STUDIES ON PROTEINOGENOUS AMINES. The discovery of the

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STUDIES ON PROTEINOGENOUS AMINES.
XIV. A MICROCHEMICAL COLORIMETRIC METHOD FOR ESTI-
MATING TYROSINE, TYRAMINE, AND OTHER PHENOLS.
BY MILTON T. HANKE AND KARL K. KOESSLER.
(Prom the Otho S. A. Sprague Memorial Institute and the Department of
Pathology, Uwiuersity of Chicago, Chicago.)
(Received for publication, October 21, 1921.)
INTRODUCTION.
The discovery of the diazonium salts by Griess in 1%X1 opened
up a most fruitful field for scientific investigation by giving the
investigator a class of chemical compounds t,hat are highly sus-
ceptible to a large variety of chemical changes. Among these
chemical reactions is one that is of particular value because it
gives rise to colored compounds; namely, the ability of diazonium
compounds to combine (couple) with imidazoles, phenols, and
amines in alkaline solutions. p-Fhenyldiazonium sulfonate,
usually called diazobenzenesulfonic acid, is particularly valuable
for calorimetric determinations because it is comparatively stable
in water solutions, it does not couple with itself to give a highly
colored compound, it is easy to prepare, the dyes formed from it
are sufficiently soluble in alkaline solutions to render the use of
a calorimeter possible, and the colors produced, usually orange to
red, are easily compared because they do not readily produce
retinal fatigue. It is not surprising then, to find that p-phenyl-
diazonium sulfonate has been used for some time as a qualita-
tive test for imidazoles, phenols, and annnes.2s 3 Up to the
present time two methods have been proposed for the use of this
diazonium salt in Dhe quantitative estimation of imidazoles;4s b
1 Griess, P., Ann. them. Pharm., 1866, cxxxvii, 39.
* Ehrlicb, P., 2. klin,. Med., 1882, v, 285; Char. nwz., 1883, viii, 140;
De?dsch. med. Woch., 1883, ix, 549; 1884, x, 419.
3 Pauly, H., 2. phy.sioZ. Chem., 1904, xiii, 508; 1905, xliv, 159.
4 W&z, RI., and Ssobolem, N., Biochem. Z., 1913-14, Iviii, 119.
6 Koessler, K. K., and Hanke, M. T., J. Biol. Chem., 1919, xxxix, 497.
235
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236 Studies on Proteinogenous Amines. XIV
but as far as is known to the authors this reagent has not been
used, heretofore, for the quantitative estimation of phenols. This
paper contains a detailed description of methods for the micro-
chemical calorimetric determination of phenol, o-, m-, and p-cresol,
p-oxyphenylacetic, p-oxyphenylpropionic, and p-oxyphenyllactic
acids, tyrosine, and tyramine.
From the standpoint of color development, these phenols can
be divided into three classes; namely,
1. Phenols in which the para position is not occupied by a
second substituent.
2. Phenols in which the para position is occupied by a second
substituent that does not contain an amino group.
3. Tyrosine and tyramine.
When the position para to the phenol radical is not occupied,
as in phenol, o-cresol, and m-cresol, the substitution with p-
phenyldiazonium sulfonate occurs, at least predominantly, in
this position.
H H
II+HO-N=N-<~)SO~~ONu-1NO~~~-N=N-&O,-OPR+H
H H H H H H
The coupling is so rapid that about 95 per cent of the color has
appeared before a reading can be taken with a Duboscq colori-
meter. The color is fully developed in about 2 minutes and it
does not change for about 3 to 5 minutes. In these casest he color
is predominantly yellow with just a dash of red.
When the position para to the phenol radical is occupied, as in
p-cresol and the aromatic hydroxy-acids, the substitution with
p-phenyldiazonium sulfonate must occur in the ortho position.
H
:: H H 0
H \H
, + HO-N = N--(_)-SOa. ONM~” N = I-?T--SO,-ONa+HtO
f
““c/HH H H H H
3 CH,
The initial rate of coupling in this case, is somewhat slower than
that of phenol, but a color of maximum intensity isnevertheless,
obtained in about 3 minutes. The colors produced are psedomi-
nantly red so that a neutral solution of Congo red can serve as a
comparison standard. These colors are stable for from 3 to 5
M. T. Hanke and K. K. Koessler 237
minutes. The stability of the color and the character of the
change that the color undergoes after it has reached its maximum
intensity is a fairly accurate index of the composition of the
aromatic hydroxy-acid that is present, if only one of these acids is
present. This is discussed in detail in the experimental section
of the paper.
Tyrosine and tyramine, in small concentrations, show an anoma-
lous behavior toward p-phenyldiazonium sulfonate in alkaline
solutions. When a solution of tyrosine or tyramine is first added
to the alkaline reagent, a pink color begins to develop promptly
as in the case of the aromatic hydroxy-acids. After about 30
seconds, however, the color changes sharply to yellow and fades.
The intensities of the yellow colors so produced are not directly
proportional to the amount of tyrosine or tyramine present.
Obviously, then, the usual procedure is worthless for the estima-
tion of either tyrosine or tyramine.
The sharp change in color from pink to yellow suggested a
chemical change, perhaps a tautomeric shift. In an effort to
stabilize the pink color, or rather to revive it after the initial
reaction had gone to completion, we added a small amount of a
concentrated solution of sodium hydroxide. The color was in-
tensified and some of the pink tint was restored; but again the
color intensities were not directly proportional to the concentra-
tion of the phenol. The phenomena noted were so similar to
those that one would expect from a compound in which a tauto-
merit equilibrium existed between a carbonyl form and an enol
form that we thought to stabilize the carbonyl derivative, which
we believed to be most highly colored, by allowing it to react with
hydroxylamine to form an oxime. The addition of hydroxylamine
hydrochloride to the alkaline reagent did not give rise to an
intensification of color; but when sodium hydroxide was added
previous to the addition of hydroxylamine, a very intense bluish
red color was produced which was directly proportional to the amount
of tyrosine or tyramine added. Briefly then, the method for esti-
mating either tyrosine or tyramine consists of
1. A reaction between tyrosine or tyramine and p-phenyldia-
zonium sulfonate in an alkaline-sodium carbonate-solution
which gives rise to a primary yellow color. This reaction is
allowed to proceed for 5 minutes.
238 Studies on Proteinogenous Amines. XIV
2. The addition of 2 cc. of 3.0 N NaOH, after the initial reaction
period of 5 minutes, which intensifies the color, stops any further
action between the phenol and the diazonium salt and converts
the diazonium salt into a sodium diazotate which cannot couple
with the hydroxylamine that is to be added later. The alkali
is allowed to act for 1 minute.
3. The addition of 0.10 cc. of a 20 per cent solution of hydroxyl-
amine hydrochloride, which gives rise to a very intense bluish
red color that is stable for at least half an hour.
An explanation of the chemical reactions involved must ac-
count for the following facts: Tyrosine and tyramine are the
only phenols, that we have examined, that give a color intensi-
fication with sodium hydroxide and hydroxylamine. Since oxy-
phenyllactic acid, which is structurally identical with tyrosine
excepting that an OH group replaces the NH2 group, does not
give a color intensification with NaOH and NH20H, the NH2
group must play a r81e in the color development with these phenols.
A number of other compounds in which a tautomeric equi-
librium exists between a carbonyl form and an enol form, for
example acetaldehyde, acetone, and acetoacetic acid, also give an
intense color under the conditions just described. The color
produced is almost identical with that obtained with tyrosine or
tyramine.
We offer the following explanation tentatively for these color
phenomena. Work is now under way to determine the correctness
of the following formulations.
Since the position para to the phenol group in tyrosine and
tyramine is occupied by an alanyl side chain, the substitution
with diazobenzenesulfonic acid must occur in the ortho position.
H H
0 :: . . . . . . . . . . .
0
H/‘\ iH+HOi -N = N-CsHd-SOyONa H/\-N = N-C&-SO,. 0 Ns
1 1. . . . . . . . . . . . .
H\/H H( ’
bH7 --Lb x HI + Hz0
H b NH2 HbNHz (4
COO Na LOO Na
Compound A-the yellow phenol form-would be in tautomeric
equilibrium with a small amount of the supposedly red quinoid form
M. T. Hanke and K. K. Koessler 239
0
::
H/\-N = Pr’-CsH4S02. 0 Na H/(= N-~-C~HISOn* 0 Na
HI )
H?J
j
CH, e AH2
HASH* HbNHz 03)
bO0 Na bO0 Na
In a solution made alkaline with sodium carbonate, practi-
cally all of the compound would exist in its phenol form A. The
addition of a strong alkali, like sodium hydroxide, would give
rise to the sodium phenate derivative of A which, because of its
high degree of dissociation, would pass more easily into the quinoid
derivative than the phenol itself.
Na
0
d
H/\-N = N-CsH,S02. 0 Na H[$= N-$-C,H,SO, .O Na
H?i H\
i
AH* +&O * CHz +NaOH
H&HZ H&NH1 0.9
LOO Na Yellow. bO0 Na Red.
The result would be an intensification of color with the influx
of red. The amount of quinoid derivative formed under the
influence of alkali must, nevertheless, be small because the color
intensification is not very great. The addition of hydroxylamine
hydrochloride to such a strongly alkaline solution would give rise
to the following reactions. -
1. NHsOH.HCI + 2 NaOH -+ Hz N-O Na + NaCl + Hz0
0 N-O Na
!I H II H
H/\= N-N-CoH,SOz.O Na H \= N-N-GHG30z. 0 Na
/
H?! H\ I
2. AH2 +HzN-ONa-+ &Hz + Hz0
HbNHa H&NH2
A00 Na LOO Na
240 Studies on Proteinogenous Amines. XIV
One would expect the quinoneoximehydrazone derivative (C)
formed in this way to be dark red. Such o-quinoneoximehydra-
zones seem not to have been studied; but the para derivatives
have been known for some time.6 They all give deep red alkaline
solutions.
o-Quinonedioxime
HF)=N-OH
which is a closely related compound, also gives a deep red alka-
line solution.’
Just why tyrosine should give a highly colored quinoneoxi-
mehydrazone while p-oxyphenyllactic acid does not give such
a highly colored derivative, is a problem for future investigation.
The role played by the amino group in the side chain of tyrosine
and tyramine is still obscure.
The reactions that probably occur when certain aldehydes and
ketones are treated with diazobenzenesulfonic acid, sodium
hydroxide, and hydroxylamine can be represented by the follow-
ing type equations.
RI
Lo RO + ILO
H
Rz- t: !k,‘;;-‘G! = N-N-CQH4S02. 0 Na+ Rz-b= N--NH-CsH.$OrONa
. . . . . . . . . .
Pale yellow.
RI
RI
Lo
+HzN-ONa-+ #=N-ONa + Hz0
Rz--b= N-~-CsHIS02. ONa RI-(: = N-N-CsH,S02. ONa
H
We believe that the red compounds formed with acetaldehyde,
acetone, and acetoacetic acid are the sodium salts of hydrazo-
xime derivatives. Van Pechmann and Wehsarg8 have prepared
a hydrazoxime derivative of methyl glyoxal which is similar to
the compound that we believe to be formed with acetone.
6 Borsche, W., Ann. &em. Pharm., 1907, ccclvii, 171.
7 Hantzsch, A., and Glover, W. H., Ber. &em. Ges., 1907, xl, 4344.
8 von Pechmann, H., and Wehsarg, K., Ber. them. Ges., 1888, xxi, 2996.
M. T. Hanke and K. K. Koessler 241
H H
C = N-OH C = N-:-&He-SO* * OH
b = N-NH-C&HE A =N-OH
AH% AH3
van Pechmann and Wehsarg’s Hydrazoxime formed from
hydrazoxime. acetone.
They give no data on the color of the compound in alkaline solu-
tions although they found the alcoholic solution to be yellow and
the sulfuric acid solution to be dark red.
EXPERIMENTAL.
Reagents Employed.
Most of the reagents employed in these estimations are identi-
cal wit,h those employed by us in the estimation of imidazoles.6
A detailed description is repeated here for the sake of completeness.
Stock Sulfanilic Acid.-Sulfanilic acid (4.5 gm.) is mixed with
45 cc. of 37 per cent hydrochloric acid (sp. gr. 1.19) in a 500 cc.
volumetric flask. Water is then added to the mark. The solid
dissolves slowly; but completely.
Stock Sodium Nitrite.-25 gm. of 90 per cent sodium nitrite
are dissolved in water and diluted to 500 cc. in a volumetric
flask.
Sodium Carbonate.-Baker and Adamson’s anhydrous sodium
carbonate (5.50 gm.) is dissolved in water and diluted to exactly
500 cc. We recommend the above grade because we have found
it to give uniform results. The purity of this carbonate is a very
important factor in the color development. Some grades of carbon-
ate contain impurities that give yellow colors of inferior intensities.
The finished carbonate solution must be preserved in a glass
vessel that has little tendency to dissolve in alkali. Pyrex glass
vessels have proved to be entirely satisfactory.
Stock Methyl Orange.-Vacuum-dried Griibler’s methyl orange
(0.5000 gm.) is dissolved in water and diluted to exactly 500 cc.
This solution keeps indefinitely.
Stock Congo Red.-Vacuum-dried Grtibler’s Congo red (2.5000
gm.) is mixed with 50 cc. of absolute alcohol in a 500 cc. volumet-
242 Studies on Proteinogenous Amines. XIV
ric flask. Water is then added to the mark. This solution
keeps indefinitely.
Stock Acid Fuchsin.-Vacuum-dried Harmer’s acid fuchsin
(2.5000 gm.) is dissolved in water and diluted to exactly 500 cc.
in a volumetric flask. The thymol-preserved solution keeps
indefinitely.
Stock Phenol Red.-Hynson, Westcott and Dunning’s phenol
red (0.0500 gm.) is dissolved in water and diluted to exactly
500 cc. The thymol-preserved aqueous solution can be kept for
at least 1 year. The alcoholic solution employed by many in-
vestigators for pH determinations deteriorates quite rapidly and
cannot be used in this work.
Standard Indicator Solutions.-For the estimation of phenol,
a solution containing 10 cc. of stock phenol red in a total aqueous
volume of 100 cc. is employed. Redistilled water should be used
for dilutions and the volumetric flask should be thoroughly rinsed
with distilled water before the phenol red solution is introduced.
It is best to prepare a fresh standard every day. The color SO
obtained matches that produced by phenol perfectly. When
this standard indicator solution has been used for comparisons the
symbol (Ph-RR) is suffixed to the reading obtained.
For the estimation of o- and m-cresol, a solution containing
5 cc. of stock methyl orange in a total aqueous volume of 500 cc.
is employed. The color so obtained matches that produced by
o- and m-cresol almost perfectly. When this standard indi-
cator solution has been used for comparisons the symbol (MO)
is suffixed to the reading obtained.
For the estimation of p-cresol, and p-oxyphenylacetic, p-oxyphenyl-
propionic, and p-oxyphenyllactic acids, a solution containing 1.00
cc. of stock Congo red in a total aqueous volume of 500 CC. is
employed. When this standard indicator solution has been used
for comparisons t,he symbol (CR) is suffixed to the reading
obtained.
For the estimation of tyrosine or tyramine, a solution containing
1 cc. of stock acid fuchsin and 1.8 cc. of stock methyl orange in a
total aqueous volume of 500 cc. is employed. When this stand-
ard indicator solution has been used for comparisons the symbol
(F-MO) is suffixed to the reading obtained.
M. T. Hanke and K. K. Koessler 243
Preparation of p-Diazobenzenesulfonic Acid Solution (The
Reagent).
1.50. cc. each of the stock sulfanilic acid and sodium nitrite
solutions are measured into a 50 cc. volumetric flask. The flask
is then immersed in an ice bath for 5 minutes. Then 6.00 cc. more
of the stock sodium nitrite solution arc added and the well mixed
solution is again allowed to lie in the ice bath for 5 minutes. Dis-
tilled water is then added to the mark and the flask returned to
the ice bath where it is kept. This reagent must not be used for
at least 15 minutes after diluting with water. We have found it
to give perfect results after 24 hours. It is best, however, to
prepare a fresh reagent every day.
Preparation of the Phenols.
Phenol.-Merck’s highest purity phenol was distilled in vacua.
The product collected boiled at 95” in a vacuum of 10 mm. of
Hg. The colorless oil solidified readily. The colorless crystals
so obtained had a melting point of 43”. The substance was as-
sumed to be 100 per cent pure phenol.
o-CresoZ.-A chemically pure product obtained from a local
supply house was doubly distilled in vacua. The colorless oil
obtained boiled at 81” at a pressure of 10 mm. and at 188” under
atmospheric pressure. The oil solidified on standing. The
colorless crystals so obtained had a melting point of 32”. It was
assumed to be 100 per cent pure o-cresol.
m-CresoZ.-A chemically pure product obtained from a local
supply house was doubly distilled in vucuo. The colorless oil
so obtained boiled at 104” under 10 mm. pressure and at 201” under
atmospheric pressure. It did not solidify. It was assumed to
be 100 per cent pure m-cresol.
p-CresoL-The p-cresol was obtained as a by-product in the
preparation of tyramine.” The substance boiled at 90-91” at a
pressure of 10 mm. and at 198”under atmospheric pressure. The
oil solidified after standing for some time. The colorless crys-
tals so obtained melted at 36”. It was assumed to be 100 per
cent pure p-cresol.
9 Koessler, K. K., and Hanke, M. T., J. Biol. Chem., 1919, xxxix, 585.
244 Studies on Proteinogenous Amines. XIV
Oxyphenylacetic A&.-This substance was obtained as a by-
product in the preparation of tyramine.g The colorless solid
had the following properties.
1. It melted at 150”.
2. It left no residue on ignition.
3. The solid (0.1000 pm.) was dissolved in 10 cc. of water and titrated
with 0.1 N NaOH using phenolphthalein as indicator. Exactly 6.59 cc. of
the alkali were required to produce the first change in the indicator. The
amount demanded by the formula is 6.58 cc. The substance was, there-
fore, 100 per cent pure.
Oxyphenylpropionic Acid.-This compound was prepared
synthetically as follows.
Cinnamic acid was reduced, with sodium amalgam, to hydro-
cinnamic acid.lO
p-Nitrophenylpropionic acid was then prepared by a modifi-
cation of the method of Beilstein and Kuhlberg.ll
StijhF prepared p-aminophenylpropionic acid by reducing the
ethyl ester of p-nitrophenylpropionic acid with zinc and hydro-
chloric acid. He isolated the zinc double salt of the acid and
treated this with acid and sodium nitrite to prepare p-oxyphenyl-
propionic acid. We reduced p-nitrophenylpropionic acid (6 gm.)
directly with zinc (14 gm.) and hydrochloric acid (50 cc. of
the 37 per cent acid) in alcoholic solution (70 cc. of 95 per cent
alcohol) adding the acid slowly and keeping the temperature
below 30”. The colorless liquid obtained, after the reaction
had been allowed to proceed for 24 hours, was treated with 23.5
cc. of 95 per cent sulfuric acid and subjected to a distillation
in vacua at 60”. The pale yellow solid so obtained was dissolved
in water (200 cc.) treated with sulfuric acid (20 cc. of the 95
per cent acid) and cooled in an ice bath. Sodium nitrite (60
cc. of a 5 per cent solution) was poured into the cold liquid.
The resulting solution was allowed to remain in the ice bath for
1 hour after which it was treated with 250 cc. of water and heated
to the boiling point. There was a copious evolution of nitrogen;
but practically no tar formation. The aqueous solution was
lo Fischer, E., Anleitung zur Darstellung organischer Praparate, Bruns-
wick, 8th edition, 1908, 39.
11 Beilstein, F., and Kuhlberg, A., Ann. them. Pharm,., 1872, clxiii, 132.
I2 Stiihr, C., Ann. them. Pharm., 1884, ccxxv, 60.