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decomposition with sulphuric acid, do not give the reaction. Schlitzen-
berger ^ found that tyrosine is absent from the putrefaction products of
gelatin. Now, Salkowski stated that gelatin does not react with Millon's
reagent. But Chittenden and Solley '^ have found that the products of
gelatin digestion give a characteristic reaction, and Pickering that pure
gelatin and gelatinoses give it in a marked manner ; thus confirming the
statement made by Millon ^ in his original memoir. Gelatin, therefore,
owes this property to something which is not tyrosine, but which, like
tyrosine, contains a hydroxybenzene nucleus.

Adamkieivicz reaction.^ — If glacial acetic acid in excess and then
concentrated sulphuric acid are added to proteid, a violet colour with
feeble fluorescence is produced. The test is by no means a certain one,
and is given by proteoses and peptones in concentrated solutions only.
It is not given by gelatin (Hammarsten).

This test is only given by the aromatic substances of Salkowski's
third (indol) group. The strong reagents added are likely to produce

'^ Journ. f. pralct. Chem., Leipzig, N.F., Bd. iii. S. 180.

2 Journ. Physiol., Cambridge and London, 1893, vol. xiv. p. 372.

2 Ztsclir. f. d. ges. Naturw., Halle, Bd. xxix. S. 506 ; Virchow's Archiv,VA.xx\i-x. S. 130.

4 Cliem. Ccntr.-Bl., Leipzig, 1879, Bd. x.

5 Ztschr.f. Biol., Munchen, Bd. xxii. S. 423.

^ Article in Wurtz' "Diet, de cliim.," 1886, Suppl. 1 A, p. 58.
'' Journ. Physiol., Cambridge and London, vol. xii. p. 23.
^ Co')n2ot. rend. Acad. d. sc, Paris, tome xxviii. p. 40.

'•^ Per. d. deutsch. chem. Gesellsch., Berlin, Bd. viii. S. 761. See also Wurster Chem.
Ztg., Cothen, Bd. xi. S. 187.


considerable change in the proteid molecule ; indol and skatol can
hardly be considered to be simple cleavage products of the proteid
molecule (see p. 29).

Liebcrmaniis tcsf^ is performed by precipitating the proteid by
alcohol, and then heating the washed precipitate with strong hydrochloric
acid. The result is a violet-blue colour. The reaction is not given by pure
peptone.^ It is also not given by any of the aromatic putrefactive products
of proteid, nor by a large number of other cleavage products of proteid
which Pickering worked with. Its cause is therefore at present unknown.

Krassers reaction.^ — Alloxan in solution stains proteid matter a
brilhant red. It reacts in the same way with asparagin, aspartic acid,
and tyrosine. The reaction is probably connected with the presence
of amido groups.

Piotrowshi s reaction} — If a few drops of dilute copper sulphate
solution are added, and then excess of strong solution of caustic soda
and potash, a violet solution is the result. If ammonia is used instead,
a blue solution is formed.

In the case of the proteoses and peptones, the result is a rose-red solu-
tion with potash^ and a reddish violet with ammonia. As the same colour
is given bv the decomposition product of urea called biuret,^ the test
is often called the Uuret reaction (2C0NoH, - NH. = C.O.NsPIj). Bim>et
yields, on decomposition, compounds which contain cyanogen ; for instance,
by heat it is split into ammonia and cyauuric acid, (0^)311303. Bim'et,
cyanuric acid, xanthine, hypoxanthine, sarcosine, hydrocyanic acid, all give
smiilar reactions to the proteids. Gnezda '' considered it probable that the
biuret reaction was due to a cyanogen radicle, and that the cyanogen in
albumin and peptone is differently combined, correspondmg to the smiilar
differences in cyanuric and hydrocyanic acid respectively. Pickering,^
however, concludes, that the radicle in question is not CN but CONH.

The related metals, nickel (Gnezda) and cobalt (Pickering) give correspond-
ing colour reactions, which may be summarised in the following table : —


Copper Sul-
phate and

Copper Sul-
phate and

Nickel Sul-
phate and

Xickel Sul-
phate and

Cobalt Sul-
phate and

Cobalt Sul-
phate and

Native proteids")
(al bu mill s, |
globulins, and -
nucleo - pro-
teids) J

Products of pro-'^
teolysis (pro- |^
teosesaiidpep- j
tones) j








Pickering found that when a cobalt salt has entered into the proteid

^ Jahresb. il. d. Fortschr. d. Thier-Chcm., Wiesbaden, Bd. xvii. S. 8 ; Chcm. Ccntr.-BL,
Leipzig, 1887, Nos. 18 and 25.

- Le Nobol, Jahresb. i'l. d. Fortschr. d. Thicr-Chem., AViesbaden, Bd. xvii. S. 3.

^ Monatsh. d. Chem.., Wien, Bd. vii. S. 673.

^ Sitzungsh. d. k. Akad. d. JFisscnsch., Wien, Bd. xxiv. S. 335.

^ Briicke, Monatsh. d. Chcm., Wien, Bd. iv.

" Wiedemann, Ann. d. Fhys. u. Chem., Leipzig, Bd. Ixxiv. S. C7.

'' Froc. Roif. Soc. London, 1889, vol. xlvii. p. 202.

" Journ. FhysioL, Cambri<lge and London, 1893, vol. xiv. p. 317.


molecule, it can be easily displaced by a nickel salt, and then in turn by a
copper salt, each yielding its characteristic colour reaction. He examined
these and other reactions in connection Avith various albuminoids as well ; the
addition of cobalt sulphate and potash to gelatin he found to produce a play
of colours in the order of those of the spectrum, commencing with violet.

Drechsel ^ has drawn attention to an old observation of Krukenberg's, that
at the boiling temperature there is in the so-called biuret reaction a reduction
of the cupric to cuprous oxide ; the latter, however, remains in solution.
Drechsel shows that the reduction also occurs at the ordinary temperature.
C. J. Martin '^ is also of opinion that the biuret reaction is a reduction.
He finds that alkali albumin dissolves cuprous oxide and forms a pink solution,
never violet or purple ; these latter colours, when the test is ordinarily performed
with copper sulphate, are due to admixture with cupric hydrate, held in solu-
tion by the proteid and not reduced. The most powerful reducing proteids are
proteoses and peptone, hence the pink biuret reaction ; whereas the native
proteids have a smaller reducing power, and the pink colour is mixed with the
blue cupric hydrate, and so the colour obtained is a violet.

J'rom the preceding study of the properties and reactions of the
proteids, it will be gathered that since many other substances give the
same tests, a proteid can only be identified by employing a large
number of its reactions. Winternitz^ recommends a combination of a
precipitant and colour reactions. The precipitant he has chiefly used
in cases of albuminuria is acetic acid and potassium ferrocyanide. The
precipitate so obtained gives the colour reactions well. This is also the
case with the precipitate produced by several other reagents, among
which may be mentioned salicylsulphonic acid,* and the halogens.^

Classification of Proteids.

It will be seen from the preceding description of the proteids, that
I have used the term proteids throughout as an equivalent for albuminovis
substances (German, Eiweisskorper) ; certain other substances (such as
haemoglobin, mucin, nucleo-proteids) named proteids, by Hammarsten,
Neumeister, and other continental writers, will be treated separately
as compound proteids.

The proteids may be divided into those of animal and those of
vegetable origin. There does not appear to be any essential difference
between these two classes, and each can be subdivided in the same
manner into sub-groups, bu.t the distinction is a convenient one in practice.

Animal proteids. — Class 1. Alhwmins. — These are proteids which
are soluble in water, in dilute saline solutions, and in saturated solutions
of sodium chloride and magnesium sulphate. They are, however, pre-
cipitated by saturating their solutions with ammonium sulphate. Their
solutions are coagulated by heat, usually at 70°-73° C. Serum albumin,
egg albumin, lact-albumin are examples.

Class 2. Globulins. — These are proteids which are insoluble in water,
soluble in dilute saline solutions, and insoluble in saturated solutions of
sodium chloride, magnesium sulphate, and in half-saturated solution of

1 Ztsclir. f. 2}^iiJsiol. Cliem.i Strassburg, 1895, Bd. xxi. S. 68.

" Private comnmnication to author.

^ Ztschr. f. physiol. Chcm., Strassburg, Bd. xv. S. 187; xvi. S. 439.

■* Pickering, loc. cit., p. 377.

^ F. G. Hopkins, Proc. Physiol. Soc, June 12, 1897.

VOL. I. — 4


ammonium sulphate. Their solutions are coagulated Ijy heat, the
temperatm'e of lieat coagulation vaiying considerably. Filirinogen,
serum globulin, globin, paramyosinogen, and myosiuogeu, crystalhn,
vitellin,^ egg globulm are examples.

The differences in solubihty of these two important classes of native
proteids may be stated in tabular form as follows : —


Albumin. i Globulin.


Dilute saline solution .....
Saturated solution of naagnesium sulphate or

sodium chloride

Half-saturated solution of ammonium sulphate
Saturated solution of ammonium sulphate






Class 3. Albuminaics. — These are proteids derived from either
albumms or globulins by the action of weak acids or alkalis. The term
has been extended to include metallic compomids of the proteids, Ijut
restrictmg it here to acid alljumui or syntonm, and alkali albmnin, the
class may be defined as consisting of proteids which are insoluble in
water, and in neutral solutions containing no salt. They are soluble in
acid or alkaline solutions, and in weak saline solutions. They are
precipitated by neutralisation, and resemble globulms in then- liehaviom-
to neutral salts. Then- solutions are not coagulated by heat.

A less soluble variety of these proteids, called Lieberkiihn's jelly,
is formed by adding strong acid or alkah respectively to undiluted
white of ecro;.

Caseinogen, formerly regarded as a member of this group, will be
studied with nucleo-proteids and with milk.

After egg albumin is treated with formaldehyde it remains solut»le
in water, but is not coagulable on heating.^

Class 4. Products of lyroteoly&u ; inotcoscs and pcptonrs. — These will
be studied in detad in connection with digestion. They can, however,
]je formed by other hydrolysiug agencies than digestive juices, such as
treatment with mineral acids, or superheated steam.^ The term proteose
for the intermediate products of hydration is a convenient general name,
which includes not only albmnoses, but also vitelloses, globuloses, caseoses,
myosinoses, and the like.

Class 5. Coajjulated 'proUids. — Tliis class includes the proteids in
which coagulation has been produced by heat, and those in which coagu-
lation has been induced by ferment action, such as fibrin, myosin, casein,
and anti-albmnid, an insoluble l:)y-product formed in gastric digestion.

Since the mdividual members of these groups have either been
described in preceding sections, or will be discussed elsewhere under
other heads, such as blood, milk, etc., they need not be further considered
in this place.

^ ^ itellin, unlike otber globulins, is not precipitated by sodium chloride. Some regard
it as a nucleo-proteid. It will be more fully discussed later.

- Blum, ZUrhr. f. jjhy.-iiol. Chem., Strassburg, 1896, Bd. xxii. S. 127 ; Berl. klin.
Wchnnchr., 1897, Bd. x.x.xiii. S. 1043.

^ On " Atmid-albumoses " (that is, those formed by superheated steam) see Xeumeister,
Zlschr. f. Biol., Mlmchen, Bd. xxvi. S. 57; Chittenden and Meara, Jourii. Physiol.,
Cambridge and London, 1894, vol. xv. p. 501.


Vegetable proteids. — The amount of proteid matter in yjlants,
especially in those which are full grown, is less than in animals. It occurs
dissolved in their juices, or in their protoplasm, or deposited in the form
of granules called aleuron grains. Plant proteids have frequently been
obtained in a crystalline form. They may be divided into the same
classes as the animal proteids.

Class 1. Albiwiins. — Small quantities of true alljumin have been
described by S. Martin ^ in the juice of the papaw fruit, and by Green ^
in the latex of several caoutchouc-yielding plants of the natural orders
Apocyneffi and Sapotacese.

Class 2. Glohulins. — These are by far the most abundant natural
proteids present in plants. This view, which was taken by Hoppe-
Seyler,^ is contrary to that of Eitthausen,* who regarded vegetable
proteids as consisting chiefly of legumin and allied substances.^

Class 3. Albuminates. — Acid and alkali albumin are formed readily
from vegetable proteids, especially from plant myosin. Legumin or
vegetable casein was used synonymously with vegetable proteid by some
of the earlier investigators,^ but it is now usually regarded as alkali
albumin, formed artificially in the extraction of the globulins by alkali.
The name conglutin was introduced by Eitthausen'^ for the more
glutinous product obtained from almonds and lupins.

Class 4. Proteoses and ■pe^ptones. — Proteoses have been described in
latex, in papaw juice, and flours of different kinds. True peptones are
not found in the circulating juices of plants. Probably the circulatmg
proteids in plant life are proteoses, hemialbumoses (Vines), though amido-
acids (leucine, tyrosine, asparagine, adenine, etc.)^ also occur. These
substances are formed by proteolytic ferments during germination. The
best known of these ferments, papain, has been investigated by Wurtz,
Martin, and others. Such ferments, as well as malting ferments, which
convert the insoluble starch of the seed into the soluble sugar, are probably
almost ubiquitous.^ In carnivorous plants, another ferment is met with
of a somewhat different character.

Class 5. Coagulated proteids. — Vegetable albumm and globulin, like
those of animal origin, are converted at a high temperature into an
insoluble heat coagulum.

With regard to the value of vegetable proteids as food, it may be stated that
as a rule they are not nearly so readily digested as animal proteids. Prausnitz ^"^

^ Journ. Physiol., Cambridge and London, vol. vi. p. 336.

^ Proc. Roy. Soc. London, vol. xl. p. 28.

3 "Physiol. Chem.," S. 75.

■^ Ztschr.f. Chem., Leipzig, Ser. 2, Bd. iv. S. 528, 541 : vi. 126; Joimi. f. pra7d. Chem.,
Leipzig, Bd. ciii. S. 65, 78, 193, 273.

^ Ritthausen defends his view in Chem. Centr.-Bl., Leipzig, 1877, S. 567, 578.

8 Einhof, Neue allrj. Journ. cl. Chem., v. A. Gehlen, 1805, Bd. vi. S. 126, 548. Dumas
and Cahours, Liebig, and others also examined this substance.

"'Ibid., Ser. 2, Bd. xxvi. S. 440.

^ E. Schulze and E. Kisser, Landw. Versuchs Stat., Berlin, Bd. xxxvi. S. 1 ;_E. Schulze,
numerous papers in Ztschr. f. i^hysiol. Chem., Strassburg. See especially Bd. xii. S. 405.

^ Gorup-Besanez, Ber. d. deutsch. chem. Gesellsch., Berlin, 1874, S. 1478 ; Krauch,
Journ. Chem. Soc., London, 1878, Abst. p. 996 ; Green, Proc. Roy. Soc. London, vol.
xli. p. 466; Thiselton Dyer's Presidential Address, Sect. D, Brit. Assoc., 1888; Hansen,
Bot. Ztg., 1886, S. 137 ; Ellenberger and Hofmeister, Centralbl. f. ar/ric. CJiem., Leipzig,
1888, S. 319. The subject of enzymes and reserve materials in plants, however,_ is now
a very large one, and it Avill be found discussed, with bibliography, in a series of articles^by
J. Reynolds Green, in Science Progress, London, vol. i. p. 342 ; ii. p. 109 ; iii. pp. 68, 376 ;
V. p. 60.

1" Ztschr.f. Biol., Miinchen, Bd. xxiv. S. 227.


experimented with beans; he found the fseces contained as much as 30"3 of the
nitrogen of the beans in an undigested condition. Beans thiis compare
unfavourably with lentils and bread, but even here there is a considerable
undigested residue. The investigations of Kutgers ^ point to the fact that this
is due rather to the admixture of vegetable proteids with cellulose and other
indigestible materials than to any peculiarity in the proteids themselves.

The foregoing brief account of the vegetable proteids may be amplified by
further consideration of some of the points raised : —

Researches on crystallised vegef able proteids. — In 1855, Hartig- pointed out
the existence of crystallised jiroteid matter in seeds. Four years later, Maschke^
obtained hexagonal plates of proteid matter by extracting Brazil nuts with
water at 40°-50° C, and evaporating the filtered extract at 40°. ISTageli'^ de-
signated such crystals as crystalloids. Weyl^ identified the crystals as vitellin.
Sachsse,^ by Maschke's method, and also by precipitating the aqueous extract
by a stream of carbonic anhydride, obtained several preparations of proteid from
Brazil nut which he analysed. The precipitate consisted of small discs,
not crystals. Schmiedeberg "^ obtained crystalline products from the car-
bonic anhydride precipitate by digesting it with magnesia solution at 35° C, and
evaporating at the same temperature. Drechsel ^ obtained hexagonal crystals,
by submitting the solution containing Schmiedeberg's magnesia compound to
dialysis against alcohol, and also by the slow evaporation of a warm sodium
chloride solution of the proteid.^ At Drechsel's suggestion, Grlibleri° applied
this method with some modifications to the proteids of squash seed, from which
he obtained octahedral crystals ; he obtained lime as well as magnesia crystalline
compounds. Eitthausen,^i by similar methods, obtained octahedra and rhombic
dodecahedra from expressed hemp cake, castor-oil seeds, and seeds of Sesamum
i7idicum. Molisch ^^ has separated by the use of ammonium sulphate a
crystalline proteid (phycocyanin) from the alga, Oscillaria leptotriclia.
Vines ^'^ found that the natural crystalloids, embedded in the ground
substance of the aleuron grains, were hexagonal rhombohedra in some plants,
and regular tetrahedra in others.

Some of the details of Vines' work are as follows : —

The aleuron grains of the peony contain an albumose and vegetable
myosin ; of the castor-oil plant, an albumose, a myosin, and vitellin ; of blue
lupin, chiefly crystalloid vitellin. He classified aleuron grains into —

1. Those soluble in water, albtnnose.

2. Those soluble in 10 per cent, sodium chloride solution —

(ff) Without crystalloids, soluble in saturated sodium chloride solution,

(Ij) With crystalloids, insoluble in saturated sodium chloride solution, myosin.

3. Those partially soluble in 10 per cent, sodium chloride solution. Some of
these are crystalloid, some insoluble, some soluble in saturated salt solution.

Vitellin is the principal constituent of egg yolk, and occurs there in the
form of semicrystalline sphserules, corresponding to the crystalloid aleuron
grains of plants. The proteids described by Valenciennes and Fremy ^^ in the

1 ZL.ichr.f. Biol., Mlinchen, Bd. xxiv. S. 251. - Bot. Ztg., 1855, S. 881.

'^ Journ. f. prukl. Chem., Leipzii;, Bd. Ixxiv. S. 436.

^Bot. MitiU., Mlinchen, 1863, Bd. i.

^ Arch. f. d. (jes. Physiol., Bonn, Bd. xii. S. 635 ; Ziscltr. f. 2)liysiol. Chem., Strassburg,
Bd. i. S. 72.

*^ "Die Farbstoffe, Kohlenliydrate und Protcinsub.stan;c," Leipzig, 1877, S. 315.

'' Ztschr. f. jjhysiol. Chem., Stiassbnrg, Bd. i. S. 205.

^ Journ.f. prakt. Chem., Leipzig, Bd. xix. S. 331.

" See Giiibler, ibid., Bd. xxiii. H. 100. i" Ibid., Bd. xxiii. S. 97.

\i Ibid., Bd. xxiii. S. 481. 32 ^^^^^ 2:tg., 1895, Bd. i. S, 131.

3* Proc. Roy. Soc. London, vol. xxviii. p. 218 ; xxx. -p. 387 ; xxxi. p. 62.
" Ann. dechim., Paris, Ser. 3, tome L ]■>. 129 ; Ann. d. Chem., Leipzig, Bd. cxxvii. S. 188.



yolks of fishes' eggs, and termed by them ichthin, ichthulin, and emydin, are
regarded by Hoppe-Seyler as doubtful chemical units, and are probably ]uixtures
of vitellin with nuclein and lecithin. Whether vitellin contains phosphorus in
its molecule or not is a moot point. Some regard it as a nucleo-proteid rather
than a globulin ; others look upon the phosphorus generally found in it as
belonging to either nuclein or lecithin, adherent to it as an impurity. The
same question arises in connection with phytovitellin (vegetable vitellin).
Recent analyses by Osborne ^ show that it contains no phosphorus, though
Sachsse, one of the earlier workers, described the presence of this element.

Proteids of flours. — Sidney Martin "^ found the principal proteids in wheat
flour to be (1) a vegetable myosin, and (2) a soluble proteose, which he
called phytalbumose.

Gluten is a mixture of two substances — ■

(a) Gluten fibrin, which is insoluble in alcohol, and formed from the
myosin ; and

(&) A proteose insoluble in water, formed from the phytalbumose. This

Fig. 11.— Crystallised vitelliu of the oat kernel. — After Osborne.

insoluble proteose is, however, soluble in strong alcohol, and gives the sticky
consistency to gluten ; it corresponds to the two substances called gliadin and
mucedin by Eitthausen.^

The existence of proteids soluble in strong, though probably not in abso-
lute, alcohol, is a remarkable occurrence, and is not unique in vegetable

Martin considered that gluten does not pre-exist in wheat-flour, but is
formed on the addition of water by a ferment action. This is supported by
the fact that washing flour with water at a low temperature {T C.) does not
lead to the formation of gluten. The ferment, however, has not been
separated, and Johannsen* has advanced certain facts that tell against the
ferment theory and in favour of the pre-existence of gluten in the flour.

^ Am. Chem. Journ., Baltimore, vol. xiv. No. 8.

^ Brit. Med. Journ., London, 1886, vol. ii. p. 10'4.

'^ Journ. f. praU. Chcm., Leipzig, Bd. Ixxiv. S. 193, 384. For otlier observations on
gluten, see Bouchardat, Compt. rend. Acad. d. sc, Paris, tome xiv. p. 962; Taddei, Gior.
fisica di Brugnatelli, vol. xii. p. 860 ; Gnnsberg, Journ. f. praJct. Chem., Leipzig, Bd.
Ixxxv. S. 213.

* Ann. agronomiqucs, Paris, tome xiv. p. 420.


Osborne ^ investigated the proteids of the oat and analysed three primary
oat proteids, one soluble in alcohol, the second a globulin, and the third a
proteid soluble in alkali only. From these, secondary proteids are obtained
by mixing the ground oats with water ; he regards the change as one pro-
duced by ferment activity.

In conjunction with Chittenden,^ he worked out in a similar way the
proteids of maize, and found there two globulins, one or more albumins, and a
proteid soluble in alcohol. These differ in solubilities, coagulating points, and
elementary composition ; one of the globulins is a vitellin, the other a myosin.
A small amount of proteose also present was regarded as artificially produced
in the processes of analysis. The proteid soluble in alcohol is called zein ;
and it, like the globulins, is converted into an insoluble modification on ad-
mixture of the flour with water.

The proteids of flax seed ^ he found to be chiefly globulin, with smaller
quantities of albumin, proteose, and peptone.

In wheat Osborne and Voorhees ■* describe five proteids : —

1. Gliadin ; a proteid soluble in alcohol, and like gelatin in some of its
other properties.

2. Glutenin ; a proteid soluble only in alkalis.

3. Edestin ; a globulin of the vitellin class.

4. Leucosin ; an albumin, which Martin described as a myosin.

5. Proteoses.

They do not agree with Martin's ferment theory of gluten formation.
O'Brien^ has arrived at a similar conclusion; he regards gluten formation as
due to hydration, though not produced by a ferment. The proteids in the
flour he describes as globulins of the myosin and vitellin type, and a
proteose which he regards as the mother substance of gluten.

Other vegetable proteids investigated by Osborne are those of the kidney
bean ^ (two globulins called phaseolin and phaselin, and proteose) ; of the
cotton seed (almost altogether proteose, with small amounts of edestin and
insoluble proteid) ; of rye (gliadin, leucosin, edestin, and proteose) ; and of
barley *" (leucosin, proteose, edestin, and hordein, an insoluble proteid,
corresponding to Ritthausen's mucedin). He also investigated the chemical
nature of diastase, and considers it is closely related to the albumin he has
named leucosin.

Researches on proteolytic ferments m lAants. — Those in the papaw plant
and in pine-apple juice are the best known, or most fully worked out.

Papain was the name given by Wurtz to the proteolytic ferment in the
juice of the papaw plant. ^ The close similarity of its action to that of

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