PRECIPITATION OF SERUM-ALBUMIN AND GLUTIN BY ALKALOIDAL REAGENTS.

BY PAUL J. HANZLIK.

(From the Laboratory of Physicochemical Biology, University of Vienna. Director: Prof. Wolfgang Pauli.)

(Received for publication, November 1, 1914.)

The study of the mechanism of the precipitation of proteins by so called alkaloidal reagents (tannin, iodine potassium iodide, po- tassium ferrocyanide, potassium mercuric iodide, tungstate, and phosphomolybdate) has received practically no attention from the standpoint of physical chemistry, especially the isoelectric point. According to Michaelis,1 the isoelectric point of serum is the point where the number of positive and negative charges of electricity held by the protein ions are about equal; that is, at this point there is a maximum of neutral particles of protein. This lies ap- proximately at the concentration of 2. 10d of acid, such as ob- tains in a mixture of equal parts (+) of & acetic acid and G sodium acetate. Certain other phenomena which are exhibited by proteins at this point are, according to Pauli and his pupils, a minimum of viscosity as shown by serum and a maximum of pre- cipitation by ethyl alcohol. It would be interesting to know the behavior of other precipitants, such as the alkaloidal reagents, with respect to this point. It might also indicate whether the mechanism of precipitation by all alkaloidal reagents is the same or different. This has hitherto been regarded as identical.

In the following experiments attempts have been made to as- certain where precipitation of serum and glutin takes place with respect to their isoelectric points, and how it is influenced by differ- ent degrees of acidity and the addition of salt. The conditions under which precipitation was observed have been expressed in a quantitative manner; that is, definite quantities of serum and glutin of known protein content were mixed with definite quanti-

1 L. Michaelis and B. Mostunski: Biochem. Ztschr., xxiv, p. 79, 1910. 2 W. Pauli: Ztschr. j. Chem. u. Ind. d. Koll., xii, p. 222, 1913.

13

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14 Precipitation of Serum-Albumin and Glutin

ties of known strengths of acids, and to this, the same quantity of the reagent of known strength was added. The total volume of the mixture was always the same. The effect of salt was studied in a similar manner.

All experiments were performed at ordinary room temperature in test-tubes. The precipitates were observed when. freshly formed and at the end of twenty-four hours. However, it is to be noted that, as a rule, the precipitates remained unchanged on standing, and the data have been compiled without reference to t,his factor. Precipitation was regarded to have taken place when 6he mixture upon the addition of the reagent became non-trans- parent. The different degrees of precipitation have been expressed

TABLE I.*

Degree of acidity represented by acetic-acetate (“bufer”) mixtures.

A ‘ib - : IH+l 0.27 X lo-4t 1.8 X 10-S PIi I I 4.57 ’ 4.74

o.o;.i-. 1 o.45;;w 1 o.22;;Fs 1 ,zT+ *In the tables, the various signs and abbreviations used have meanings as follows: the plus

sign (+) = precipitate present; the minus sign (-) = no precipitate; sl = slight; st = strong; m&y = maximum; (?) = precipitate doubtful; tr = trace. The order in which the solutions were 6xed and the quantities used are appended aa footnotes to each table.

t Calculated.

in ordinary terms as indicated in the various tables. The reagents were previously rendered neutral to litmus by the addition of sodium hydroxide, and the strengths of these were usually 5 per cent. The horse serum and glutin used in these experiments had been previously dialyzed for a period of five to six weeks, and were practically salt-free. The end concentration represented in the different experiments will be found appended to each table. The standard “buffer” mixtures of acetic acid and sodium acetate were prepared according to Sorensen,3 and the degrees of acidity that. t,hey represent are shown in Table I. In my work the concen-

3 Sorenson: Ergebn. d. Physiol., xii, p. 393, 1912.

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Paul J. Hanzlik 15

tration of sodium acetate in all experiments is always the same, only the variation in the acetic acid being present. The different concentrations of acetic and hydrochloric acids were prepared by dilution of standard solutions in the usual manner, and the con- centrations of these are indicated in the various tables.

1. Precipitation of serum and glutin in mixtures of acetic acid and sodium acetate.

In mixtures of acetic acid and sodium acetate with proteins, the dissociated hydrogen ions are not bound by the protein, but

TABLE II.

Precipitation of serum and glutin by alkaloidal reagents in the acetic-acetate mixtures.

-

-

- S i - I- + + f

-

L

Pcm.4ss1UY MEBCURIC IODIDE, i PER CENT s lG + + Sl + + :: + /+ + /+ +q+ + 81 - -

_‘-

I

-

- I s - _- + : + 81 + -I-?

G I -I- + st + leas st + least

- - -

-

I

S _- + + + f msx + 81 + sl -

-

L

7

POTASSIUM BEIlRO-

CY.&.nDE, 5 PER CENl

S G __

+ st + st + +

TANNn-4, 1 PER CENT

S T + 81 + sl -t + St Z? - -

G

: +

+ + st +

“S” refers to horse serum; “G” to glutin. Order of mixing solutions: aceticgcetate mixture 1 cc.. water 1 CO., reagent 1 drop and serum (0.5 per cent) orglutin (0.5 per cent) 1 drop.

are made available for the precipitation reaction with the alka- loidal reagents. It is thus possible to ascertain if precipitation is concerned (1) with neutral particles of protein, (2) with positive protein ions, or (3) whether it depends upon the liberation of the free acid of the reagents. With this in view, experiments with horse serum and glutin were made according to the method de- scribed above, the details of which are appended to the results pre- sented in Table II.

From this it is to be seen that the precipitation of serum by the following reagents, iodine potassium iodide, potassium mercuric iodide, potassium ferrocyanide, sodium tungstate, and phos-

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16 Precipitation of Serum-Albumin and Glutin

phomolybdate, takes place above the isoelectric point, and in the direction of higher concentrations of acetic acid, and that it in- creases as the acidity increases. Precipitation by the different reagents begins practically with the same mixture of acid and acetate, and the small variations which occur are probably within experimental error. Inasmuch as the dissociated acid is not bound to the protein ion under these conditions, it would seem to indicate that a certain amount of free and excess of acid is neces- sary for the formation of the insoluble protein compounds.

On the other hand, with tannin, the maximum of precipitation occurs at about the isoelectric point, and then diminishes on either side of it; that is, with either increased or decreased concen- trations of free acid. This would indicate that the mechanism of precipitation by tannin is different from that of the other alka- loidal reagents used.

With glutin, precipitation takes practically the same course as with serum. No precipitation was observed with iodine and po- tassium iodide and the ferrocyanide. Tannin exhibits practically the same differences from the other reagents as with serum.

2. Precipitation of serum and glutin by alcohols in mixtures of acetic acid and acetate.

From the preceding section it appears that the mechanism of the precipitation of proteins by tannin is different from that with the other reagents. It is possible that the mechanism is similar to that of certain alcohols; for ethyl alcohol also produces a max- imum of precipitation at the isoelectric point. This analogy was tested out by observing the precipitation of serum and glutin by different alcohols with respect to the isoelectric point. The dat.a are presented in Table III.

It is seen that the maximum of precipitation of serum by t,he different alcohols used occurs at about the isoelectric point; i.e., with mixtures of one to two (3) and one to four (2) parts of acetic acid and acetate, respectively. With the exception of propyl alcohol, the precipitates diminish in intensity on either side of this point. There appeared to be no ‘difference in the pre- cipitates produced by propyl alcohol. It is also to be noted that

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Paul J. Hanzlik 17

TABLE III.

Precipitation of serum bu alcohols in the acetic-acetate mixtures.

4 + - ...................... f - ...................... 9 - ...................... l-5 1 .................... +? + ...................... + + ...................... + st + ....................... + st + ...................... + less st

1 iq . . . . . . . . . . . . . . . , . . . + still less st

Serum and water (equal parts). . . . . . . + least st / -

- -

-

+? + + St

+ St + less st

+ least st

+ least st

- I - - - - -

+ tr + St

+ less st + still less

st -I- tr

+ st + St

+ St

+ tr Order of mixing solutions: horse emurn (0.5 per cen?J 1 cc., acetic-acetate mixture 1. cc., and

alcoho! reagent 1 00.

serum alone without the acid-acetate mixture gave slight precip- itates with all alcohols except propyl alcohol. The data strongly indicate that the mechanism of precipitation by tannin more closely resembles the alcohols than the other alkaloidal reagents.

The results obtained with glutin (Table IV) lead to practically, the same conclusions.

TABLE IV.

Precipitation of glutin by alcohols in mixtures of acetic acid and acetate.

f - ...................... i ...................... +

+ less st - + + St + -I- i + St + + + v. sl -

m . . . . . . . . . . . . . . . . . . . . . 1 iI. . . . . . . .

1 j - I

7 . . . . + *. . . . . -

Order of mixing mlutiom: glutin (0.5 per cent) 0.5 cc., acetic-acetate mixture 0.5 CC., water 1 cc.. and alcohol 2 cc.

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18 Precipitation of Serum-Albumin and Glutin

3. Precipitation of serum and glutin in acids.

Thus far it is seen that the precipitation of serum and glutin by the various reagents occurs when acid protein is present; that is, it takes place in the presence of a concentration of hydrogen ions higher than the isoelectric point, and, apparently, is aug- mented when these are in excess. However, it still remains to be shown whether precipitation depends upon the liberation of the acid of the reagent, or the formation of protein salts with added acid and the reaction of these with the reagent.

According to Pauli, the dissociated hydrogen ions of such acids as acetic or hydrochloric are bound by protein with the formation of dissociated protein ions with positive electric charges. The number of these charges increases with the addition of more acid until all of the protein is, saturated. This occurs even in the lowest concentrations of acid. Such bound hydrogen ions do not become available for other reactions unless the quantity of protein remains constant and an excess of acid is added. If, then, precipitation in a mixture of protein and acid by a reagent occurs only during the phase of excess acid and not when protein ions only are pres- ent, the conclusion must be drawn that precipitation depends upon the presence of free acid and not upon dissociated protein ions alone; that is, the precipitate is a combination of the free acid of the salt with the protein or acid protein. This, indeed, is the case with iodine potassium iodide, mercuric iodide, ferrocyanide, tung- state, and phosphomolybdate, and to a certain extent with tannin.

The experiments were performed by adding small and constant quantities of serum and glutin to different concentrations of acetic and hydrochloric acids and noting where precipitation occurred by the further addition of constant quantities of different reagents. The data obtained, as well as the details in which the experiments were performed, are presented in Table V.

It is seen that in the lowest concentrations of both acids, where dissociated protein ions occur, no precipitation of serum with any of the reagents took place. Precipitation occurred only with the higher concentrations; that is, in the presence of an excess of acid. The beginning of precipitation with the different reagents varies, and small but negligible variations are to be noted in the same reagent with different acids. With tannin, a maximum of pre-

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Paul J. Hanzlik I9

TABLE V.

Precipitation of serum and glutin by alkaloidal reaaents in dilute acids.

+? + 81

;:

- -

-

+ 81 + 81 +

-

-

-

+ 81 + +

-I-- I- I-. - - - I- - - fsl - - - + +? - - +st + - - + St -k? - - +1ess st + ? - fsl +1essst - -+s1+ - +s1 +1essst + - + + St +kast -

5.10-6 7.5.1w lO.lO-5 1.5.10-r 2.5.1W 4.10-r 5.10-4 10.10-C 1.25.10-1 2.5.103 5.10-r

7.5.10-s lO.lO-6 1.5.10-r 2.5.10-r 5.10-4 lO.lW 1.25.10-s

2.5.10-Z

5.1w

- -- - -- - -- + - : ++ + + + + + 1st + + + + : ++ f, + +

-

-

-

-

-

-.

-

-

+ sl

-

+ sl

- -- - -- - -- - f- + - + : - + f-

+ + +s1

+ + +s1

- + +s1 + +more + + still + more

+.&II + more

+

- _- - _-

fsl - -

+I31 - -

fmore - +s1

Order of mixing ~011 Iti ions: acid I cc., water 1 cc., glutin or home serum (0.5 per Cent) 1 CC., and reagent 1 drop.

cipitation in both acids occurred at the concentration of 2.5.10 -4. In the higher concentrations the precipitates dissolved. This was also true of iodine and potassium iodide, which at no time gave absolutely distinct precipitates. This phenomenon perhaps de- pends upon the formation of soluble acid salts and requires further investigation.

With glutin, practically the same results were obtained as with serum.

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20 Precipitation of Serum:Albumin and Glutin

It is to be concluded that the precipitation of serum and glutin in hydrochloric and acetic acids by iodine potassium iodide, mer- curic iodide, ferrocyanide, tungstate, and phosphomolybdate, takes place only in the presence of an excess of free acid and depends upon the formation of insoluble compounds with the free acid of the salt.

4. The e$ect of salts on the prec&tation of serum and glutin by alkaloidal reagents.

The addition of salt to an acid might conceivably alter the pre- cipitation reaction by alkaloidal reagents and alcohols. This was studied in the following manner: To constant and definite con- centrations of glutin and serum in the same volumes of acetic and hydrochloric acids of different concentrations were added con- stant volumes of potassium chloride and potassium sulphocyanide, and finally to this mixture constant and definite quantities of the reagents. Each series of experiments with the different salts was compared with a series without salt; that is, with distilled water as a blank. The results from all of the experiments are to be found in Tables VI, VII, and VIII.

From these data it is seen that the two salts, chloride and sul- phocyanide, exerted practically no influence upon the formation of the precipitate by the various reagents used. Practically no precipitation occurred in any ca.se until the concentration of 4. 10m4 was reached. The small variations which occurred fall within the experimental error. This was true of both serum and glutin.

With tannin, precipitation in the presence of the chloride and sulphocyanide occurred in the lowest concentrations of the hydro- chloric acid and before 4.10 -4. Thus a small difference from the other reagents is indicated.

With alcohols, the addition of salts also had practically no in- fluence upon the precipitation. The slight variations exhibited by the sulphocyanide are too small to permit the drawing of any conclusions. The same can be said of glutin in acetic acid-ace- tate mixtures (Table IX).

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TABL

E VI

.

Effec

t of

salts

on

the

pr

ecipi

tation

of

seru

m by

alk

aloida

l re

agen

ts.

I -

RIO

/ I- + + + + +

*t

+ + +

.- -

: + + + *t

+ + +

---

:sm

- - - +?

+ + + + + - - - -

BODI

UY

PHO

S-

PxKn

!“NGs

T*TE

, 5

PER

CENT

- I TA

NNIN

, 1

PER

CENT

-- i

+ i

+

+ I

+ +

i +

+ 1

+

KC1

-.- - - : + + + +

_

- + + + + : +

Hz0

+ 81

+ +

St

+ St

+

less

st +

still

less

8 +

still

less

s +

still

lee.

7 8

+ le

as

st

+ 51

+ +

st +

St

+ le

as

st +

less

st

: + 81

KC1

3 _ + + + :*I

-

! -

I - +

sl i- + + + 18

1 -I-

? -

CSCN

- + + + + st

+ St

+ + - - -t-

+ + st

+ st

+ + sl

HZ0 - - + : + + + + - - - + 1

---

KC1 - +

sl

+ sl

+ + i- - - - - + +

HZ0

Hz0

KC1 - - + 81

+ + -t’

+ st

3?

I-

-i .- I

KC1 - - + + +?

+ + - - - - + -L? -

- - :s

cn

;SCN

KC

1 CS

CN

;SCN

- - - + *l

+ + + + *t

I

I ~.

--

1 .- t t t t _-

-

- - + sl

+ *I

+ + + + sl

4-T

+ -+ + + sl

+ s’

+

- - - - + sl

+ + + st

- +*

+ 8’

+ - - - -t?

+ *I

:; - - - -

2.5.

10-h

4.

10-r

5.10

-4

10.1

0-4

1.25

.1~3

z 2.

5.10

-3

5.10

-a

WCE

TIC

*a

7.5.

10-s

10

.10-

S 1.

5.10

-4

2.5.

10-d

4.

1cw

5.lcP

’

lO.llv

1.25

.10-

3 2.

5.10

-s

5.lck

3

D I-

- - - - - +s +* +S

- - - + 8’

: -L

Or

der

of

mixi

ng

solu

tions

: ac

id

1 cc

.. $,

sa

lt so

lutio

n (K

C1

or K

SCN)

1

cc.,

hors

e se

rum

(0

.5

per

cent

) 1

drop

. an

d re

agen

t 1

drop

.

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TABL

E VI

I.

Effec

t of

salts

on

the

pr

ecipi

tation

of

utin

bv

alkalo

idal

reag

ents.

KC1

1SCN

KC

1 KC

1 KS

CN

+ 81

+

81

+

--I

--

--

-!-

KCI

KC1

KC1

2.10

-a

1 .lO

W

2.10

-s

7.5.

10-G

10

.10-

S 1.

5.10

-4

2.5.

104

5.10

-a

10.1

0-r

1.25

.10-

8 k

2.5.

10-s

5.

10-a

-

- +

81

+ sl

-I?

+

- + 81

- - - - + sl

- - 4-1

_-

.- 2.

10-s

1.

10-S

2.

104

7.5.

10-6

10

.10-

6 1.

5.1w

2.

5.10

-a

5.10

-4

lO.lO

- 4

1.25

.10-

s 2.

5.10

-a

5.10

-1

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- +

81

- -

+ 81

+

81

+ -

- -

- I

+ 81

+

81

+ +

81

+ sl

+ sl

- -

- __

Or

der

of m

ixing

eo

lutio

ns:

acid

1

cc.,

wate

r or

+?

salt

(KC1

or

KS

CN)

solu

tion

1 cc

., gl

utin

(0

.5 p

er

cent

) 1

drop

, an

d re

agen

t 1

drop

.

Ha0

+ + : 1 + + + + + +?

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Paul J. Hanzlik

TABLE VIII.

Effect of salts on the precipitation of serum and glutin by alcohols.

i ‘I -i- I:

END CONcENTRdTION

cm HCl 8

3 P i -

+ + - - -

8

1.25.W’ - - - 0.2.10-4 - - - +a1 0.3.10-r - - +a1 0.4.1~’ +s1 - + sl f? + 81 fsl - 1.25.10-4 + +st - + St + + St i-at - 0.62.10-a +a1 + - - - f? - 1.25.10-a - + - - -

0.62.W* - - - - - - - 1.25.10-2 - - - - - - 0.62.10-’ - - - - - - - 1.25.W-1 - - - - - -

_~ Acmxc ACID

0.5.10-4 - - 1.25.10-r - + 81 + st - 0.62.10-8 + sl +? + 1.25.1W + :1 ::i

+

0.62.10-* - - - - 1.25.10-o + - 0.62.lW - - 1.25.10-’ - -

order of mixing mlutions: acid 0.5 cc.. glutin (0.5 per cent) 0.5 cc., water or N/5 ssk (KC1 or KSCN) solution 1 cc., and alcohol reagent 2 cc.

TABLE IX.

E$ect of sulphoqanide on the precipitation of glutin by alcohols in acetic- acetate mixtures.

N/10 *.CETIC *CID BESORCIN,~ PHENOL, HYDROOUWONE, PBOPYL 5 PEB CENT 5 PER CENT 5 PER CENT d‘coHOL

N/10 SODImd *cJvlwm Hz0 KSCN Ha0 KSCN Hz0 KSCN I&O KSCN _________~~---

t . . . . . . . . . . . . . . . . . . . . . . +s1 +st - + - + + - i . . . . . . . . . . . . . . . . . . . + st + St’ + st + + st + sl + st + ? + . . . . . + st + sl + st - - - +st +? * . . . . . . . . .._.__........ +s1 - - - - - + +,.. . - . - - - - - - 1; $+i . . . . . . . . . . .._...._.. - - - - - - - +?

Order of mixing solutions: 2 per cent glutin (1:4) 0.5 DC., acetic-acetate mixture 0.5 cc., : KSCN 1 OC,, and alcohol 2 cc. The end concentration of KSCN is &; of glutin 0.5 per cent.

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24 Precipitation of Serum-Albumin and Glutin

5. E$ect of concentration of protein on the precipitation.

This was tested out by using two different concentrations of serum, 0.017 per cent and 1 per cent (as end concentrations of protein) in constant volumes of hydrochloric acid of different strengths and precipitated by the further addition of one drop of t,he reagent. The data have been placed in Table X.

As the results with the different concentrations of serum are practically identical, it is to be concluded that wide differences in protein content have no marked influence on the precipitation.

TARLEl X.

Ejtfect of concentration of serum-albumin on the precipitation by

_-

7.5.10;s 1O.W-’ 1.5.10-4 2.5j.10-4 5.104 ld.iW 1.3.10-3 2.5.10-a 5.104

I

-

T

.017 %,

-

-

+ +

I I

-

- - + sl + + + -

-- - /- -- _ - ++ - - ++ - - + +s1 - I- + +st - i- + +st +s1!- + + st + sl + sl

” j+st +st~+sy ,I

-- i- -

I- -

- -

+ 81 + 81 + 81 +st + + st max +

Order of mixing solutions: horse serum 1 cc., acid 1 cc., and reagent 1 drop. The per- centages under “serum-albumin” represent end concentrations of albumin.

i

_. 1

% 0.017 1

% %

SUMMARY.

The mechanism of the precipitation of dialyzed horse serum and glutin,by tannin is different from that of certain precipitants com- monly known ?s “alkaloidal reagents.” With these a certain amount of free acid (hydrogen ion concentration) is necessary for bhe formation of the complex insoluble compounds.

Tannin behaves like certain alcohols, e.g., resorcin, phenol, hydroquinone, and propyl alcohol, since the maximum of precip- itation in both cases corresponds to the isoelectric point in serum- albumin and glutin.

Precipitation of serum is uninfluenced by wide differences of concentration and the addition of such neutral salts as chloride and sulphocyanide.

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Paul J. HanzlikREAGENTS

AND GLUTIN BY ALKALOIDAL PRECIPITATION OF SERUM-ALBUMIN

1915, 20:13-24.J. Biol. Chem. 

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