Vol XIV Now 6
`
`1930,'
`
`-EDITED, FOR--'TH-E BIOCHEMICAL, SOCIETY.
`BY.
`CHARES RO13ERT'HARINOTON
`-AND
`-ARTHU -H-ARDEN
`
`2
`
`ED-,,ITORA COMITTEE
`PaorG.]~AGE
`SIRJ F.G
`OKINS~
`PRoF_ V HBACKMA
`Si EBE
`MR J. A. GARDNER
`Pe.W RAMSDE
`SIR E.J RUSLL
`
`CAMBRIDGE UNIVERSITY PRESS
`J~~~ U~LNnoi:Fete Lane,EC4
`
`ower Sre,Londo'n,
`I(.~R*xs& >C Lt. 16
`niversit oChcgo rs
`CHJCAO~> Th~
`:(Aginu fo-r thei United States)
`
`.
`
`Toyo Mruzen Company,`Ltd.
`
`-
`
`1930=-,AU
`
`..-
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`--I
`
`Elysium Health Exhibit 1012
`Page 1 of 26
`
`

`

`Vol XIV Now 6
`
`1930,'
`
`-EDITED, FOR--'TH-E BIOCHEMICAL, SOCIETY.
`BY.
`CHARES RO13ERT'HARINOTON
`-AND
`-ARTHU -H-ARDEN
`
`2
`
`ED-,,ITORA COMITTEE
`PaorG.]~AGE
`SIRJ F.G
`OKINS~
`PRoF_ V HBACKMA
`Si EBE
`MR J. A. GARDNER
`Pe.W RAMSDE
`SIR E.J RUSLL
`
`CAMBRIDGE UNIVERSITY PRESS
`J~~~ U~LNnoi:Fete Lane,EC4
`
`ower Sre,Londo'n,
`I(.~R*xs& >C Lt. 16
`niversit oChcgo rs
`CHJCAO~> Th~
`:(Aginu fo-r thei United States)
`
`.
`
`Toyo Mruzen Company,`Ltd.
`
`-
`
`1930=-,AU
`
`..-
`
`--I
`
`Elysium Health Exhibit 1012
`Page 2 of 26
`
`

`

`d ypublished
`The Biochemical ouand is
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`Elysium Health Exhibit 1012
`Page 3 of 26
`
`

`

`CLXXVII. THE COLORIMETRIC DETERMINATION
`OF THE TYROSINE AND TRYPTOPHAN CONTENT
`OF VARIOUS CRUDE PROTEIN CONCENTRATES.
`
`By WILLIAM DOUGLAS MCFARLANE
`AND HUGH LEHMAN FULMER.
`From the Biochemical Laboratory, Ontario Agricultural
`College, Guelph, Canada.
`(Received July 29th, 1930.)
`
`IN the course of investigations on poultry nutrition, it was desired to know the
`tyrosine and tryptophan content of the protein of buttermilk powder, fish meal,
`cod-liver meal, and two abattoir by-products, meat meal and tankage. A rapid
`and reliable method for the estimation of tyrosine and tryptophan in food-
`stuffs would be an extremely valuable asset, not only in regard to our par-
`ticular problem, but in a multitude of nutritional investigations.
`At the present time it is doubtful whether any of the known methods for
`determining the tyrosine, and particularly the tryptophan, content of purified
`proteins are absolutely reliable. Onslow [1924], summarising the values given
`for the tryptophan content of caseinogen by different investigators, has pointed
`out the great discrepancies in the results obtained. Though the colorimetric
`methods have sometimes given low values, and are open to the usual objections
`to colorimetric methods in general, they are the only methods which could be
`satisfactorily applied to the determination of tyrosine and tryptophan in a
`routine way, such as we contemplated.
`It was hoped that after making as thorough a purification of these materials
`as was possible, results, of sufficient accuracy to compare the tyrosine and
`tryptophan content of one material with another, might be obtained by colori-
`metric methods. Fiirth and Lieben [1921] and Ide [1921], using a colorimetric
`method based on Voisenet's reaction [1905], have determined the tryptophan
`content of many of the common foodstuffs. The unreliability of the Voisenet
`formaldehyde reaction has been shown by Hunter and Borsook [1923], who
`state that glycyltryptophan gives a far deeper and redder colour than pure
`tryptophan. This reaction is thus affected both as to hue and intensity, not
`only by the presence of tryptophan, but by the mode of combination of
`tryptophan in the protein molecule. Kretz [1922] in endeavouring to use this
`method for localising tryptophan in plant cells has concluded that it did not
`give accurate results because of the susceptibility of the reaction to many
`destructive influences. The colorimetric method of Folin and Ciocalteu [1927]
`
`Elysium Health Exhibit 1012
`Page 4 of 26
`
`

`

`1602
`
`W. D. McFARLANE AND H. L. FULMER
`
`for determining tyrosine and tryptophan commended itself. The two deter-
`minations are combined into one procedure, which is reasonably rapid, and
`the accuracy has been subjected to considerable study in so far as its applica-
`tion to purified protein is concerned. Whether accurate determinations of the
`tyrosine and tryptophan content of these crude protein materials could be
`made by this method, or by other methods discussed in the text, remained to
`be determined.
`
`EXPERIMENTAL.
`Representative samples of these protein supplements were prepared for
`analysis as follows. They were ground in a Wiley mill to pass through a 100
`mesh sieve and then dried in vacuo at 1000 and 5 mm. for 24 hours. The dried
`samples were then extracted with sodium-dried ether in a large Soxhlet ex-
`tractor until fat-free, which usually took about 48 hours. The residues were
`placed in 250 cc. centrifuge bottles and extracted with two volumes of absolute
`alcohol by shaking for 12 hours. After centrifuging, the alcohol was decanted
`and the extraction repeated three times as before. The residues were again dried
`in the vacuum oven and finally stored in tightly stoppered bottles. All sub-
`sequent weighing of samples for analysis was made by weighing the stoppered
`tube before and after the transfer. Total nitrogen was determined (Kjeldahl)
`and all subsequent determinations are expressed as % of the total crude
`protein (N x 6.25). The tyrosine and tryptophan determinations were first'
`made by Folin and Ciocalteu's method. A sample of purified caseinogen on
`repeated analysis was consistently found to contain 6-62 % tyrosine and 1-37 %
`tryptophan. The values obtained by Folin and Ciocalteu for a highly purified
`caseinogen were 6-55 % tyrosine and 1-40 % tryptophan.
`In each case a weighed quantity of the protein material representing the
`equivalent of 1 g. of crude protein was used in duplicate determinations. The
`alkali digests, excepting the caseinogen digest, were highly coloured and in the
`subsequent determinations of tyrosine it was found to be impossible to make
`accurate colour comparisons against the standard solution in the colorimeter.
`It was then decided to decolorise the hydrolysates by shaking with 1 g.
`of kaolin for 12 hours and centrifuging. By similar treatment of standard
`solutions of tyrosine and tryptophan it was determined that no loss of these
`amino-acids by absorption took place. While norite and animal charcoal made
`much better decolorising agents, a very considerable loss, particularly of
`tyrosine, was found to take place. The results of the determinations on the
`decolorised hydrolysates, expressed as % of the total crude protein (N x 6.25),
`were as follows:
`
`Meat meal
`Tankage
`Fish meal
`Cod-liver meal
`Buttermilk powder
`
`Tyrosine
`2-93
`3-02
`2-61
`
`5-46
`
`Tryptophan
`1-34
`0-67
`1-30
`2-04
`1-80
`
`Elysium Health Exhibit 1012
`Page 5 of 26
`
`

`

`DETERMINATION OF TYROSINE AND TRYPTOPHAN 1603
`
`With the phenol reagent (tryptophan) the colour comparison with the un-
`known and the standard was excellent in every case and duplicate determinations
`gave practically the same results. With Millon's reagent (tyrosine) the colour
`comparison was not perfect, the unknown having a yellowish red colour and
`being slightly cloudy, while the standard was bright red in colour and perfectly
`clear. The readings were made as carefully as possible but leave some doubt as
`to their accuracy. It was found impossible to determine the tyrosine content
`of the cod-liver meal protein by this procedure as, on boiling the tyrosine-con-
`taining solution with HgSO4 and cooling, the solution became very cloudy.
`Careful regulation of the acidity of the solution at this point, varying the
`acidity in repeated determinations, or carefully avoiding undue delay in
`making the colour comparisons, did not prevent this fine grey cloudy precipi-
`tate from forming with the other hydrolysates. Making the solution (after
`heating with HgSO4) up to volume with N H2SO4 instead of water slightly
`improved the readings, especially in the case of the fish meal hydrolysate.
`Hanke [1928], in a criticism of Folin and Ciocalteu's method for determining
`tyrosine, maintains that tyrosine cannot be determined by this method when
`cystine is present. Cystine gives a precipitate and this precipitate contains
`tyrosine. He has proposed a method for determining tyrosine which consists
`in hydrolysing the protein with H2SO4 and then removing the H2SO4 with
`baryta. The tyrosine is precipitated, even from a mixture of amino-acids and
`such other substances as might be present in a protein hydrolysate, by boiling
`in acetic acid solution with mercuric acetate and treating the mixture with
`NaCl. The mercury is removed by H2S and the tyrosine determined in the
`filtrate colorimetrically with Millon's reagent, as in the Folin and Ciocalteu
`procedure. More accurate determinations of tyrosine in these crude protein
`materials might be expected by actual precipitation of the tyrosine in the
`hydrolysate, rather than by a colorimetric determination in the presence of
`all possible interfering substances.
`The tyrosine content of these proteins was next determined by Hanke's
`method with the following results:
`
`r
`
`A
`
`% tyrosine
`Folin and
`Ciocalteu's method
`6-62
`2-93
`3-02
`
`Hanke's
`method
`Caseinogen
`6-59
`2-35
`Meat meal
`2.51
`Tankage
`Cod-liver meal
`3-60
`Fish meal
`2-61
`2-64
`Buttermilk powder
`5-28
`5-46
`In every case perfect colorimeter readings were obtained by this method.
`Excepting in the fish meal proteins, the tyrosine determination by Folin and
`Ciocalteu's method was, in every case, higher than that obtained by Hanke's
`method. Higher values were almost invariably obtained by Hanke, using
`Folin and Ciocalteu's procedure on purified proteins.
`The tyrosine content of meat meal, tankage, fish meal and cod-liver meal is
`
`Elysium Health Exhibit 1012
`Page 6 of 26
`
`

`

`1604
`
`W. D. McFARLANE AND H. L. FULMER
`
`much lower than that of the milk proteins. Ingvaldsen [1929] reports for fish
`meals from different sources and processed at different temperatures a tyrosine
`content of the proteins (N x 6.25) varying from 3-85 to 4-43 % determined by
`Folin and Ciocalteu's method. He does not report encountering any difficulties
`in the application of this method to fish meal hydrolysates. With the possible
`exception of tankage, these protein materials appear to be well supplied with
`tryptophan when compared with the value for caseinogen. The question still
`remains as to whether these figures represent the actual tryptophan content of
`these materials.
`Hanke [1928] states that " a crude protein hydrolysate, either acid or alka-
`line, appears to contain something other than tyrosine or tryptophan that
`reacts with both the phenol reagent and Millon reagent of Folin and Ciocalteu.
`This may be one substance or there may be several; but whatever it is, it does
`not react in an identical manner with both reagents." In criticising the use of
`the phenol reagent for determining tryptophan, Kraus [1925] has pointed out
`that indole and skatole give a blue colour with the phenol reagent so that de-
`composition products of tryptophan would also be included.
`Indole and
`skatole, however, are extremely soluble in ether, and were they present in the
`original materials as the result of putrefaction they would have been removed
`in the preparation of these products for analysis. It is possible, however, that
`in the alkali-digestion of these crude protein materials considerable reduction
`of tryptophan may take place. Indole and skatole, according to Kraus, may
`be removed quantitatively from the hydrolysates by extraction with toluene.
`The original decolorised hydrolysates were next extracted with toluene by
`continuous shaking for 12 hours, allowed to stand for 2 hours in separating
`funnels and then separated from the toluene. That tyrosine and tryptophan are
`not removed from solution by toluene was determined by a similar treatment
`of standard solutions. The tyrosine and tryptophan contents of the toluene-
`extracted hydrolysates were next determined by Folin and Ciocalteu's method
`with the following results:
`In original
`After extraction of
`decolorised hydrolysate
`hydrolysate with toluene
`Tryptophan
`1-37
`1-34
`0-67
`1-30
`2-04
`1-80
`
`Tyrosine
`6-55
`2-31
`2-92
`1-83
`
`5-40
`
`Tyrosine
`6-62
`2-93
`3-02
`2-61
`-
`5-46
`
`Tryptophan
`1-23
`0-90
`0 59
`1-17
`0-77
`1-51
`
`Caseinogen
`Meat meal
`Tankage
`Fish meal
`Cod-liver meal
`Buttermilk powder
`A considerable reduction in the values for the tryptophan and tyrosine
`content in all cases was found. A reduction in the apparent tryptophan content
`of cod-liver meal of 1-27 % was very surprising. If the substances giving the
`reactions of tryptophan (i.e. precipitated in acid solution with HgSO4 and
`giving a blue colour with the phenol reagent) but removable with toluene, are
`indole and skatole, it would seem that they must have been produced during
`the alkali-digestion of the protein material.
`
`Elysium Health Exhibit 1012
`Page 7 of 26
`
`

`

`DETERMINATION OF TYROSINE AND TRYPTOPHAN 1605
`
`It was next decided to determine if tyrosine and tryptophan added to these
`protein materials could be recovered quantitatively in the alkali hydrolysates.
`Weighed quantities of Pfanstiehl C.P. tyrosine and tryptophan were added to
`weighed quantities of the prepared meat meal and fish meal as used in the
`previous determinations. The alkali-digestion and the subsequent determina-
`tion of tyrosine and tryptophan were carried out as before. The results were
`as follows:
`
`Meat meal
`Fish meal
`
`Tyrosine
`A,
`
`Added
`(mg.)
`45 0
`40-1
`
`Recovered
`(mg.)
`40 3
`44 0
`
`Tryptophan
`
`A
`
`Added
`(mg.)
`4-0
`6-4
`
`Recovered
`(mg.)
`3-37
`7 0
`
`It will be observed that in some cases more tyrosine and tryptophan was
`recovered than had been added. This can only be explained by the slight
`improvement in the readings obtained where tyrosine and tryptophan had
`been added. In these readings the colour of the unknown was more nearly
`proportional to the colour developed by the standard solution. The results,
`however, indicate that there was no appreciable destruction of added tyrosine
`or tryptophan during the alkali-digestion of these two crude protein materials.
`Results obtained after extraction of the meat meal and fish meal hydrolysate
`with toluene gave practically the same recovery of added tyrosine and trypto-
`phan as recorded above. Evidently, only negligible amounts of decomposition
`products of tryptophan or tyrosine soluble in toluene were formed as the
`result of alkali-digestion.
`The next endeavour was to determine what the substance (or substances)
`present in the alkali hydrolysates of these materials was, which gave the re-
`actions of tryptophan but which could be extracted by toluene. The toluene
`extracts of all the hydrolysates gave a blue colour of varying intensity with the
`phenol reagent. The blue colour was particularly deep in the case of the toluene
`extract of the cod-liver meal hydrolysate, and least in the toluene extract of
`the caseinogen hydrolysate. Evidently the unknown substance was present in
`the greatest amount in the hydrolysate of cod-liver meal. That the original
`cod-liver meal contained no indole was determined by applying Bergeim's
`[Hawk and Bergeim, 1927] method for the quantitative determination of
`indole in faeces. Negative results were also obtained on the alkali hydrolysate
`of cod-liver meal.
`The toluene extract of these hydrolysates gives Millon's reaction, which
`indicates that this unknown substance may be phenolic. Negative results were
`obtained for free and conjugated phenols by Tisdall's [1920] modification of the
`Folin and Denis method. Uric acid gives a blue colour with the phenol re-
`agent; it is soluble in NaOH but is precipitated by H2SO4. However, sufficient
`remains in solution to give a fairly heavy precipitate with HgSO4. While uric
`acid was found to be insoluble in toluene it may be that some other purine
`base, or some pyrimidine base, set free by decomposition of nucleoprotein is
`
`Elysium Health Exhibit 1012
`Page 8 of 26
`
`

`

`1606
`
`W. D. McFARLANE AND H. L. FULMER
`
`contributing to the blue colour given by tryptophan. The actual purine content
`of the ox-liver as reported by Vogel [1911] is only 0-099 %.
`While the colour given by tyrosine with the phenol reagent is a different
`shade of blue from that given by tryptophan, the colour given by indole is
`identical with that given by tryptophan. The comparative solubilities of indole
`did not indicate that, even if indole were present in the hydrolysates, it would
`increase the tryptophan reading. To confirm this 0-0833 g. indole was added
`to 0-9327 g. caseinogen and the determination of tryptophan carried out in the
`usual manner. A reading representing 2-46 % tryptophan was obtained while
`caseinogen contains 1-37 % tryptophan. The apparent tryptophan content of
`caseinogen was increased by 1-09 % by adding 8&9 % indole. The presence of
`indole in the hydrolysates would, undoubtedly, increase the tryptophan
`reading. The colour given by Millon's reagent, in determining tyrosine in
`presence of indole, was not rose-pink but a brownish yellow, and was somewhat
`typical of the colour given by the crude protein hydrolysates with Millon's
`reagent. Inorganic substances, such as ferrous iron or any sulphite, produce a
`deep blue colour with the phenol reagent.
`It would seem, as Gortner and
`Holm [1920] have pointed out, that any substance which is easily oxidised will
`react with the phenol reagent and produce a blue colour.
`That some substance is present in these alkali hydrolysates which shows
`the reactions of tryptophan, but which evidently is not this amino-acid, has
`been reported by Hanke [1928] and by Kraus [1925], independently, and is con-
`firmed by the above findings. The evidence does not point to this substance as
`being indole as Kraus has concluded. Until this unknown chromogenic sub-
`stance has been identified, the results obtained after extraction of these crude
`protein hydrolysates with toluene would appear to represent the true tyrosine
`and tryptophan content of these protein materials.
`An endeavour was made to determine the tryptophan content of these
`materials by the method of Komm [1926], based upon the reaction of trypto-
`phan with p-dimethylaminobenzaldehyde. A value for caseinogen of 2-39 %
`tryptophan was obtained. While the tryptophan standard gave a uniform blue
`colour, the colour given by caseinogen was more of a purplish tinge. The colour
`comparison was not perfect and as a result the figure for caseinogen is probably
`too high. All the other determinations were a complete failure, owing to the
`deep brown colour produced on adding concentrated H2SO4 which prevented
`any possible colour comparison.
`
`THE TYROSINE AND TRYPTOPHAN CONTENT OF THESE CRUDE PROTEIN.
`MATERIALS AS DETERMINED BY ENZYMIC HYDROLYSIS.
`A weighed quantity of the crude protein material (approximately the
`equivalent of 1 g. crude protein) was digested for 10 hours at 370 in 50 cc.
`0-1 N HCI containing 0-2 % pepsin. The digest was neutralised with 5 cc. N
`NaOH and 5 cc. of a 6 % solution of trypsin in 041 N NaOH added. Digestion
`was then continued for 24 hours. Toluene was added as a preservative. The
`
`Elysium Health Exhibit 1012
`Page 9 of 26
`
`

`

`DETERMINATION OF TYROSINE AND TRYPTOPHAN 1607
`
`enzyme activity was destroyed by heating on a water-bath for 5 minutes
`at 75°-80°. The digests were then acidified with H2SO4, diluted to 100 cc. and
`filtered. The addition of a small amount of kaolin greatly facilitated obtaining
`a clear filtrate. Tyrosine and tryptophan determinations were then made on
`an 8 cc. aliquot of the filtrate by Folin and Ciocalteu's procedure. Correction
`was made for the ty-rosine and tryptophan content of the enzyme blank, ob-
`tained by adding known amounts of standard solutions of tyrosine and trypto-
`phan to the blank pepsin-trypsin digest and so determining the tyrosine and
`tryptophan content of the latter by difference. The following results were
`obtained:
`
`Amino-N % Tyrosine % Tryptophan %
`1-54
`8-31
`Caseinogen
`7 59
`Buttermilk powder
`1-25*
`6-36
`7-60
`Meat meal
`3-02
`4 97
`0.95
`Tankage
`0-88
`2-42
`4-06
`Cod-liver meal
`0-77
`2-25
`1-38
`2-92
`5-43
`Fish meal
`* This solution was persistently cloudy and accurate colour comparison was extremely
`difficult. The result is, therefore, very doubtful.
`
`A much higher reading was obtained for caseinogen by this method than
`was obtained by alkali-hydrolysis. The results obtained for meat meal were
`somewhat the same as before, while the tryptophan content of tankage was
`slightly higher. Fish meal would seem to contain considerably more tryptophan
`than meat meal and tankage. It was interesting to find that the tryptophan
`content of cod-liver meal, as determined by this method, was exactly the same
`as that obtained after extracting the alkali hydrolysate with toluene. This may
`have been simply a coincidence, but the fact that the results were very much
`of the same order suggests strongly that the unknown chromogenic substance
`present in the alkali hydrolysate of cod-liver meal in particular was formed
`during alkali-digestion.
`Ragins [1928, 1] has recently reported the perfecting of her method for the
`determination of tryptophan by means of the vanillin-HCl method, so that it
`may be applied to proteins. She claims that this reaction for tryptophan is more
`sensitive than other colour reactions and that it is more specific. The method
`has been applied with evident success to the tryptic hydrolysates of highly
`purified proteins. An endeavour to determine the tryptophan content of these
`crude protein materials by this method and to compare the results with those
`obtained by Folin and Ciocalteu's method on the same hydrolysate was next
`made.
`1 g. of the protein material and 0-25 g. u.s.p. trypsin were weighed into
`100 cc. Erlenmeyer flasks containing 25 cc. 0-4 % Na2CO3. The solutions were
`preserved with toluene and incubated for 5 days at 37°. The enzyme was de-
`stroyed by heating at 75°-80° on the water-bath for 5 minutes. The digest was
`then acidified with 1 cc. 14 N H2SO4, diluted to 100 cc. and filtered. Trypto-
`phan determinations were then made on an 8 cc. aliquot of the digest by Folin
`and Ciocalteu's method and on 3 cc. by the vanillin-HOl method.
`Biocbem. 1930 xxirv
`
`102
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`

`1608
`
`W. D. McFARLANE AND H. L. FULMER
`
`Tryptophan %
`A
`Phenol
`reagent
`1-25
`
`Vanillin-
`HCI
`1-30
`
`0-52*
`
`-*
`1-21*
`0.59*
`1-87
`
`-
`
`1-73
`
`Caseinogen
`
`Meat meal
`
`Tankage
`Cod-liver meal
`Fish meal
`Buttermilk powder
`
`Remarks on colour with
`vanillin-HCl reagent
`Not identical with the standard but good colour
`comparison
`Reddish blue colour, impossible to match but
`qualitative indication that tryptophan was present
`Red colour-no indication of blue colour
`Deep brownish red colour
`Reddish blue colour as with meat meal digest
`Colour identical with that developed with caseinogen
`digest
`* 2 cc. standard tryptophan solution (0-59 mg. tryptophan) were added to 8 cc. of the digest
`and tryptophan determined by difference. In the tankage digest the same amount of tryptophan
`was recovered as was added. The filtrate from the HgSO4 precipitate in every case gave a negative
`test with the Hopkins-Cole reagent.
`
`Tryptophan determinations by the phenol reagent in the tryptic digest of
`meat meal and fish meal were much lower than the previous results obtained
`by peptic-tryptic digestion. The preliminary digestion with pepsin resulted in
`a very considerable increase in the amount of tryptophan liberated. The
`negative result with the tankage digest, and more particularly the higher value
`obtained for cod-liver meal, would seem to be impossible to interpret.
`Except in the case of the milk products, the results with the vanillin-HCl
`reagent were a uniform failure even when the determinations were repeated
`after decolorising the hydrolysates with kaolin. Some other substance present
`was found to develop a red colour with the reagent which made colorimeter
`readings impossible. The result for caseinogen is in fair agreement with that
`obtained by Ragins, i.e. 1-23 % tryptophan.
`Waterman and Jones [1921, 1922] have found their determinations of the
`relative digestibility of proteins in vitro to correspond with protein digestibility
`trials in vivo. It was next decided to repeat these digestions by their method
`and to determine the tryptophan content.
`0-5 g. of the protein material was digested for 14 hours at 370 in 50 cc.
`041 N HCI containing 0-2 % pepsin'. The digest was neutralised with 5 cc. N
`NaOH and 5 cc. of a 6 % solution of trypsin1 in 0-1 N NaOH added. Digestion
`was continued for 21 hours. The enzyme was destroyed by heating on a water-
`bath for 5 minutes at 75°-80°. The digest was then acidified with 1 cc. 14 N
`H2SO4, diluted to 100 cc. and filtered. A 3 cc. aliquot was taken for the amino-
`nitrogen determinations (Van Slyke) and a 5 cc. aliquot for the tryptophan
`determinations by the vanillin-HCl procedure. Tryptophan in concentrations
`of less than 0-2 mg. in a 5 cc. aliquot cannot be precipitated quantitatively
`under the conditions described. Therefore, 2 cc. of the standard tryptophan
`solution (the same amount as the standard) were added to each digest and
`tryptophan determined by difference, allowance being made for the tryptophan
`determined in the blank enzyme digest. The colours developed by the digests
`were much the same as already described; the interfering red colour making
`1 Products of the Digestive Ferments Co., Detroit, Mich.
`
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`
`

`

`DETERMINATION OF TYROSINE AND TRYPTOPHAN 1609
`
`It was found that by
`accurate comparison with the standard impossible.
`placing a blue-green (minus red) filter, which only admitted light of wave
`., between the colorimeter lamp and the Duboscq
`lengths 3800 A.-5800
`colorimeter excellent colorimeter readings could be obtained. The results were
`as follows:
`
`Caseinogen
`Buttermilk powder
`Meat meal
`Tankage
`Fish meal
`
`Amino-N %
`4 99
`3-18
`3-96
`2-86
`4-08
`
`Tryptophan %
`1-72
`2-95
`1-78
`0-92
`1-62
`
`The results obtained were much higher than in all previous determinations
`but, comparatively, they fell into the same order as before. Preliminary diges-
`tion of these crude protein materials with pepsin, followed by trypsin, and
`limitation of the duration of digestion have given higher values for tryptophan
`liberated. Ragins [1928, 2] has found that treating the purified proteins which
`she has studied (caseinogen, edestin and squash seed-globulin) first with pepsin
`and then with trypsin, does not hasten or increase the liberation of tryptophan;
`also that only the enzymes in u.s.p. pancreatin liberate tryptophan from the
`proteins studied. Frankel [1916] found that proteins after having been acted
`upon by pepsin are much more readily digested by trypsin or erepsin. No
`definite interpretation can be placed on the results obtained with these crude
`protein materials until much more comprehensive experiments, determining
`the rate at which tryptophan is liberated with changing time of peptic and
`tryptic digestion, have been made. Excepting caseinogen and buttermilk
`powder, a considerable amount of these crude protein materials remains in
`suspension during enzyme digestion, so that accurate results cannot be
`obtained by removing aliquots as digestion proceeds.
`
`SUMMARY.
`1. The tyrosine and tryptophan content of the proteins of buttermilk
`powder has been found to be much higher than that of the other crude protein
`materials investigated. No very appreciable difference in the tyrosine content
`of fish meal, cod-liver meal, meat meal and tankage was found.
`2. Conflicting results as to the tryptophan content of the proteins of fish
`meal and meat meal, depending upon the method of determination, have been
`found. In general, the tryptophan content of fish meal was found to be higher
`than that of meat meal. The tryptophan content of tankage has invariably
`been found to be much lower than that of fish meal and meat meal.
`3. The limitations in the methods described for determining tryptophan
`leave some doubt as to the actual tryptophan content of any of these materials,
`particularly in the case of the cod-liver meal proteins. The alkali digest of these
`crude protein materials contains some substance, or substances, precipitable
`by HgSO4 and giving a blue colour with the phenol reagent which, unlike
`tryptophan, is soluble in toluene. This unknown chromogenic substance does
`102-2
`
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`

`1610
`
`W. D. McFARLANE AND H. L. FULMER
`
`not appear to be indole as Kraus has concluded. The results obtained after
`extraction of the alkali hydrolysates with toluene would appear to represent
`the true tyrosine and tryptophan content of these protein concentrates.
`
`REFERENCES.
`
`Folin and Ciocalteu (1927). J. Biol. Chem. 73, 627.
`Frankel (1916). J. Biol. Chem. 26, 31.
`Furth and Lieben (1921). Biochem. Z. 122, 58.
`Gortner and Holm (1920). J. Amer. Chem. Soc. 62, 632.
`Hanke (1928). J. Biol. Chem. 79, 587.
`Hawk and Bergeim (1927). Practical physiological chemistry, 9th ed., p. 328.
`Hunter and Borsook (1923). J. Biol. Chem. 57, 507.
`Ide (1921). Tokio Z. Exp. Med. 24, 166.
`Ingvaldsen (1929). Canadian Chem. Metallurgy, 13, 97.
`Komm (1926). Z. phy8iol. Chem. 156, 35.
`Kraus (1925). J. Biol. Chem. 63, 157.
`Kretz (1922). Biochem. Z. 130, 86.
`Onslow (1924). Biochem. J. 18, 63.
`Ragins (1928, 1). J. Biol. Chem. 80, 543.
`-
`(1928, 2). J. Biol. Chem. 80, 551.
`Tisdall (1920). J. Biol. Chem. 44, 409.
`Vogel (1911). Miinch. med. Woch. 58, 2433.
`Voisenet (1905). Bull. Soc. Chim. 33, 1198.
`Waterman and Jones (1921). J. Biol. Chem. 46, 9; 47, 285.
`(1922). J. Biol. Chem. 52, 357.
`-
`
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`

`INDEX
`
`Accessory "B" factor, further evidence for a
`third (Reader)
`77
`Acid-base balance of cattle urine, influence of
`foodstuffs on (Warth and Ayyar)
`1595
`Acidosis, lactic, in avitaminosis B, brain locali-
`sation of, and relation of, to symptoms
`(Kinnersley and Peters)
`711
`Acids, fatty, of butter, variations in, due to
`changes in seasonal and feeding conditions
`(Hilditch and Sleightholme)
`1098
`Acids, fatty, of glycerides of fish-liver oils
`(Guha, Hilditch and Lovern)
`266
`ADAIR, G. S. and ROBINSON, M. E. The specific
`refraction increments of serum-albumin and
`serum-globulin
`993
`ADAIR, G. S. and ROBINSON,