THE QUANTITATIVE ESTIMATION OF TYROSINE AND HISTIDINE IN PROTEIN.

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1 THE QUANTITATIVE ESTIMATION OF TYROSINE AND HISTIDINE IN PROTEIN. A METHOD FOR ESTIMATING TYRAMINE IN PROTEIN- CONTAINING MIXTURES. BY MILTON T. HANKE. (From the Otho S. A. Sprague Memorial Institute and the Department of Pathology, University of Chicago, Chicago.) (Received for publication, September 12, 1925.) INTRODUCTION. In Dr. Koessler and I published a colorimetric method for the quantitative estimation and separation of phenols, including tyrosine and tyramine. The method is based upon the color that is produced when phenols are allowed to react in alkaline (Na 2 COs) solution with diazotized sulfanilic acid. The phenols tried, other than tyrosine and tyramine, give colors varying from yellow to red and the intensity of these colors is directly proportional to the amount of phenol introduced. This is not the case with tyrosine and tyramine. 2 An evanescent pink color appears which rapidly fades and becomes yellow. If, at this time, the liquid is treated with a solution of sodium hydroxide and then with a small amount of hydroxylamine hydrochloride, a brilliant violet color develops which reaches its maximum intensity within a few seconds and remains unchanged for 15 or more minutes. The amount of color so produced is directly proportional to the concentration of tyrosine or tyramine present. A search for interfering substances revealed the fact that tyrosine and tyramine are the only common phenols that give this reaction and that beside these two phenols, the reaction is obtained only with cer- I Hanke, M. T., and Koessler, K. K., J. Biol. Chem., 1922, 1, 235, See, however, Fiirth, O., Biochem. Z., 1924, cxlvi, 259. Using larger concentrations of tyrosine, the color production seems to be fairly proportional to the concentration of tyrosine. 475

2 476 Estimation of Tyrosine tain volatile or easily decomposable enolic compounds such as acetone, acetoacetic acid or ester, and acetaldehyde. The method is not applicable to the estimation of tyrosine or tyramine in protein and protein-containing mixtures because when other amino acids are present in sufficient quantity, they give a yellow color with the test reagent which masks the color produced by tyrosine or tyramine. This reaction, which is so characteristic for tyrosine and tyramine, would serve as an ideal method of estimation in mixtures if the compounds could be at least partially freed from impurities before subjecting them to the colorimetric procedure. Phenols have long been known to give insoluble compounds when they are heated, in aqueous solution, with mercuric acetate, the products being mercurized phenols with the mercury attached to a carbon atom of the benzene ring. A search of the literature revealed the fact that what one might presume to be such a compound, had been prepared 3 to which the formula C 9 H903NHg had been assigned. This compound was said to be insoluble in water and was used, therapeutically, after it had been dissolved in dilute' alkali. Attempts were made, therefore, to ascertain under what conditions an insoluble tyrosine mercury compound could be prepared and this has been accomplished. The tyrosine-containing mixture must first be freed from histidine, because histidine is precipitated, to some extent, by mercury under the conditions to be described. This can be most accurately accomplished by precipitating with silver sulfate and barium hydroxide. The histidine-free filtrate is freed from silver and barium and evaporated to dryness. The residue is suspended in 75 cc. of water, treated with 1 cc. of glacial acetic acid and 3.5 gm. of mercuric acetate, and boiled for 10 minutes under a reflux condenser or heated for 1 hour on the steam bath. If a quantity of pure tyrosine less than 100 mg. is present, a clear solution is obtained that remains clear when cooled. With protein hydrolysates a slight precipitate is always obtained which contains some, but not all, of the tyrosine. The cooled liquid, or mixture, is treated with 7.5 gm. of sodium chloride which precipitates the tyrosine as a fairly heavy amorphous white solid. The mixture is separated by centrifugalization and the insoluble resis British patent No. 11,302 and U. S. patent Nos. 1,167,622 and 1,180,694.

3 M. T. Hanke 477 due washed once with a small amount of a 10 per cent solution of sodium chloride. The clear supernatant liquids are discarded. They contain not more than 0.1 mg. of tyrosine. The mercury compound is dissolved in hot 20 per cent HC1, the solution diluted with water and freed from mercury by saturation with hydrogen sulfide. The mixture is filtered. The clear and usually colorless filtrate is freed from HC1 by evaporation on the water bath. The residue is nearly pure tyrosine chloride. The amount of tyrosine present can be most conveniently estimated by the colorimetric process of Hanke and Koessler. 1 The process thus briefly described fulfills all of the requirements for an exact determination. The tyrosine is first removed from everything that can possibly interfere with its colorimetric estimation and it is then estimated by an accurate colorimetric process. Before proceeding to a detailed description of the method and the results obtained with this method, something should be said about the tyrosine mercury compound. When properly isolated it has the molecular formula CgHgHg 2 Cl203N for which three structural formulae can be written; namely, H HgCI i H O O O C1Hg- -HgCl H nh H H H H HUH H ih H1C H2C H 2 C HCNII 2 HCNH 2 ii l HU - NXHgCi NK COOH COOHgCI COOH I. II. III. The compound is formed rapidly only when the reaction liquid is heated. When washed exhaustively with water it loses a part or all of its chlorine. It gives a clear solution when boiled for a few minutes with N' H 2 SO 4, which remains clear when the liquid is cooled. Hydrogen sulfide precipitates themercury quantitatively from this liquid. When the solid is suspended in water and the mixture saturated with hydrogen sulfide, the mercury is removed quantitatively.

4 478 Estimation of Tyrosine These facts prove conclusively that the mercury atoms cannot be attached to carbon, which eliminates formula I. Formula II looks improbable. The fact that the compound is formed readily only when the reaction mixture is heated does not suggest salt formation. Such a compound would not be expected to exist in an acid solution because of the depressing action of the H ion on the reactivity of the phenolic hydrogen ion. The addition of NaCl, which converts the mercury into NaHgC3, reduces the concentration of the mercury ion almost to nil, which makes salt formation almost impossible. Formula III represents the configuration of the compound that is precipitated under the conditions of the experiment. It is a derivative of Millon's base. H H HgOH N I HgOH H It might be called p-hydroxyphenyl, a-dichloromercuramino propionic acid or tyrosino-dimercuric chloride; but I shall refer to it subsequently as tyrosino-mercuric chloride and to its derivatives as tyrosino-mercury compounds. Since the a amino group is common to all amino acids, one would expect that entirely similar compounds are formed with the other amino acids. They certainly do not precipitate to any marked extent, hence they may be soluble. Leucine-O.1 gm.-when treated as above precipitates to only a very slight extent. EXPERIMENTAL. I. The Precipitation of Tyrosine and Tyramine Dimercuric Chloride. A. How Long Must Tyrosine Be Heated with Mercuric Acetate to Obtain a Quantitative Yield of Tyrosine Mercuric Chloride? Seven reaction mixtures were prepared containing: 0.05 gm. tyrosine cc. water " glacial acetic acid. 3.5 gm. mercuric acetate.

5 M, T. Hanke 479 Five of the flasks were attached to reflux condensers and the contents boiled for 5 minutes, 30 minutes, 1 hour, 2 hours, and 3 hours respectively. One flask was placed on the steam bath and heated for 1 hour. One flask was not heated. The treatment in this one case is separately described. The liquids that had been boiled for 2 and 3 hours, respectively, were slightly colored and contained a small amount of metallic mercury. The others were clear and nearly colorless. The reaction liquids were cooled and treated with 7.5 gm. of sodium chloride which produced an immediate, flocculent but heavy, white precipitate. The mixtures were placed in the ice chest overnight, transferred to centrifuge tubes with 20 cc. of a 10 per cent solution of sodium chloride, and centrifuged. The solid separates readily. The clear supernatant liquid was removed by decantation and the solid washed once with 10 cc. of a 10 per cent solution of sodium chloride. The washed solid was transferred back to the boiling flask with 25 cc. of 20 per cent hydrochloric acid and heated on the water bath. A clear solution was obtained after heating for 30 minutes. Water-50 cc.-was then added. The liquid was saturated with hydrogen sulfide, the mixture heated on the water bath for 30 minutes, and filtered through a small folded filter. The filtrate and washings were collected in a glass dish and evaporated until all of the liquid had been removed. The pale brown residue was dissolved in water, diluted to 500 cc., and its tyrosine content estimated colorimetrically by the method of Hanke and Koessler.' Exactly 0.05 gin. of tyrosine was recovered in every case which is 100 per cent of the amount originally introduced. The Unheated Reaction Liquid.-In this case the tyrosine was dissolved in the water and acetic acid by boiling. The solution remained clear when it was cooled. It was then treated with 3.5 gm. of mercuric acetate and allowed to stand at room temperature for 30 minutes. The subsequent treatment with sodium chloride gave an opalescent solution but no precipitate. I feel justified in concluding that the reaction does not occur, to any appreciable extent, in 30 minutes at room temperature. Heat seems to be essential for the reaction; but the time of heating is, within certain limits, a matter of minor significance. Protracted boiling seems to produce a slight decomposition which is evidenced by the fact

6 480 Estimation of Tyrosine that a small amount of metallic mercury separates out. In all of our subsequent experiments, therefore, the reaction liquid has either been boiled for 10 minutes or heated on the water bath for 1 hour. B. How Small an Amount of Tyrosine or Tyramine Mercuric Chloride Can Be Quantitatively Precipitated? Seven flasks were prepared as in A excepting that they contained, respectively, 10, 5, 2, and 1 mg. of tyrosine, and 10, 2, and 1 mg. of tyramine chloride. The liquids were boiled for 10 minutes and subjected to chemical treatment as given in A. The results are given in Table I. TABLE 1. Amount introduced. Amount recovered. mg Tyrosine Tyramine mg. The loss of tyrosine or tyramine by this method is 0.1 to 0.2 mg. under the conditions of the experiment. This probably represents the limit of solubility of the compound. That perfect recoveries were obtained with the 5 and 10 mg. samples is not surprising because the limit of accuracy of the colorimetric methods is about 1.5 to 2 per cent. Since, in the average tyrosine determination, quantities considerably in excess of 5 mg. are determined, this solubility correction can be ignored entirely. C. Is Tyrosine Mercuric Chloride Soluble in a Solution That Contains Amino Acids? Are Insoluble Mercury Derivatives Obtained with Other Amino Acids? Is Any Tyrosine Precipitated before the Sodium Chloride Is Added? A mixture was prepared containing 0.1 gm. of each of the following amino acids: glycine, alanine, a-amino-n-valerianic acid,

7 M. T. Hanke 481 valine, leucine, iso-leucine, aspartic acid, glutamic acid, phenylalanine, and tyrosine. The mixture was treated with 75 cc. of water and 1 cc. of glacial acetic acid and boiled under a reflux condenser until a clear solution had been obtained. Mercuric acetate-3.5 gm.-was then added and the boiling continued for 10 minutes. A precipitate formed during the first 30 seconds which increased in amount as the boiling continued; it was never large. The mixture was cooled, filtered through a very small filter, and the precipitate washed with 25 cc. of water. The Precipitate.-This was suspended, paper and all, in 25 cc. of 20 per cent HCl and heated for 1 hour on the water bath. It was then diluted, saturated with hydrogen sulfide, etc., as described in A. The crystalline solid finally obtained was dissolved in water and diluted to 500 cc. It contained colorimetricc determination) mg. of tyrosine. The Filtrate.-This was treated with 10 gm. of sodium chloride, etc., as described in A. The solid finally obtained was dissolved in water and diluted to 1000 cc. It contained 56.2 mg. of tyrosine. The total recovery of tyrosine was, therefore, mg. which is per cent of the amount originally introduced. A somewhat similar experiment was carried out on hydrolyzed gelatin with and without the addition of tyrosine. Of the added tyrosine, 99 per cent was recovered over and above that present in the gelatin. Conclusions.-Tyrosine can be quantitatively precipitated from a mixture of amino acids as tyrosine mercury derivative. Other amino acids are precipitated to only a very small extent, possibly to some extent as components of the tyrosine mercury complex and to some extent as individual mercury compounds. The amount of the other amino acids that are precipitated is too small to interfere in any way with the colorimetric estimation of tyrosine. D. To What Extent Do Chlorides and Sulfates Interfere with the Formation of Tyrosine Mercuric Chloride? The process to be described directly for estimating histidine and tyrosine in protein involves an adjustment to remove barium with sulfuric acid and silver with hydrochloric acid. The work is facilitated if the liquid can be so adjusted that it contains a slight excess of sulfate and chloride ions. It was necessary, therefore,

8 482 Estimation of Tyrosine to prove that these two ions do not interfere with the formation of the mercury compound. Four solutions were prepared as in A containing 0.05 gm. of tyrosine. These were then treated with 0.45 and 0.9 gm. of sodium chloride and 0.71 and 1.42 gm. of sodium sulfate, respectively. The liquids were boiled for 10 minutes and otherwise treated and analyzed as in A. 100 per cent of the tyrosine originally introduced was recovered in every case. Sodium chloride and sodium sulfate do not interfere with the formation of the tyrosine mercuric derivative within the limits prescribed by this experiment. II. Composition of the Tyrosine Mercury Compound. Preparation of the Compound. Tyrosine.-1.0 gm. was mixed with 750 cc. of water, 10 cc. of glacial acetic acid, and 35 gm. of mercuric acetate. The mixture was boiled for 5 minutes under a reflux condenser. The liquid was cooled in an ice bath, filtered from a small insoluble residue, and the clear filtrate treated with 75 gm. of sodium chloride. The mixture was centrifuged. The insoluble residue was washed ten times, by centrifugalization, with a 10 per cent solution of sodium chloride using about 50 cc. of the salt solution each time. The final washings did not contain mercury. The solid was then washed twice, rapidly, with distilled water to remove the sodium chloride. The perfectly white solid so obtained was dried in vacuo for 4 days over phosphorous pentoxide. It was then removed from the centrifuge tube, pulverized, transferred to a weighing bottle, and dried to constant weight in vacuo. Nitrogen.-Of the compound, gm. was analyzed for nitrogen by the Kjeldahl process. The ammonia so liberated neutralized 2.96 cc. of 0.2 N HCI. This is equivalent to gm. of N per gm. of compound = 2.12 per cent N. Mercury.-Of the compound, gm. were mixed with 175 cc. of normal sulfuric acid and boiled under a reflux condenser until the solid had dissolved completely. The hot liquid was saturated with hydrogen sulfide, the mixture heated for 30 minutes on the water bath, and filtered through a small, weighed filter paper. The thoroughly washed solid, dried to constant weight at 110 C., weighed gm. This is equivalent to gm. of mercury per gm. of compound = 61.2 per cent Hg.

9 M. T. Hanke 483 Chlorine.-The filtrate from the HgS was concentrated on the water bath to a volume of about 150 cc. This was then diluted to exactly 200 cc. Of this solution two 50 cc. portions were titrated for chlorine with 0.1 N silver nitrate using the process of Volhard. 4 The entire 200 cc. of solution contained gm. of chloride ion which is equivalent to gm. of chlorine per gm. of compound = per cent Cl. Sodium.-Of the above solution, 50 cc. were digested with concentrated sulfuric acid to destroy the tyrosine. The liquid was evaporated in a platinum crucible. A residue was not obtained. Sodium was absent. This was checked by repeatedly extracting 0.8 gm. of the dry mercury compound with water, which should dissolve any NaCl that might be present. The extracts left no residue on evaporation. These figures agree perfectly with the percentage composition of three possible formulae. Hg H H O O O ClHg HgCl H H HUH kh H- H H H H 2 C H 2 C H 2 C I I I / g-c1 HCNH 2 HCNH 2 HC N - C I I I \Hg - C1 COOH COOHgCI COOH I. II. III. Theoretical values. Determined values. per cent per cent Nitrogen Mercury Chlorine Location of the Mercury.-This has already been adequately discussed in the introduction. Formula III represents the configuration of the molecule. 4 Volhard, J., Ann. Chem., 1878, exc, 1.

10 484 Estimation of Tyrosine III. The Quantitative Estimation of Histidine and Tyrosine in Protein. The Method in Detail. Of the vacuum-dried protein, 1 to 3 gm. are mixed with 100 ccof water and 15 cc. of 95 per cent sulfuric acid in a 300 cc. flask- The flask is attached to a reflux condenser by means of a ground glass joint. The mixture is boiled for 24 hours, cooled, transferred to a 3000 cc. flask, diluted to 1500 cc., heated on the water bath, and treated with a hot solution of 89 gm. of barium hydroxide in 500 cc. of water. The mixture is then adjusted with dilute solutions of barium hydroxide and sulfuric acid until it contains a slight excess of the sulfate ion. Digest for several hours and filter, while hot, on a Biichner funnel. The barium sulfate precipitate is thoroughly washed with hot water. The filtrate is transferred to a glass dish and concentrated on the steam bath. Silver Precipitation. Separation of Histidine and Tyrosine. The semisolid residue is transferred to a 1000 cc. flask with 50 cc. of water. Silver sulfate-600 cc. of an 0.8 per cent solutionis added and the liquid thoroughly mixed. It is then treated with a warm solution of 30 gm. of barium hydroxide in 100 cc. of water. The brown mixture is transferred, immediately, to an ice bath where it is allowed to settle for half an hour. Under these conditions the mixture remains brown; there is no noticeable deposition of metallic silver. The cold mixture is centrifuged. This effects a sharp separation in a cold medium in a minimum space of time. The clear, colorless, supernatant liquid is decanted into a 2000 cc. flask and immediately acidified with 20 per cent sulfuric acid. 5 The residue is washed once, by centrifugalization, with 50 cc. of a cold, saturated solution of barium hydroxide. The washings are added to the main bulk of the silver filtrate. This silver filtrate contains all of the tyrosine and is discussed later under the caption "Silver filtrate. Estimation of tyrosine." 6 This liquid still contains a small amount of silver ion which invariably leads to the formation of a silver mirror and a loss of tyrosine as the solution warms up. Oxidation does not occur in the acidified liquid.

11 M. T. Hanke 485 The Silver Precipitate. Estimation of Histidine. The silver precipitate is transferred to a 500 cc. flask with 60 cc. of N sulfuric acid and 150 cc. of water. The mixture so obtained is treated with 3 cc. of 37 per cent HCI, agitated until it becomes homogeneous, and finally warmed on the steam bath, for 15 to 30 minutes, to complete the conversion of the silver compounds into silver chloride. When the conversion is complete, the silver chloride becomes unmistakably homogeneous and it is usually light colored. Filter (folded filter) into a 500 or 1000 cc. volumetric flask, wash the precipitate thoroughly with water until the washings are free from chlorides, neutralize the filtrate with 20 per cent NaOH, and dilute to volume. Histidine is estimated, colorimetrically, in this liquid using the method of Koessler and Hanke.? I have never encountered any difficulty in this fraction from interfering substances. The colors come up brilliantly and the values are practically identical with those previously obtained by the phosphotungstate method.' The phosphotungstate method involves adding a correction for the solubility of histidine phosphotungstate. Histidine silver seems to be quite insoluble; still the values are identical with those obtained with the phosphotungstate method before correcting for solubility. The divergence is never over 0.2 per cent. It is well to remember that silver, and phosphotungstic acid, precipitate purines as well as histidine and that certain of the purines give a diazo reaction. Purines would, however, not be expected to occur in proteins other than nucleoproteins. Silver Filtrate. Estimation of Tyrosine. As has already been stated, the silver filtrate must be acidified with sulfuric acid before it gets warm to avoid oxidation of tyrosine by the silver which is invariably present in small amounts. A sufficient excess of sulfuric acid is added to cause the barium sulfate to settle readily. Normal hydrochloric acid is then added, drop by drop, until a precipitate of silver chloride is no longer obtained. The liquid should now give a faint test for chloride ion. It is heated on the steam bath and adjusted with barium hydroxide and sulfuric acid until a slight excess of sulfate ion is present. 6 Koessler, K. K., and Hanke, M. T., J. Biol. Chem., 1919, xxxix, 497, 521, Hanke, M. T., and Koessler, K. K., J. Biol. Chem., 1920, xliii, 527.

12 486 Estimation of Tyrosine Digest for several hours, filter, transfer filtrate and washings to a glass dish, and concentrate on the steam bath. Precipitation of Tyrosine as Tyrosine Mercury Compound.- The semisolid residue, which usually contains well defined crystals of tyrosine, is transferred to a 300 cc. flask with 75 cc. of water. Glacial acetic acid-1 cc.-and mercuric acetate-3.5 gm.-are added and the mixture either boiled for 10 minutes under a reflux condenser or heated for 1 hour on the steam bath without a reflux. A small amount of precipitate is always obtained. The mixture is cooled, treated with 7.5 gm. of sodium chloride, and placed in the ice chest for 2 hours. Transfer to a centrifuge tube with 20 cc. of a 10 per cent solution of sodium chloride. Centrifuge for 5 minutes at high speed. Decant and discard the clear supernatant liquid. The solid is washed once with 25 cc. of a 10 per cent solution of sodium chloride. The washed solid is transferred back quantitatively, to the boiling flask with 25 to 50 cc. of hot 20 per cent HC1. The mixture is heated on the steam bath for 30 minutes. Most, and frequently all, of the solid passes into solution. The liquid is treated with 50 cc. of water, saturated with hydrogen sulfide, heated on the water bath for 30 minutes, and filtered through a small folded filter. Filtrate and washings are collected in a glass dish and evaporated on the steam bath until all of the liquid has been removed. The pale brown crystalline residue is dissolved in water, diluted to 500 cc., and its tyrosine content estimated, colorimetrically.' The histidine and tyrosine values obtained by this method for a number of proteins, are given in the following article. Note on the Estimation of Tyramine in Protein-Containing Mixtures. The mercury compound of tyramine is just as insoluble as that of tyrosine and it is formed under identical conditions. It probably has a chemical constitution analogous to that of the tyrosine compound. This compound can, therefore, be used as a means of separating tyramine from amino acids and other interfering substances that might occur in protein-containing mixtures. The method is briefly as follows: The mixture is hydrolyzed' with 20 per cent HCl and freed from water and acid by distillation in vacuo. Ammonia and humin 8 This hydrolysis is always a laborious process because it dissolves so many things that could be eliminated by filtration at the outset. It seems,

13 M. T. Hanke 487 are removed with barium hydroxide. Barium is removed with sulfuric acid. The concentrated aqueous solution of the residue is then treated with sodium hydroxide (final concentration of the alkali should be 30 per cent) and centrifuged. The clear supernatant liquid is exhaustively extracted with amyl alcohol. Amines are removed from the amyl alcohol with sulfuric acid. This solution, which contains all of the histamine and tyramine and small amounts of the corresponding amino acids, is freed from sulfuric acid, concentrated, realkalinized, and reextracted with amyl alcohol. This time the amino acids remain, quantitatively, in the aqueous layer. The amines are in the amyl alcohol, from which they are extracted with normal sulfuric acid. The liquid is neutralized with barium hydroxide, the filtrate from the barium sulfate concentrated and subjected to a silver precipitation. This precipitates the histamine as silver salt and leaves tyramine in solution. Histamine is then estimated as previously described. 9 Tyramine is precipitated with mercuric acetate and finally estimated colorimetrically. SUMMARY. When tyrosine is boiled with mercuric acetate under suitable conditions, and the resulting clear solution treated with sodium chloride, a white solid is obtained that has the formula: H 0 H2C /HgCl HC - N HC \HgCl COOH however, to be essential because aqueous or alcoholic extracts of feces or tissue, in which one would expect the amines to dissolve, do not always contain the amines. The latter are largely attached to the insoluble matter either by adsorption or in a more stable form of combination. To obtain a reasonably quantitative yield of histamine or tyramine it is, at least for the present, necessary to decompose the insoluble matter as far as possible by hydrolysis. 9 Hanke, M. T., and Koessler, K. K., J. Biol. Chem., 1920, xliii, 543.

14 488 Estimation of Tyrosine This compound is very insoluble in water and in a mixture of amino acids. It can serve, therefore, as a means of separating tyrosine more or less completely from the other amino acids that are formed by the acid hydrolysis of a protein. The solid can be dissolved in dilute sulfuric acid or in 20 per cent HC1. Mercury can be quantitatively removed from the compound by passing hydrogen sulfide into its suspension in water or its solution in acids. The resulting solution contains all but a mere trace of the tyrosine that was originally present in the substance under investigation and not much of anything else. The exact concentration of tyrosine can then be most accurately estimated by the process of Hanke and Koessler.' Protein hydrolysates contain amino acids that are, to a small extent, precipitated with the tyrosine. With the exception of histidine, these amino acids in such small quantities do not interfere with the colorimetric estimation of tyrosine. The complete analytical process is, briefly, as follows: The protein is hydrolyzed with sulfuric acid. Histidine is precipitated as silver complex and its amount estimated colorimetrically. The filtrate from the histidine silver is boiled with mercuric acetate and treated with sodium chloride which precipitates tyrosinomercuric chloride. The precipitate is dissolved in 20 per cent HC1, freed from mercury with hydrogen sulfide, and the tyrosine estimated in the filtrate from the mercuric sulfide. A method for estimating tyramine in protein-containing matter is briefly outlined.

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