitate tyrosine both from pure solutions and from hydrolysates. Hanke’s statement on the subject is not altogether satisfactory and his analytical data are too meager, since they are supposed to prove the most important point in his paper. We have deemed it worth while, therefore,

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0. Folin and A. D. Marenzi

to make some experiments of our own on the precipitation and re- covery of tyrosine from pure tyrosine solutions, by Hanke’s method. Our results are recorded in Table I.

The figures in Table I speak for themselves. But we might call particular attention to the lack of uniformity in the absolute losses of tyrosine. Practically every experiment gives a different loss even when the same amount of tyrosine is taken. It may be pertinent to recall in this connection that Hanke now believes that the tyrosine content of casein is a variable, so that, in its tyro- sine content, the casein from one cow is different from the casein obtained from another, and, in addition, that old casein samples contain less tyrosine than when first prepared. One would neces- sarily expect to encounter both larger and more variable losses from protein hydrolysates than from pure tyrosine solutions or from artificial mixtures of pure amino acids, by his precipitation process.

Hanke checked up the losses of tyrosine by determining the tyro- sine content of the filtrates from the tyrosine mercury precipi- tates, so that the losses when working with pure solutions could readily be int.erpreted as representing merely the solubility of the tyrosine precipitate. Our experience does not confirm this obser- vation. Our filtrates yielded only insignificant traces of tyrosine, so that in our work the lost tyrosine had just disappeared. It is to be noted that Hanke when working with casein hydrolysates obtained exactly the same sort of tyrosine disappearance which we have obtained from pure tyrosine solutions. His explanation of the phenomenon seems rather romantic (3).

There is one observation in Hanke’s paper for which we must not fail to give him credit; namely, the fact that cystine interferes with the direct determination of tyrosine by the reaction of Folin and Ciocalteu. This seemed of interest to us because our plan of work included a critical study of cystine determinations in acid protein hydrolysates by the method of Folin and Looney. Cystine is destroyed when protein is hydrolyzed with sodium hydroxide just as tryptophane is destroyed by acid hydrolysis. But just as the decomposition products of tryptophane might interfere with the subsequent determination of tyrosine with the phenol reagent by giving some color with it, so the residue from the cystine may interfere with the tyrosine determinations in the new method:

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Tyrosine and Tryptophane of Protein

because the residue, like undecomposed cystine, gives a precipitate and turbidities with the mercury reagent.

In the Folin-Ciocalteu paper, due attention was called to the fact that there is danger of getting turbidities when working with protein hydrolysates. It was not recognized that these turbidities were due to the original presence of cystine. The method as described provides, however, not only for the removal of the tryptophane, but also for the removal of most of the decomposed cystine, and if the method is carried out exactly as described, with due attention to the acidities of the reagents, turbidities will rarely be encountered. The precipitate which cystine and the cystine derivative give with mercuric sulfate is the more soluble the greater the acidity. In the Folin-Ciocalteu method, the cystine derivative and the tryptophane are precipitated from a total soIution of 12 cc., having an acidity of a little more than 3 N, and the final reading of color is made on 100 cc. of solution whose acidity is that of a normal acid. These conditions were empirically found to yield clear deep red solutions with the protein hydrolysates. But it is possible that the selected conditions provide a little too small a margin of error though this margin was intentionally made small, because the greater the acidity of the final solution the more does the color change from red to orange. The directions also call for an immediate color comparison. It is not really essential that the colors obtained should contain the least possible shade of yellow, for the somewhat more orange-colored solutions obtained at an acidity of about 1.2 N acid also give a perfect proportionality for widely different quantities of tyrosine. By adding 9 cc. of 7 N

acid to the standard and the unknown where the original direc- tions call for 6 cc. (p. 139 of the Folin-Ciocalteu paper) the sug- gested acidity is obtained and the tendency to get turbidities is practically excluded. By the additional change of the heating time from 15 minutes to 5 minutes, all turbidity due to cystine is completely excluded.

In connection with the present research we have, of course, taken cystine into account, as was not done by Folin and Ciocalteu, and have shown that neither the cystine as found in acid hydroly- sates, nor added cystine, nor alkali-decomposition products of cystine interfere with our tyrosine and tryptophane determina- tions.

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0. Folin and A. D. Marenzi 93

In other words, the Folin-Ciocalteu process for the determin- ation of tyrosine can be applied to protein hydrolysates obtained by the help of sulfuric acid. The mercuric sulfate precipitation then removes the cystine so completely that it does not interfere with the tyrosine determination. In this case, one simply precipi- tates the cystine in exactly the same manner as the tryptophane is precipitated from the alkaline hydrolysates. And it may be remarked in passing that the tyrosine values will be the same whether one uses an acid or an alkali hydrolysate. In the present research, we encountered some difficulty due to cystine when work- ing with serum albumin. This protein contains fully 6 per cent of cystine, a value almost approaching the cystine contents of the keratins, and with the alkali hydrolysates from this protein we did encounter some troublesome turbidities in the tyrosine determina- tions until we had discovered the twofold remedy suggested above.

II. Development and Description of Micro Method.

As already indicated, we are here trying to solve the problem of determining both tyrosine and tryptophane in 0.1 gm. of protein. In pursuit of this object, we have replaced Kjeldahl flasks with test-tubes and have substituted the water bath for direct boiling in connection with the hydrolysis. A temperature of about 100” is, of course, much less effective than is boiling with a 20 per cent sodium hydroxide solution, and we were rather skeptical as to whether the projected process would work. And as a matter of fact it does not work with casein. It is remarkable that casein should be so much more resistant to alkaline hydrolyses than other proteins, but the fact that it cannot be used in this process is perhaps of minor importance, since this protein is always available in large quantities.

Our micro method probably could be adapted to casein by substituting gentle boiling in a metal bath or an oil bath for the lower temperature of the steam bath, if one wanted, for example, to make a comparative study of the caseins obtained from different animals, but as we are not now contem- plating such a study here, we have not included casein in this work.

Since our micro methods are in principle identical with the original methods of Folin and Ciocalteu we shall confine ourselves to concise descriptions of the methods as actually used.

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94 Tyrosine and Tryptophane of Protein

Hydrolysis.-Transfer to the bottom of a clean, dry Pyrex test- tube (150 mm. X 16 mm.) about 100 mg. of dried protein mate- rial. Add 2 cc. of 20 per cent sodium hydroxide solution. Shake gently with a little heating until the protein has dissolved. Close the mouth of the test-tube with a cork wrapped in tin-foil and carrying as condenser a glass tube about 50 cm. long. Heat in a steam bath or boiling water bath for 12 to 18 hours. The time required for complete hydrolysis differs for different proteins. Albumins are hydrolyzed in from 12 to 14 hours, but globulins require from 16 to 18 hours. At the end of the hydrolysis, add immediately to the hot solution 3 cc. of 7 N sulfuric acid. It is essential that the silica should here be obtained as a precipitate and not as a colloidal solution. After cooling, transfer the hy- drolysate to an accurately graduated 25 cc. test-tube or volumetric flask by repeated washings with small quantities of water. Dilute to volume with water, add 0.2 gm. to 0.5 gm. of kaolin, shake well, and filter. A small filter (9 cm.) should be used, and the funnel must be covered with a watch-glass to prevent evaporation, be- cause the filtration is slow. 20 cc. of the filtrate are taken for the determinations.

Determination.-Transfer 20 cc. of the filtrate to a conical cen- trifuge tube, capacity 50 cc. Add 4 cc. of a solution containing 15 per cent of mercuric sulfate in 6 N sulfuric acid. The mercury solution must be added, drop by drop, from a distance of not less than 3 cm. Perfect mixing is thus secured without any stirring. Set aside for 2 to 3 hours. 2 hours are required for the complete precipitation of the tryptophane. Centrifuge at a fairly high speed for 5 to 10 minutes. We have now the tryptophane with a little tyrosine (and nearly all of the cystine derivative) in the sediment and the tyrosine in the supernatant solution.

Decant into a 100 cc. volumetric flask and wash the edge of the centrifuge tube with 1 cc. of 0.1 N sulfuric acid.

Next transfer 10 cc. of a 1.5 per cent mercuric sulfate solution in 2 N sulfuric acid and stir with a glass rod; rinse the rod with 2 cc. of the same solution, and let the mixture act for 10 minutes. Centrifuge as before, and add the wash liquid to the main solution in the volumetric flask, again rinsing off the lip of the centrifuge tube with 1 cc. of 0.1 N sulfuric acid.

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In order to remove the surplus mercury and the slight traces of tyrosine which may still be in the centrifuge tube, we wash the precipitate once more, this time with 0.1 N sulfuric acid. Add 10 cc. of the acid, stir, and wash the stirring rod with 2 cc. of the 0.1 N

acid. Centrifuge as before and transfer the supernatant liquid to the volumetric flask.

For the determination of the tyrosine in the volumetric flask, it is first necessary to transfer to another 100 cc. volumetric flask 4 mg. of tyrosine plus the same amounts of water, acid, and mercuric sulfate as are contained in the unknown solution; that is to say (1) 4 cc. of a solution of tyrosine in 2 N sulfuric acid and con- taining 1 mg. of tyrosine per cc., (2) 16 cc. of water, (3) 4 cc. of the 15 per cent mercuric sulfate solution, (4) 12 cc. of the 1.5 per cent mercuric sulfate solution, (5) 14 cc. of 0.1 N sulfuric acid. Fi- nally add 6 cc. of 7 N sulfuric acid to the contents of each volumetric flask. The acid in each flask should be equivalent to just about 100 cc. of N HzSOd.

It may be remarked here that we are adding only 6 cc. of 7 N

H&04, although we recommend the addition of 9 cc. of 7 N acid at the same stage in the original Folin-Ciocalteu process. The difference is due to the fact that the tryptophane is not precipi- tated at the same degree of acidity in the two procedures.

Heat the contents of the two flasks simultaneously in boiling water for 5 minutes. Folin and Ciocalteu prescribe a heating period of 15 minutes but 5 minutes are ample. Cool the flasks. Add, with shaking, 1 cc. of 2 per cent sodium nitrite solution to each flask and after 2 minutes waiting dilute to volume, mix, and make the color comparison without undue delay.

4 X 20, or 80, divided by the calorimetric reading obtained (when the standard is set at 20 mm.) gives in mg. the amount of tyrosine present in the 20 cc. of hydrolysate which were taken for analysis.

Some supplementary remarks should be made. 1. The acidity of the unknown and the standard, after dilution

to 100 cc., should be very nearly the same. The maximum per- missible difference is 10 per cent; that is, if the acidity of thestand- ard is normal, that of the unknown must be betweenO.g~and 1.1 N.

2. Perceptible fading begins within 30 minut,es.

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96 Tyrosine and Tryptophane of Protein

3. In the Folin-Ciocalteu reaction for tyrosine, 15 minutes is unnecessarily prolonged for the heating period for the preliminary production of the mercury compound. That a heating time of 5 minutes is adequate is shown by the calorimetric readings re- corded in Table II.

4. In connection with the determination of tyrosine described above, it might be pointed out that in this case one uses practically the whole of the hydrolysate. It is, therefore, rather more impor- tant than in the Folin-Ciocalteu method that no errors are made in

TABLE II.

Showing That a 6 Minute Heating Period Is Adeauate in the Folin-Ciocalteu Reaction for Tyrosine:

Tyrosine. Heating time. Calorimetric reading.

ml.

4 4 4 4 4 4 4 4

min.

15 10 10 5 5 3 3 2

20.0 20.0 20.0 19.9 20.0 20.7 20.6 23.0

In each experiment the contents of a flask which had been heated for 15 minutes were used as the standard.

following the directions and that the different required solutions are available when the hydrolysate is ready.

The stock solution for all the acids can be a 14 N solution, and a 14 N solution accurate enough for the purpose is obtained by dilut- ing 400 cc. of the pure concentrated acid (sp. gr. 1.82) to a volume of 1 liter. For additional details, such as the purification of tyro- sine, of mercuric sulfate, etc., the reader is referred to the Folin- Ciocalteu paper.

5. For the determination of tyrosine in amino acid mixtures obtained by acid hydrolysis, it is essential that the hydrolysis should be made with sulfuric acid as for cystine determinations. Almost every one must know that one cannot apply Millon’s reaction for tyrosine in the presence of much chloride, and the

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same sort of interference by chlorides is, of course, encountered in the Folin-Ciocalteu reaction. This interference is presumably due to the formation of chlorine. Hydrochloric acid, therefore, cannot be used for the protein hydrolysis.

Determination of Tryptophane.-Two slightly different methods were described by Folin and Ciocalteu for the determination of the tryptophane in the centrifuge tube. In this paper we shall describe only the one which seems a little more convenient and which is the one actually used in the analyses reported below.

Add 10 cc. of N HCl to the precipitate in the centrifuge tube and heat in boiling water for 30 minutes. It is quite necessary to heat for this length of time to break up the mercury compound of tryp- tophane. A trace of insoluble material may remain in the tube; this unknown material does not give a color with the phenol rea- gent. Cool the heated solution, and filter it through a small filter, accompanied by thorough washing into a 100 cc. volumetric flask. The total volume in the flask should be about 60 cc.

Transfer 1 mg. of tyrosine to another 100 cc. volumetric flask, and dilute to a volume of about 60 cc. Add to each flask 25 cc. of saturated sodium carbonate solution, which has not been in contact with rubber, mix, add 5 cc. of the phenol reagent, de- scribed by Folin and Ciocalteu, to each flask, mix again, and let stand for 30 minutes. At the end of this time add 2 or 3 cc. of 5 per cent sodium cyanide to each solution. Dilute to volume, mix, and make the color comparison with the standard, set at 20 mm.

The cyanide does not take part in the reaction, as it is not introduced until after the phenol reagent has been destroyed by the alkali; it is used only to dissolve the precipitated mercury salts.

1 X 20 divided by the calorimeter reading in mm., and the result multiplied by the factor 0.843 gives in mg. the amount of tryptophane present in 20 cc. of hydrolysate. The factor 0.843 represents the color value of tryptophane in terms of tyrosine as was explained in the original paper.

III. Analyses of Diferent Proteins by the Micro Method.

The tyrosine and tryptophane values given in this section are not intended to represent revisions of the values reported by Folin and Ciocalteu. The small differences encountered may be due, to a certain extent, to some slight variations in the technique as, for

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98 Tyrosine and Tryptophane of Protein

example, the clearing of all the hydrolysates with kaolin. The proteins examined were obtained from Dr. Edwin J. Cohn, unless otherwise stated.

Our first experiments were made with commercial egg albumin. These were intended only to show the dependability of the meth- ods, in the sense of giving substantially the same values in succes-

TABLE III.

Commercial Egg Albumin.

Determination No.

Average.. . . . . . . . . . . . .

Tyrosine. Tryptophane.

per cent per cent

3.92 1.37 4.05 1.28 4.06 1.23

3.98 1.35 3.91 1.26 3.94 1.31

3.98 1.30

TABLE IV,

Crystallized Egg Albumin (Preparation II, 1926).

Determination No. Tyrosine. Tryptophane.

per cent per cent 1 4.03 1.11

2 4.02 1.19 3 4.04 1.17

Average.. . . . . . . . . . . . . 4.03 1.16

sive repetitions. The figures obtained are given in Table III. The figures for crystallized albumin are given in Table IV.

Another sample of egg albumin crystallized three times gave the figures shown in Table V.

Serum AZ&min.-Two samples of this crystallized protein were analyzed, Tables VI and VII,

In the analyses recorded in Table VIII is represented one pair of determinations (the second) where the process failed to give per- fect separation between the tyrosine and the tryptophane. The

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tyrosine value, 3.74 per cent, is too high and the corresponding tryptophane value, 1.21 per cent, is too low. Those figures, perhaps, should not go into the record, but it has seemed worth

TABLE V.

Crystallized Egg Albumin (Sample; Ferry-Wyman, 1988).

Determination No. Tyrosine.

per cent

1 3.90

2 3.88 3 3.94

4 3.99

Tryptophane.

per cent

1.09 1.24

1.23 1.24

Average.. . . . . . . . 3.93 I

1.20

TABLE VI.

Twice Crystallized Serum Albumin (Sample; Wyman, November, 1988).

Determination No. I Tyrosine.

per cent

4.51 4.66 4.66

4.81

Tryptophane.

per cent

0.52 0.54 0.52

0.54

Average.. . . . . . . . . . 4.66 I 0.53

TABLE VII.

Three Times Crystallized Serum Albumin (Sample; Ferry, March, 1927).

Determination No.

1

2 3

Tyrosine.

per cent 4.75 4.62

4.66

I Tryptophane.

per cent

0.51 0.51

0.54

Average.. . . . . . . . . 4.67 I

0.52

while to show how such errors can be discovered and eliminated. Out of scores of analyses, this error is the only one found where we failed to obtain seemingly complete separation of tryptophane from the tyrosine.

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100 Tyrosine and Tryptophane of Protein

Musck Globulin.-This protein probably has not been analyzed before for tryptophane and tyrosine. It may be worth recording that the need for a micro method came to one of us in connection with a request from Dr. Cohn for an analysis of a sample of muscle

TABLE VIII.

Cottonseed Globulin from Dr. Osborne’s Laboratory.

Determination No. Tyrosine. Tryptophane. -

per cent per cent 3.68 1.44

3.74 1.21 3.55 1.39

3.59 1.33

TABLE IX.

Muscle Globulin, Not Entirely Pure (Sample; E&all).

Determination No. I Tyrosine. I Tryptophane.

1 3.93 1.55

2 3.91 1.57

TABLE X.

Highly Purified Muscle Globulin (Sample; Edsall).

Determination No. Tyrosine.

per cent

Average.. . . . . . . . . . . 3.92 I 0.98

3.92 0.98 4.00 1.03

3.96 0.97

3.82 0.99

Tryptophane.

per cent

globulin. The sample submitted in the form of suspension proved quite inadequate for the regular macro method.

The first small sample submitted probably was not very pure. It gave the values recorded in Table IX, while the purified sample gave the figures recorded in Table X.

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Gliadin.-We have no less than twenty separate pairs of tyro- sine and tryptophane determinations on six different preparations

TABLE XI.

Gliadin. Six Di.fferent Samples.

Tyrosine. Tryptophane.

per cent Osborne Sample 1.. ............................ 3.39

“ “ 2. ............................. 3.29 Dill “ 1. .............................. 3.37

“ “ 2 .............................. 2.97 Cohn “ lc ............................ 3.21

I‘ “ 2b ............................ 3.27

per cent 0.84 0.83 0.83 0.74

0.73 0.78

TABLE XII.

Edeslin. sample; Osborne (6.9 Per Cent Moisture).

Determination No. Tyrosine. Tryptophane.

per cent per cent 1 4.13 1.34 2 4.01 1.32 3 3.96 1.44 4 3.91 1.38

Correctedaverage.. . . 4.28 1.46

TABLE XIII.

Hempseed Edestin, Not Crystallized (3.8 Per Cent Moisture). -

Determination No. I

Tyrosine. I

Tryptophane.

per cent per cent

1 4.47 1.44 2 4.43 1.40 3 4.32 1.46 4 4.32 1.44 5 4.43 1.34 6 4.34 1.34

Corrected average.. . 4.54 1.45

The corresponding figures reported by Folin and Ciocalteu are 4.50 and 1.50.

of gliadin. The repetitions gave perfectly uniform results. It, therefore, seems superfluous to record the individual analyses,

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102 Tyrosine and Tryptophane of Protein

and we give only the averages in Table XI. Folin and Ciocalteu obtained an average of 3.1 per cent of tyrosine and 0.84 per cent of tryptophane. We have no explanation to offer for the rather large discrepancy between the tyrosine figures of Folin and Cio- calteu, and those now obtained. Such differences, of course, could occur if one or the other pair of investigators had used a

TABLE XIV.

Hemoglobin. Crystallized Sample.

Determination No. Tyrosine. Tryptophane.

per cent per cent 1 3.08 1.27 2 3.18 1.30 3 3.17 1.28 4 3.20 1.27 5 3.11 1.26

Average.. . . . . . . . . . . . 3.15 1.28

Determination No.

Average.. . . . . . . . . . .

TABLE XV.

Zein.

Tyrosine. Tryptophsne.

per cent per cent 5.80 0.19 5.89 0.20 5.88 0.20 5.94 0.20

5.88 0.20

slightly incorrect standard solution of tyrosine, but we are reason- ably certain that this has not been the case.

The analytical figures for edestin and for hemogIobin are recorded in Tables XII to XIV.

For the tryptophane determination in zein, Table XV, half quantities of the reagents were taken and the final volume was 50 cc., just as in the original method.

BIBLIOGRAPHY.

1. Folin, O., andCiocalteu, V., J. Biol. Chem., 73,627 (1927). 2. Hanke, M. T., J. Biol. Chem., 79,587 (1928).

3. Hanke, M. T., J. Biol. Chem., 79,605 (1928).

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Otto Folin and A. D. MarenziGRAM OF PROTEIN

DETERMINATIONS IN ONE-TENTH TYROSINE AND TRYPTOPHANE

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