A STUDY OF THE BIHDIHO BETWEEN SULFADIAZINE
AND SERUM PROTEINS

By
Leonard A. Mattauo
Fellow, National Institute of Health

A THESIS
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of
DOCTOR OP PHILOSOPHY

Department of Bacteriology and Public Health

I9U8

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A Study of the Binding Between Sulfadiazine
and Serum Proteins

I.
II.

Introduotion...................... .

IV.
V.
VI.

. .........1

Experiments and Results.......... ............... ........... .3
A.

Bactericidal aotivity................................3

B.

Isolation and idontifioation of serum proteins. . . . . . . 6

C.
III.

........ .

1.

Precipitation between pH 9*2 and ?.0.............6

2.

Crystal formation at pH 9*2................... ..2L{.

3.

Precipitation at pH i|,.0.........................29

Solubility of sulfadiazine..........................35

Discuss ion.......
Summary.

...*..38
................. .

Acknowledgment ...*......... ......... .

..i|7

Literature Cited....................................

..1)0

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A STUDY OF THE BINDING BETWEEN SULFADIAZINE
AND SERUM PROTEINS

The results of a large number of experimenbs conducted in this
laboratory have shown that serum proteins rwaoved by acid precipit­
ation of sulfadiazine from a solution of sodium sulfadiazine and
bovine Brucella anti-serum have bactericidal properties against
Brucella organisms in the presence of complement and traces of sodium
sulfadiazine.

In vitro experiments have shown that the drug in

dilutions of 1 *10° to 1 :10° in liquid culture medium retard growth
for only a 2!+ hour periodo

In view of the differences in the growth

inhibiting of the drug alone and in the presence of antibody and
complement, it became of interest in this laboratory to investigate
the binding between the drug and the various protein components of
serum.

An investigation of this nature may serve to throw light

upon probable "reactions” between sulfa compounds and blood serum
constituents when these drugs are used as therapeutic agents in humem
and animal bacterial diseases.
Studies of this problem strongly indicate that sulfadiazine
binds mainly with the globulin fraction of serum in an alkaline range
and that the binding is through the free amino groups of the proteins.
Practically no binding occurs with the free carboxyl groups of the
protein.

Evidence has also been obtained that reaction of sulfadia­

zine with serum in an acid range results in binding of the drug to
the albumin fraction,
Davis (6 , 7» 8 ) had demonstrated that sulfathiazole binds with

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-2-

the albumin fraction of (NH*)g SO4 separated human serum and that
there is practically no binding to the globulin fraction at pH
The conditions under -which his experiments were conducted differ
from the conditions used in this laboratory.

Davis used

SO4,

separated serum, -whereas the results obtained in this laboratory
were from whole serum.

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“5”

EXPERIMENTS AND RESULTS

A.

Baoterloidal activity.
The addition of dilute phosphoric acid to a clear solution of

sodium sulfadiazine dissolved in bovine serum results in the pre­
cipitation of a white, paste-like substance.

Examination under a

microscope shows that the precipitate is of a crystalline structure,
but this structure differs from the crystalline structure of pure
sulfadiazine freshly precipitated from an aqueous solution of sodium
sulfadiazine with dilute phosphoric acid.

Investigation of this

paste-like precipitate revealed that serum proteins are removed from
the solution with the precipitated sulfadiazine.
Protein thus removed from a Brucella anti-serum has very high
bactericidal activity against Brucella organisms in the presence of
fresh normal rabbit serum to serve as complement and traces of
sodium sulfadiazine (9).
in the following fashion:

This anti-serum protein fraction is prepared
1 .0 gram of sodium sulfadiazine is dis­

solved in 5*0 ml. of Brucella anti-serum,
with a pH of about 9»2,

A clear solution results

Dilute phosphoric acid (*15N) is added

dropwise while the solution is stirred with a mechanical stirrer.
Precipitation occurs immediately with the first few drops of the acid.
Addition of the acid is continued until the pH of the solution is
lowered to pH 7*0,

The solution is filtered and the precipitate

washed with 5 to 6 portions of .86^ saline*

The precipitate is

dispersed in a small amount of saline and .IN sodium hydroxide is
added dropwise to the well stirred slurry.

The precipitate grad­

ually dissolves and at pH 10,0 a clear, pale yellow solution usually
results.

The solution is dialyzed against physiological saline at

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l4.“C. to remove the sodium sulfadiazine.
A representative sample which was used for a bactericidal test
was prepared as given in the procedure above.

The dialysate was

filtered through a D8 Hermann sterilizing pad in a Seitz filter.
The concentration of the sodium sulfadiazine, as determined by the
Bratton and Marshall (3) method, was found to be 8,0 mg/ml.

The

protein nitrogen content (semi-micro Ejeldahl) was ,53 mg/ml.

Two

series of two-fold dilutions were made of the sterile solution in
5 ml. amounts of fortified tryptose medium in test tubes.

The

fortified tryptose medium contains tryptose, glucose, thiamine hydro­
chloride and sodium chloride.
indicated in Table I.

The concentration in each dilution is

To each dilution of one series, 0.2 ml. of

fresh, normal rabbit serum was added as complement in order to meas­
ure the activity of the protein nitrogen.

Each dilution in both

series was then inoculated with 10® Bruoalia abortus cells.

The

tubes were shaken and incubated at 37* 0 . for 72 hours.
It will be noted from the results that the solution containing
only sodium sulfadiazine and the anti-serum protein fraction had
only a temporary inhibiting effect on the growth of the organisms.
The maximum inhibiting dilution was no higher than that of sodium
sulfadiazine alone.

The addition of complement to the dilutions of

the anti-serum protein fraction and sulfadiazine created a bacteri­
cidal complex which caused the death of the organisms in all the
dilutions except the last.

As will be demonstrated later, the major

portion of this anti-serum protein fraction contains a high per­
centage ofy-globulln, and, therefore, contains Brucella antibodies.

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Table I
Bactericidal action of Wa-oulfadiazine and protein removed from Brucella anti-serum*

8

"O
(O '

3"

i3
CD

3
3.
"

Complsinent
added
to each
tube (*•)

Concentration of agents in tubes, % 10
I'laSD
Protein Nitrogen

80

ho

20

3 .3

1 .6

0 .8

10

o,h

jEr7"m§&I#i

5

2 .5

0 .2

0 .1

Bacteria
control

1 .2 5

0 .6 2

0 .0 5

0.0 2 5

5 ml. of
medium

CD

Incubation
period
hours

CD

■D
O
Q .
C
a
O
3
■D
O

Degree of gravth, turbidity

2h

-

-

-

-

-

~

-

1+

2.

72

-

-

-

-

“

-

-

2+

6+

2)4

-

-

-

-

-

1+

2+

2+

2+

72

1|+

h*

L+

h^

4+

6+

6+

6+

0,2 ml.

CD
Q .

None
■D
CD

(/)
o'

3

10®
(a)
(b)
- =

Br* abortus added to each dilution and controls.
Fresh, normal rabbit serum*
0*2 ml* of fresh, norroal rabbit serum added to control tube,
no visible growth* + = degree of grmvth.

-6-

B.

Isolation and identifioation of serum proteins.
1«

Precipitation between pH 9*2 and 7.0.
Because of the bactericidal effect of the mixture of the protein

fraction, sodium sulfadiazine and complement, it become of interest
to determine the nature of the proteins removed with the precipita­
tion of the sulfadiazine, as discussed above.
Preliminary work on the problem was to investigate the quantity
of protein removed with varying portions of sodium sulfadiazine
dissolved in 5*0 ml. of the serum.

A series of samples was set up

using 0 .2 gms., 0 .5 gms., 0 .6 gms., 1 .2 5 gms., 1 .7 5 gms*, and 2 .2 5
gms. each of which was dissolved in 5 .0 ml. of serum.

Dilute (.15N)

phosphoric acid was added dropwise to the agitated solutions.

The

pH of each solution was gradually lowered from the initial pH of
9.2 to pH 7*0»

The samples were filtered through 2-^1 'Whatman filter

papers placed in a Buohner Funnel.
several times with

,Q6% saline

The precipitates were rinsed

to remove the soluble substances.

They were then removed from the filter papers, dispersed in saline
and re-filtered and rinsed.

The precipitates were placed in the

original beakers and dissolved while agitated with .1 N sodium
hydroxide to

10.0,

The resulting solutions were clear or slight­

ly opalescent and pale yellow.
as possible.

This work was done as quantitatively

The alkaline solutions were placed in cellophane tubing

and dialyzed at i4.*C. against ,869^ saline for several days with daily
saline changes to completely remove the sodium sulfadiazine.

The

sodium sulfadiazine was removed so that the protein nitrogen value
determined by semi-micro Kjeldahl method was direct and not by
difference since sodium sulfadiazine contains 20.6^ nitrogen.

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After

-7-

dialyais the volume of eaoh solution was measured, a semi-mioro
Kjeldahl detemination made, and the presence of sodium sulfadiasine
checked by the Bratton, and Marshall procedure*

The total nitrogen

value was calculated on the basis of the total volume of the
solution and then multiplied by 6.S5 to convert to grams of protein.
The protein removed for eaoh weight of sodium sulfadiazine used is
recorded in Tables II, 11^ and IT.
used;

Three different bovine sera were

(8 . ^ protein) contained Brucella agglutinins in a titer

of 1 *10,000, #lf,|l).l (7 *5^ protein) came from an immunized cow of a
low agglutinin titer, and
infected cow*

(6 *9^ protein) was from a non-

More protein was removed from #991 because of the

higher y -globulin content present in this serum.

The reason for

this conclusion will become more apparent later.

Pigs* 1, 2, and 3

illustrate graphically the results given in the tables mentioned
above.

The curves in eaoh case show a steep slope up to about 0*8

gram sodium sulfadiazine*

In faot, this portion of the curve is

almost a straight line.
With eaoh drop of dilute phosphoric acid that is added to a
solution of sodium sulfadiazine and serum, sulfadiazine precipitates
and also removes protein from the serum*
gradually decreases*

The pH of the solution

Three samples of 1*0 gram sodium sulfadiazine

and 5*0 ml. of #991 serum were set up to determine the amount of
protein removed by sulfadiazine with the addition of dilute phos­
phoric acid when the final pH of each solution was 8*5» 8*0, and
7*5 respectively*

The procedure was essentially as given above.

The data in Table 7 illustrate that the quantity of protein nitrogen
removed varies with the final pH of the solution.

The ratio of

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—8«

Table II
Protein removed from a Brucella anti-serum (No. 991
bovine) during precipitation of sulfadiazine.

1

2

3

k

5

6

Ml. serum 8.5
per cent protein

5.0

5.0

5.0

5.0

5.0

5 .0

Gm. NaSD

0.20

0 .5 0

0.80

1.25

1.75

2 .2 5

Gm. protein removed

O.OUé

0 .1 1 0

0.163

0.209

0 .2J49

0 .2 5 2

Sample No.

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-9-

Table III
Protein removed from a bovine seinim (Eo. liii+l,
immunized) during precipitation of sulfadiazine,

Sample No.

1

2

5

h

5

6

5.0

5.0

5.0

5.0

5.0

5.0

Gm. NaSD

0.20

0 .5 0

0.80

1 .2 5

1.75

2.25

Gm. protein removed

0.01+5

0.097

0.1U2

0.171

0.180

0.197

Ml, serum 7*5
per cent protein

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—10“

Table IV
Protoin removed from a norziial bovine serum
(No. Ihh3) during precipitation of sulfadiazine.

1

2

3

Ml. serum
6 .9 per cent protein

5 .0

5 .0

5 .0

Gm. NaSD

1 .2 5

1 .7 5

2 .2 5

Gm. protein removed

0 .171+

0 .1 9 1

0 .2 0 1

Sample îTo.

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0.20

G
O
-P
O
G

•H

G
o

tn

0.10

0.5

1.0

1.5

Orama Sodium Sulfadiazine
Pig. 1. Serum protein removed from solutions of 5.0 ml. No*
991 serum with varying quantities of sodium sulfadiazine.

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umjGs Burea(}

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

0.25

0.2

2

t
Iu

0.151

o

cn

m

05

0

1.5
1.0
Grams Sodium Sulfadiazine

2.0

2.6

Fig. 3. Serum protein removed from solutions of 5.0 ml. No.
1443 serum with varying quantities of sodium sulfadiazine.

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-11-

mg, îîa SD ppte'd

is quite oonatant.

One mg. of sodium sulfa-

diasino precipitated removes on the average .0)3 mg. of protein
nitrogen.

The results are illustrated graphically in Fig. it.

The

graph of serum protein nitrogen removed plotted against pH forms a
smooth ourve and shows clearly that the serum protein nitrogen re­
moved is a function of the final pH.
Identification of the components of the total protein removed
by the addition of acid to a. solution sulfadiazine and serum was
determined electrophoretically with the aid of the Tiselius (19)
apparatus as modified by Longsworth and mclnnea (i))*

In order to

obtain sufficient serum protein for eaoh determination, gO.O ml.
samples of serum wore used*

To separate 5^.0 ml. samples of 1^^991

serum was added 5 .0, 8 .0 , 12.5, and 22«5 grams of sodium sulfadiazine.
Each sample was treated with dilute (.15N) phosphoric acid to pH
7.0, filtered, and the precipitate rinsed with .86^ saline.
filtrates were saved.

The

The precipitates were dissolved with .IN

sodium hydroxide to pH 10*0,

The resulting solutions were dialyzed

at ^"C. against 10 liters of .86^ saline for 9 days with daily
saline changes to remove the sodium sulfadiazine.

To increase the

protein percentage of the solutions to slightly above 2 .0^ for
electrophoresis studies, the solutions were concentrated by rapid
evaporation in cellophane tubing under an over-head fan at 37*C.
After evaporation, sodium sulfadiazine and nitrogen determinations
were made.

The samples which had been treated with 12.5

22,5

mgs, of sodium sulfadiazine were diluted to 2 .0^ with barbital buffer
of pH 8 .6 and ,1 ionic strength and equilibrated by dialysis against
this buffer.

The other two samples which had been treated with 5*0

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•>12—

Table V
Protoin removed from No. 991 serum during the precipitation of
sulfadiazine between the pH figures as indicated in the table.

Sample No.

1

2

5

Ml. serum

5.0

5.0

5 .0

Gm. NaSD

1-0

1.0

1 .0

Initial pH

9.2

9.2

9.2

Final pH

8.5

8 .0

7 .5

578.7

7 5 0 J:

7 7 7 .0

18.0

2 3 .5

2 7 .0

NaSD content, mg.
Protein N, mg.
mg. protein N
1.0 mg. NaSD

.0 3 1

.0 3 2

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.0 3 5

CD

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(
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0.25
CD

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0.02

3"
.
3
CD

f

H
%

0.019
CD

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3
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i

0.01

CD

Q.

■D

0.005

CD

(
(/
/)
)

9.8

9.0

8.0

8.5
PH

Flg, 4 . Sérum protein nitrogen removed from solutions of 5.0
ml. No. 991 serum and 1.0 gm. sodium sulfadiazine by t’
.ie pre­
cipitation of sulfadiazine from tne iiitial pH 9.2 to fc.5, 8.0
and 7.5 respectively.

7.5

-13-

and 8*0 grams sodium sulfadiazine respectively were discarded after
the total protein nitrogen removed had been determined.
Sodium sulfadiazine was determined from the filtrate solutions
and was found to vary from 0,20 to 0*2i). grams in each of the four
filtrates.

This means that the precipitation of the sulfadiazine

from its sodium salt is practically 100^ at pH 7*0.

The filtrates

for electrophoretic studies were prepared and treated in the same
way as the precipitate solutions.

Electrophoretic scanning patterns

(13) were made on all of the filtrates.
In Table VI are recorded the grams of protein removed in the
precipitate and the grams of protein that remained in the filtrate*
The recovery of total proteins as compared to proteins originally
present in the

ml, of serum varied from 9 0 ,6 to 9^,3^.

Prob­

able losses occurred in the precipitate, washings of the precipitate,
losses in the dialysing tubing, etc.

The shape of the curves in

Fig. 5 shows that protein is removed proportionately to the quantity
of sodium sulfadiazine used*
The electrophoretic patterns furnish a visual picture of protein
components that are removed during the precipitation of the sulfa­
diazine from its sodium salt with acid, and the protein components
that remain behind in the filtrate.

By comparing patterns of the

filtrate with that of the original untreated serum it is obvious
that the precipitation of the sulfadiazine removes protein mainly
from the globulin region, especially the y-globulin.

This is more

evident in the patterns of the proteins removed vrf.th the precipitate.
Figs, 6, 7» fi^nd 8 demonstrate graphically the results recorded in
Tables VI and VIII-XIV, which were calculated from the descending

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-llt-

Table VI
Protein removed from a Brucella anti-serun
(No. 991 bovine) during precipitation of sulfadiazine

Sample No.
Ml, serum

1
5 0 .0

Gm. protein in serum

2
5 0 .0

I+.2L

h

3
5 0 .0

5 0 .0

h>2)4

If.2)4

Gm. NaSD

5 .0

8.0

Gm. protein removed
■with ppte.

0.99

1.33

1 .7 5

2 .i;0

Gm. protein in filtrate

2.99

2.52

2 .1 3

1.L3

Per cent recovery

91.0

1 2 .5

91.6

2 2 ,5

90.6

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4 .5

4.0

3.5

3.0

filtrat*
fi

m

2.5

u

0

o*

1

CoO

2.0

ê

1.0

0.5

0

5.0

10.0
15.0
Crama Sodium Sulfadiazine

20.0

26.0

Pig. 5. Serum protein removed and serum protein in the filtrate
from solutions of 50.0 ml, lîo. 991 serum and varying quantities
of sodium sulfadiazine.

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-15-

eleotrophoretio patterns.

The ounres in Pig. 6, drawn from the

data obtained from the filtrates, represent the manner in whioh the
protein components of the aemra remain in the filtrate with increas­
ing amounts of sodium sulfadiazine.

The y-globulin curve has the

steepest slope, thus demonstrating that the sulfadiazine precipitate
removes Y "globulin in preference to the other protein ocmponents in
the serum.

The a and P-globulin curves show that these components

are also removed, but not nearly as rapid as the Y-globulin.
albumin curve has a peculiar shape.

The

The curve starts with a rather

steep slope, then tends to straighten out somewhat, and finally,
beyond 8.0 grams of sodium sulfadiazine has a slope about as steep
as the Y-globulin curve.

Figs. li| and 15 also show that the albumin

content in the precipitate increases as the sodium sulfadiazine is
increased from 12.5 grams to 22.5 grams.

This can also bo seen from

the albumin curve in Pig. 8, in whioh the grams of albumin in the
precipitate was plotted against grams of sodium sulfadiazine.

The

a, P and y-globulin components present in precipitate were added to­
gether and plotted as shown in Fig, 8.

In comparison, the globulins,

mainly the Y-globulin, are removed from the serum to a much greater
extent than the albumin fraction.

The electrophoretic pattern.

Pig. 9* illustrates the original No. 991 serum before treatment
with sodium sulfadiazine.

The electrophoretic patterns of the

filtrates. Pigs. 10, 11, 12, and 13 show a progressive decrease in
the globulin region.
A similar set of results were obtained from the use of ^1?4|.3
serum.
those of

The curves in Pig. l6 and 1? have the same general trend as

499^ serum.

It is of interest to note that the intersection

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-16-

Table VII
Protein removed from a normal bovine serum
(No. 1/443) during precipitation of sulfadiazine.

Sample No.
Ml. serum

1
5 0 .0

2

3

4

5 0 .0

50-O

5 0 ,0
3 .4 3

Gm. protein in serum

3J^3

3.43

Gm. NaSD

5 .0

8 .0

protein removed
with ppte.

0.97

1.21

1 .5 0

1 .9 5

Gm. protein in filtrate

2 .3 7

2 .0 6

1.77

1 .2 4

Per cent recovery

97.2

9 5 .4

3 .4 5
1 2 .5

9 5 .4

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2 2 .5

9 3 .0

-17-

Table VIII

Data obtained from calculation of descending
electrophoretic pattern. Fig. 9* No. 99^ untreated serum.

Serum protein
Per cent from pattern
Gm. protein/ 5 0 ml. serum

A

a

57.2

li+.O

1 .5 8

0 .5 9

P
9 .9
0 .4 2

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Y
58.9
1 .6 5

-18.

Table DC
Data obtained from calculation of descending
electrophoretic pattern of filtrate. Fig. 10.

Serum protein
Per cent from pattern
Gm. protein
Per cent based on
90.0 ml. serum

A

a

P

Y

h 5-5

8.5

9.8

36.2

0.26

0.29

6.0

6.9

1.36
32.1

1.03
25.5

Filtrate from 50 ml. No. 991 serum treated with 5»0 gm. NaSD.
Protein in filtrate = 2.99 gm. or ? 0.6 per cent protein remained
in filtrate.

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-19-

Table X
Data obtained from calculation of descending
electrophoretic pattern of filtrate. Fig. 11.

Serum protein
Per cent from pattern
Gm. protein
Per cent based on
50.0 ml. serum

A

a

P

Y

52.2

7 .U

9.5

50.9

0.19

0.21;

1.52
31.1

5.6

0.79
16.U

Filtrate from $0 ml. No. 991 serum treated with 8.0 gm.
NaSD. Protein in filtrate = 2,52 gm. or 59»5 per cent
protein remained in filtrate.

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-20-

Table XI
Data obtained from calculation of descending
electrophoretic pattern of filtrate. Fig. 12,

Serum protein
Per cent from pattern
Gm. protein
Per cent based on
50.0 ml. serum

A

a

p

Y

57.5

7.1

8.5

26.9

0,15

0.18

3.5

i+*3

1.22

28.9

0.57
13.5

Filtrate from $0 ml. Ko. 991 serum treated with 12.5 gni. NaSD,
Protein in filtrate = 2.13 gm, or 50-3 per cent protein remained
in filtrate.

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-21-

Table XII
Data obtained from calculation of descending
electrophoretic pattern of filtrate. Fig, 13,

Serum protein
Per cent from pattern
Gm. protein
Per cent based on
50.0 ml. serum

A
65.1
0.90
21.3

a

P

Y

6.9

9.8

20.2

0.10

0.14

0.29

2.5

5.5

6.8

Filtrate from ^0 ml. No. 991 serum treated with 22,5 gm.
NaSD. Protein in filtrate = l.i-i-5 gm. or 55.9 per cent
protein remained in filtrate.

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-22-

Table XIII
Data obtained from calculation of descending
electrophoretic pattern of precipitate. Fig. li|..

Serum protein
Par cent from pattern

A

10.0

Gm. protein

0.17

Per cent based on
50 ml. serum

If.l

a,(3,

and y

90.0
1.58
57.2

Precipitate from 50 ml. No. 991 serun treated with 12.5 gm.
NaSD. Protein in precipitate = 1.75 gm. or i-i-1.3 per cent
of the protein in the serum v;as removed.

R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.

"23—

Table XIV
Düba ouLuineu froiL calculation of descending
electrophoretic pattern of precipitate. Fig. I5.

Serum protein
Per cent from pattern
Qm. protein
Per cent based on
50 ml. serum

A
16.5
O.i^O

a,

P,

and

Y

83.5
2.00
U 7..3

Precipitate from $0 ml. No. 991 serum treated with 22.5 gm.
NaSD. Protein in precipitate = 2 .1|. gm. or 56 .7 per cent of
the protein in the serum was removed.

R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.

CD

■D
O

Q.
C

g

Q.

■D
CD

2.0
C/)
C/)

CD

8

■D
(O '

1.5

3
3.
"
CD

1.0

CD

■D
O
Q.
C
a
O
3
"O

4>
CO
0}

o

CD

Q.

0.5

■D
CD

(
(/
/)
)

TÏÏ7Ô

IF T o

2070

25.0

Grama Sodium Sulfadiazine
Pig. 6. Serum protein components in the filtrates from solutions
of 50.0 ml. No. 991 serum and varying quantities of sodium sulfa­
diazine.

3.0

2.5

2.0

o

o

L

CL,

1.5

- and Y" globulins

k
o
1.0

0.5

TÔ7Ô

TSTÔ

20.0

25.0

Grams Serum Sulfadiazine
Pig. 7. a-, p- and y-globullns In the filtrates from No. 991
serum plotted against varying quantities of sodium
sulfadiazine.

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2.5

a -, p-and v" '.lobuiliis
2.0

1.5
O

u
IX,

t.
/
1.0

u

o

0.5

albumin

10.0

15.0

20.0

25.0

Grams Sodium Sulfadiazine
Fig. 8. a-, p- and y-globulins and albumin removed from
Solutions of 50.0 ml. No. 9y 1 serum with, varying quantities of
sodium sulfadiazine.

R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.

M /

R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.

Pig. 10. Composition of filtrate from a solution
of 30 ml. No. 991 serum and $.0 gm. sodium
sulfadiazine after precipitation of sulfadiazine.
Electrophoresis for 9»000 seconds at 5*77 volts
per om. Protein cono. 2 per owt.

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A

Fig, 11, Composition of filtrate from a
solution of 50 ml. No, 991 serum and 8,0
gm, sodium sulfadiazine after precipitation
of sulfadiazine. Electrophoresis for 9*000
seconds at 5*9^ volts per cm. Protein oonc,
2 per cent.

R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.

h

■ = * d i

Flr\ 12. Composition of filtrat© from a
solution of 50 m l . No. 991 sérum and 12.F
gm. sodium sulfadiazine after precipitation
of sulfadiazine. Electrophoresis for 9*000
seconds at 5*S3 volts per cm. Protein o o n c .
2 per cent.

R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.

F

Fig* 13* Composition of filtrate from a
solution of 50 ml- No. 991 serum and 22.3 gm.
sodium sulfadiazine after precipitation of
sulfadiazine. Electrophoresis for 9»000
seconds at 3.88 volts per cm. Protein
cono. 2 per cent.

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r,i*. «

Fig. 111. Composition of protein fraction
fron' a solution of 50 ml. No. 991 serum
and 12.5 gm. sodium sulfadiazine removed
by precipitation of sulfadiazine.
Electrophoresis for 9,000 seconds at 5.80
volts per cm. Protein cono. 2 per cent.

Fit*. 15. Composition of protein fraction
frcm a solution of 50 ml. No. 991 serum
and 22.5 gm. sodium sulfadiazine removed
by precipitation of sulfadiazine. Elect­
rophoresis for 9,000 seconds at 6.01 volts
per cm. Protein conc. 2 per cent.

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-2U-

of the curves in Fig. 5 and Pig, l6 occurs at about I5 grams sodium
sulfadiazine.

This point represents the number of grams of sodium

sulfadiazine needed to divide the grams of protein recovered equally
between the precipitate and the filtrate.

Pigs. 18, I9 , 20, and 21

illustrate the removal of protein from the globulin region with in­
creasing quantities of sodium sulfadiazine,

2.

Crystal formation at pH 9.2.
If a solution of sodium sulfadiazine and serum is allowed to

stand at
material forms.

or at room temperature or at 37*C., crystalline
A reaction takes place between the sulfadiazine

anion and the serum proteins.

The crystalline material forms more

rapidly at 37*0. than at lower temperatures since the rate of reac­
tion is faster at the higher temperature.

After preliminary inves­

tigations, it was found that the crystals consisted of sulfadiazine
and serum proteins.

The nature of the proteins was determined.

In order to isolate sufficient serum protein by precipitation
for an electrophoretic study, a large test sample was prepared.

To

300 ml, of i|f99l serum was added sixty grams of sodium sulfadiazine.
After I4. to 5 hours storage at

crystals began to form.

The

crystals were filtered after 12 days and stored in saline solution.
The filtrate was again placed at 1|*C. for further reaction to occur.
More crystals occurred after

2h hours.

after a few weeks reaction period.

The crystals were filtered

The crystalline material was

combined and rinsed free of soluble materials with ,86^ saline.

No

further cryatalization occurred in the serum filtrate, even after
the addition of 5 grams of sodium sulfadiazine.

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»25“

Table XV
Data obtained from calculation of descending electrophoretic
pattern. Fig. 13. Ko. Ik h 3 untreated senxm.

Serum protein
Per cent from pattern
Gm, protein/50 ml. serum

A

a

Wi.O

li+.B

1.51

0.51

P

and y

ill.2
l.i|2

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“26-

Tabls XVI

Data obtained from calculation of descending
electrophoretic pattern. Fig, 19.

Serum protein
Per cent from pattern
Gm, protein
Per cent based on
50 ml, serum

A

57
1.36
59.5

a

7.6

P

11,8

0.18

0.28

5.2

8.2

Y
23.5
0,56
16,2

Filtrate from $0 ml. Wo. lJ|i|3 serum treated with 5»0 gm.
WaSD. Protein in filtrate = 2.37 gm., or 68,9 per cent
protein re/mined in filtrate.

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-27-

Table XVII
Data obtained from calculation of descending
electrophoretic pattern of filtrate. Fig. 20 .

Serum protein
Per cent from pattern
Gm. protein
Per cent based
on 50 ml. serum

A

67.2:
1.19
34.0

a
5.6

P
11.2

Y
15.8

0.10

0.20

0.28

2.3

3.8

8.2

Filtrate from 50 ml. No. lIjll-J serum treated with 12,5 gnis.
NaSD. Protein in filtrate = 1*77 gm., or 51-o per cent
protein remained in filtrate.

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-28-

Table XVIII
Data obtained from calculation of descending
electrophoretic pattern of filtrate. Pig. 21 ,

Serum protein
Per cent from pattern
Gm, protein
Per cent based
on 50 ml. serum

A

a

72.U

5.0

0.90

26,1

P

11.5

Y
11.3

0.06

o.lU

o .ih

1.8

U .1

U.i

Filtrate from $0 ml. No. ll|l|.3 serum treated with 22.5 gm.
îlaSD. Protein in filtrate = 1 .2l|. gm., or 56 .1 per cent
protein remained in filtrate.

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3.0 j—

filtrate

.a
4
2.0

1.5
k
o
precipitate

1.0

0.5

5.0

10.0

15.0

20.0

26.0

Grama Sodium Sulfadiazine
Fig, 16. Serum proteir. removed arid serum protein in the
filtrate from solutions of 50.0 ml. ;.'o. 1443 serum and
varying quantities of sodium sulfadiazine.

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2.0

1.5

albumin

L,
®
CO

1.0

u
o
0.5

0

5.0

10.0

15.0

20.0

Grams Sodium Sulfadiazine
Pig. 17.
Serum protein components in tne I’ilLrates from
solutions of 50.0 ml. No. 1443 serun and varying quantities
of sodium sulfadiazine.

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É

CL<r
Fig. 18. Bovine serum Ho. Ii4l3Electrophoresis for 9»OCX) seconds at 5*57
volts per cm. Protein oonc. 2 per cent#

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Fig. 19. Composition of filtrate from a
solution of 50 ml. No. liili.) serum and 5.0
gm. sodium sulfadiazine after precipitation
of sulfadiazine. Electrophoresis for 9*000
seconds at 5*?7 volts per cm. Protein cono.
2 per cent.

À
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.

Fig. 20. Composition of filtrate from a
solution of 50 ml. No. Ikh3 serum and 12.5
gm. sodium sulfadiazine after precipitation
of sulfadiazine. Electrophoresis for 9*000
seconds at 5*^J^ volts per cm. Protein conc.
2 per cent.

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i

f

Pig. 21. Composition of filtrate from a
solution of 50 Ell. No. li+1+5 serum and 22.5
gm. sodium sulfadiazine after precipitation
of sulfadiazine. Electrophoresis for 9*000
seconds at 5*^2 t o Its per cm. Protein conc.
2 per cent.

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Â

y

CL<r

■i

Plg# 22* Composition of protein fraction
from a solution of 300 ml* No. 991 and 60
gm. sodium sulfadiazine removed by crystal­
lization of sulfadiazine-protein complex at
l4.®C. Electrophoresis for 9»000 seconds at
3*96 volts per cm. Protein oonc. O.9O per
cent*

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i

-29-

The crystals, which went into solution with difficulty, were
dissolved with .IN NaOH up to pH 10.C.

The resulting pale yellow

solution was placed in cellophane tuhing and dialyzed against ten
liters

,66% saline

for 6 days with daily saline changes to remove

the sodium sulfadiazine .
0*37 grams.

The protein content of the solution was

The solution was concentrated to 0.9C^ protein and

equilibrated by dialysis against barbital buffer in preparation for
eleotrophoretio analysis*

An electrophoretic scanning pattern.

Pig. 22, revealed that the proteins removed from the serum consisted
of

97*3% P-globulin

and 2*7^ y -globulin.

This serum protein component

designated as p-globulin had a mobility between a- and p-globulin*
It is possible that P-globulin has a greater mobility when other
proteins are not present.

Under the conditions of this experiment,

the sulfadiazine anion had an affinity for p-globulin.

The quantity

of p-globulin removed, was very small.

The original 300 ml. of

serum contained 25*5 grams of protein.

The amount of protein removed

from the serum was 0*57 gram, or only

of the total protein.

Of the protein removed,

3*

l,hl% was

P-globulin and O . C ^ was T-globulin.

FreeIpitation at pH I4..O*
During the study of this problem, it was thought that the reac­

tion between sulfadiazine and serum proteins was influenced by the
charge of the protein molecules*

The isoelectric points (15) of

bovine serum proteins are*

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i

-30-

albumin

k*h5

a -globulin

i;,98

p -globulin

5,28

Y -globulin

6,02

Since the precipitation studies had been carried on between pH 9,2
to 7,0, which is above the isoelectric points of the serum proteins,
it was considered of interest to determine what the results would be
if the charges on the serum proteins were reversed,

A sample was

precipitated at pH I(.,0, which is below the isoelectric points of the
proteins,

To 50

ml, of f991 Brucella anti-serum was added ,15N phosphoric

acid to lower the pH from 8,6 to l4.,0.

Ten grams of sodium sulfa­

diazine was dissolved in 25O ml, of distilled water.

To the well

agitated serum at pH U, ,15N phosphoric acid and the sodium sulfa­
diazine solution from separate burettes were added simultaneously*
During the entire addition of the two reagents, the serum solution
was maintained at pH i4,0.

At this pH, the sulfadiazine precipitated

with the removal of sei-um proteins.

The solution was filtered

through two #1 Whatman filter papers.

The precipitate was rinsed

with 25 ml, portions of 0.86^ saline and these rinsings were allowed
to drain in the original filtrate.

The filtrate was saved.

After

more complete rinsing of the precipitate, it was dispersed in 0.86^
saline and dissolved with ,1N sodium hydroxide to pH 10.0,

The

alkaline solution was dialyzed at i|®C. against 0.86^ saline to remove
the sodium sulfadiazine and finally concentrated to increase the
protein percentage of the solution.
were determined,

Nitrogen and sodium sulfadiazine

A 2,0^ protein solution was prepared for electro-

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-31-

phoretic study.

The sample was equilibrated against barbital buffer

at pH 8.6 and .1 ionic strength.
The filtrate was made alkaline to pH 10,0.

The sodium sulfa­

diazine in the total filtrate was found to be 90 mg.

The solution

was dialyzed, concentrated and prepared for an electrophoretic
determination.

The original untreated serum sample was also analyzed

ele ct rophoretic ally.
A comparison of the electrophoretic pattern of the proteins
removed from the serum. Fig, 2lj., with that of the proteins remaining
in the filtrate. Fig, 25, reveals that the major portion of thep ,
y-globulins was not removed with the precipitate.

It will be

recalled that precipitation of sulfadiazine in the presence of serum
between pH 9*2 to 7*0 removes the P, y -globulins,

It is noted with

reference to Tables XIX, XX, XXI, and the patterns of the filtrate
and precipitated proteins. Figs, 23,

and 25, that the total

a-globulin fraction increased, while the p, y-globulins and albumin
fractions decreased.

The reason for this discrepancy is unknown, but

one may speculate that during the treatment at pH ij.,0, the mobilities
of a portion of the P, y-globulins and albumin approached the mobil­
ities of the a-globulins,

The albumin fraction was found to be about

equally divided betvreen the filtrate and the precipitate, whereas
the p, y -fraction in the filtrate was

6,6fo,

and in the precipitate,

Discussion of the charges on the protein and possible reac­

tion with the sulfadiazine will be given after some pertinent data
have been presented.

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â

-32-

Table XIX
Data obtained from calculation of descending electrophoretic
pattern. Fig. 25 . No. 991 untreated serum.

Serum protein
Per cent from pattern
Gm. protein/5 0 ml. serum

A
2 6 .0
1.12

a
17 .a
0 .77

P and Y
5 6 .2
2.1+5

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-33-

Table XX
Data obtained from calculation of descending
elsctrophoretic pattern of filtrate. Fig. 2i|.

Serum protein
Per cent from pattern
Gm,

protein

Per cent based on
5 0 .0 ml. serum

A

a

18.0

7 .5

0.47
1 1 .0

0.20
4.6

P and

y

7 4 .5
1.97
4 5 .5

Filtrate from. 50 ml. No. 991 serum treated with 10.0 gm.
NaSD at pH i+.O. Protein in filtrate = 2,614 gHi., or 6l.l
per cent protein remained in filtrate.

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i

“3U“

Table XXI
Data obtained from calculation of descsnding
electrophoretic pattern of precipitate. Fig. 25-

Serum protein
Per cent from pattern
Gm, protein
Per cent based on
5 0 .0 ml. serum

A

a

5 0 .9

4 9 .8

0 .5 0
10.6

0 .7 4
17.1

p

and y
19.3
0 .2 9
6.6

Precipitate from 50 ml. No, 99I serum treated with 10.0 gm.
NaSD at pH i+.O. Protein in precipitate = 1.49 gm*» or 34.5
per cent of the protein in the serum was removed.

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Fig, ?3» Bovine aarujn No. 991» Elactrophor08is for 9*000 aeecndB at 6,33 volts per on.
Protein cone, 2 per cent.

Fig, 21*. Compoeition of filtrate from a solution
of 50 ml. No, 991 serum and 10 gm. sodium sulfa­
diazine after precipitation of sulfadiazine at pH
U.O, Electrophoresis for 9*000 seconds at 5-93
volts per cm. Protein conc. 2 per cent.

Fig, 25, Composition of protein fraction from
a solution of 50 ml. No. 991 serum and 10 gm,
sodium sulfadiazine removed by precipitation of
sulfadiazine at pH 1*.0. Electrophoresis for
9,000 seconds at 6.20 volts per cm. Protein
conc, 2 per cent.

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-35-

C*

Solubility of Sulfadiazine.
During the course of this investigation, a method was developed

to measure the relative "solubility** or reaction of sulfadiazine.
This reaction was tested in a number of solutions and these results
have strongly indicated a possible reaction between sulfadiazine
and proteins.

Sulfadiazine was found to be **aoluble*’ in aqueous

solutions of compounds of various structures.

On the basis of these

structures and the degree of "solubility**, conclusions have been
drawn as to reaction between sulfadiazine and proteins.
One molar solution of the compounds was made up in a phosphate
buffer.

The buffer (7) was made up as follows:

,15 mol. sodium

chloride, ,005 mol, dipotassium acid phosphate, and ,005 mol.
potassium dihydrogen phosphate are dissolved in distilled water
and made up to one liter.

One fortieth of a gram molecular weight

of the compound was dissolved in 10 ml, of the phosphate buffer.
The solution was adjusted to pH 7*7 with ,1N sodium hydroxide.

The

solution was diluted to 25*0 ml. with phosphate buffer previously
adjusted to pH 7*7,

The resultant pH of the solution was 7*7*

To

this solution, placed in a 50 ml. Erlenmeyer flask, was added a
slight excess of sulfadiazine powder.

The solution was shaken on a

Burrel Shaker for four hours at a moderate shaking speed.
period of four hours was found to be optimum.
excess sulfadiazine

A shaking

After filtering the

on a number 50 Whatman filter paper, a sulfa­

diazine determination was made on the filtrate.

The sulfadiazine

found in the filtrate was a measure of the "solulibitÿ* or reaction.
The "solubility" was calculated in milligrams of sulfadiadine reacted
in 100 ml, of solution.

à
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-36-

Solubilities of sulfadiazine in the sera and the distilled
■water given in Table XXII were determined by adjusting the pH as
specified in the Table, adding the
four hours and filtering*

sulfadiazine and shaking for

The sulfadiazine was determined from the

filtrate*
The solubility or reaction of sulfadlagine was greatest in
solutions at pH 7*7 of n-butyl amine, lysine, and arginine.

The

solubility of sulfadiazine in the other amino acid solutions at
this pH was considerably lower.

At pH 6,0, the isoelectric point

of glycine and y-globulin, the solubility of sulfadiazine in a
glycine solution and in ^ 9 1 and #lijlj.3 sera fell off sharply.
Solubility in the two sera was also determined at pH 3*0, the iso­
electric point of a-globulin, which is below the isoelectric points
of (3 and y-globulins and above the isoelectric point of albumin.

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I

-37-

Table XXII
Solubility of sulfadiazine in 1.0 molar solutions of various
substances. Solubility calculated in
—
100 ml. solution.

Substance

Solubility
pH 7 .7 '

No. 991 serum
No. II1J4.I serum

1U2

Distilled water

Solubility
pH 6.0

Solubility
pH 5 .0

18

10

16

9

Isoelectric
Points

7 .5

n-butyl amine

171

Lysine

210

9*7k

Arginine

230

10.76
10

8

Buffer

ifl

Urea

itif

Tyrosine

IfS

5.66

Histidine

62

7.99

Acetic acid

63

Threonine

67

Glutamic acid

79

Glycine

80

3 .2 2
8 .5

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9.97

É

-58-

DISCUSSION

Amino aoiôs are amphoterio compounds.

Because of the carboxyl

and amino group within the amino acid molecule, amino acids can
exist in an aqueous solution as dipolar ions (17).

A proton shift

between the carboxyl and the amino group causes the following trans­
formation (16) in glycine, for example*

Ma-CHg-COO” , 2

HHa-CEa-COO” ^

CE'

NHa-CHa-COOfi

"~cr

Negative charge
Strongly
alkaline

Zero charge
Isoelectric

Positive charge
Strongly acid

Glutamic acid, which contains three dissociating groups, may exist
in several different forms, depending upon the pH of the solution*

C0 0 “
I
(OHg)a
I

COO"
I
H*
^ _

HC-HHj3 OE
COO"

Negative charge
Strongly alkaline

COOH

COOH
1
H * . (CH.).

I
(CH.).
I

(OHs)s
^

EC-NEg-E
COOH

Zero charge
Isoelectric

^

EC-HHg-H
COO"

Zero charge
Isoelectric

^

CH

^

HC-NHg-H
COOH

Positive charge
Strongly acid

Lysine, which also contains three dissociating groups, may exist in
several different forms, depending upon the pH of the solution*

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-39-

Ma

HEa-H

(CHs )4
I
HC-IÎH3

H

(CH«)4
I

OH

H C -NH a

coc"

NÏÏ,a
^

HHe -H

(CHa),

H

H C -N H a-H ''

(CH«)4
I
HC-îîHa-H

OH

I

I

1

COO*

COO'

COOH

In Table XXII are given the pH of the isoelectric points (it) of
these amino acids.

It will be noted that the isoelectric points of

lysine and arginine are pH

9»7k end

"solubilities" were conducted at pH

10,76 respectively.

7»7* which

Since the

is below the isoeleo-

trio points of these two amino acids, then lysine and arginine each
had a net positive charge.

Histidine with an isoelectric point of

7.59 was either neutral or had a very slight net negative charge.
The other amino acids listed in Table XXII had net negative charges*
Solubility in a glycine solution was also conducted at the isoelectric
point, pH 6,0,

The solubility of sulfadiazine at this pH was slightly

lower in the glycine solution than in the buffer.
have a net charge of zero*

The glycine would

The slightly lower solubility than in the

buffer may be due to a salting out effect.
The chemical structure of the compounds listed in Table XXII
are given below*
KHa
I
C = 0
I
NH b

CHb -CH(NHe )-COOH
I

C

/\\
EC
II
EG

CH
I
CH
\ //
C

EC = C-CH2-CH(M e )-C0QH
I
I
HN
N
\ //

C
H

ÔH

Urea

Tyrosine

Histidine

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-l+O-

CH3-CH(0H)-CE(lffi[a)-C0CH

Threonine

CHg (CHs)3lIHj3

n-Butyl amine

COCE
I
(CHs)«
I
HC-HHg
I
COOH

CHg^COOH
I
KHb

Glutamic Acid

Glycine

Mg
I
(CHg)*
I
HC-NHa
I

HgNC - HH

Lysine

Arginine

I

NH
(CHa)a
I
nCSKa
I
COOH

Comparison of the structure of these compounds with the degree
of solubility of sulfadiazine in 1.0 M solutions of these compounds
indicates that certain radicals may he responsible for the increase
in the solubility of sulfadiazine.

Lysine, arginine and n-butyl

amine have a profound effect on the solubility.

Each of these

compounds contains a group which confers a positively charged region
within the molecules in a slightly alkaline range.

At pH 7*7 lysine

and arginine have positively charged regions at the6 -amino group,
and the guanidinium group respectively.
Prom the data presented it can be concluded that the reaction
between amino acids and sulfadiazine is due to the net positive
charge residing in the vicinity of the "free" amino groups.

The

The active point of lysine is thef -amino group, and the active

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4^1-

point of arginine may be the amino group in the guanidinium radical.
The ionization of sulfadiazine in an aqueous solution may be
represented by the following equation:

H
H
C=C

/

\

HaN-O

0

II 9

I

0-S-N-C

\

/
c— C
H
H

II
0

H H
C= C

N=C5H

I

/

CH--- tH,N-C

II

II

N=CH

II -

I

C-S-H-C

\

N — CH

0

\
/

II

0— 0
H
H

0

II

I

+

CH <■ H

II

ÏÏ— CH

In this form it is entirely possible that the sulfadiazine anion
unites with the positively charged lysine and arginine through the
amino groups.
Boll and Roblin (l) present data to support the hjrpothesis that
the sulfadiazine anion exists in solution, as shcwrn above,

Eumler

et al, (10, 11, and 12) claim that the active form in solution, based
on the resonance theory, is *

H
H
C=C
0“
N = CH
f
/
\
I H I
I
EgH= C
C= S-N-C
CH

\

/ I I
C=
H

C
H

0

II

II

N—

CH

On this basis, it would mean that the positive amino group
the negative oxygen.

L

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binds to

•it2-

Amino aoids oan be looked upon as small molecular weight
proteins as far as charge effects are concerned (18),

Reactions of

proteins are, in effect, reactions of the side groupings of the
protein.

Proteins are made up of amino acids linked to one another

through the alpha carboxyl group and the alpha amino group (5 ).

A

hypothetical polypeptide with the side groups is given below*

H

H

I

I

O

II

H

H

I

O

I

— H — C— C— H - C -

I
I

H

I

H

I

O

III

H

I

H

O

I

'I

C— N - C — C— R - C - C

i

I

i
I

CHg

CHg

CHg

CHg

CHg

C =C

CHg

I
CHg

CHQH

—

CHg

I

CE,

I

I

I

NHa

OH

HH

C =N H

I
HHg

Lysine

Glutamic acid

Threonine

Arginine

Because of the presence of "free" amino active points in protein
molecules, there is a strong indication that the binding of the
sulfadiazine anion to the protein occurs through these amino groups.
The F -amino groups from lysine residues, and the guanidinium
groups from arginine residues are considered as carriers of positive
charges at reactions acid to pH 8,

The imidiazole groups of histidine.

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É

-i+3-

and any a-amino groups present in the molecule, carry positive charges
7fhen acid to pH 6 , and are uncharged when alkaline to pH 8 ,5 or 9 (2).
Thus, under the conditions which the various experiments wore conduct­
ed, reaction between proteins and the sulfadiazine anion occurred
mainly through the f -amino groups from lysine and the guanidinium
groups from arginine.
TOien serum proteins are surrounded by an alkaline medium,
albumin has a greater negative charge than y-globulin.
tric points of albumin and y-globulin are

k»h3 &nd

The isoelec­

6 ,0 2 respectively,

and thus, the pH of the alkaline medium is furthsr removed from the
isoelectric point of the albumin than it is from the isoelectric
point of the y-globulin.

a- and |3-globulin isoelectric points are

and 5*28 respectively, and, therefore, have intermediate nega­
tivity in reference to albumin and y-globulin.

Conversely, of the

serum proteins in an alkaline medium, y-globulin has the greatest
positive charge, followed by P-globulin, a-globulin and finally
albumin (ll;).

The net charge of the serum proteins is negative, but

positive charges do reside in the moleculee, such as in the lysine
and arginine residues.
The eleotrophoretio patterns of the precipitation of sulfadia­
zine in the presence of serum between pH 9*2 and 7*0 show that
sulfadiazine preferentially binds with y-globulin.

The graphs of

the serum protein components of the filtrates. Pig. 6 and Fig. 1?,
illustrate that y -globulin is readily removed during the precipita­
tion of the sulfadiazine,

a-, p-globulins and albumin are also

removed, but not nearly as much as y -globulin.

The sulfadiazine

anion combines with the positively charged portions of the serum

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-1414-

proteiiis .

Since y-globulin has the greatest positive charge, it

binds with sulfadiazine more readily.

With a-, P-globulins and

albumin serum oomponents, which have a smaller degree of positive
charges, there is less binding with the sulfadiazine anion.
In the experiments performed by Davis, human serum was sepa­
rated into fractions with varying quantities of

80$.

eleotrophoretio analysis was determined for eaoh fraction.

In
These

fractions were dialyzed against a phosphate buffer solution which
contained sulfathiazole.

After complete equilibration the drug

conoentration was found to be greater in the cellophane bag con­
taining the protein solution.

He found that the drug concentration

paralleled the albumin concentration.
out at pH

These experiments were carried

From this he oonoluded that sulfathiazole was bound

to the albumin.

He also found that there was practically no binding

to Y-globulin.
It was found in this laboratory that when whole serum is frac­
tionated and the fractions dispersed in a suitable buffer, they do
not seem to react the same as when they are present in the whole
serum*

For example, albumin in serum is surrounded by et-, p-, and

Y-globulins.
globulins.

There are protein interactions between albumin and the
The positive charges of albumin oombine with the negative

charges of the globulins, and vice versa.
act among themselves.

The globulins also inter­

Thus fractionated serum does not include

these protein interactions and results obtained with fractions do not
give the true picture.
With precipitation at pH ij..O, the electrophoretic patterns
illustrate that p — and y —globulin remained in the filtrate and
albumin and a-globulin wore removed with the precipitate.

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It was

â

-U5“

also demonstrated that a serum protein component, ithloh has an eleo­
trophoretio mobility between p -, and a,-globulins and was designated
as P-globulin, was removed from a mixture of serum and sodium sulfa­
diazine at pH 9«2.

On the basis of the charges carried by the

various serum proteins, these facts are difficult to interpret.
Apparently the charges on the proteins aro not the only factors that
are involved in the binding of the proteins to sulfadiazine anions.

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-it6-

SUMMARY

Serum proteins isolated by acid precipitation of sulfadiazine
between pH 9*2 and 7*0 in the presence of Brucella anti-serum were
found to be bactericidal against Brucella abortus in the presence
of fresh normal rabbit serum to serve as complement and traces of
sodium sulfadiazine.
The above serum proteins were identified by electrophoretic
analysis and found to consist of globulins, mainly Y-globulin,
small amount of albumin was also present.

A

The sulfadiazine anion

combined with the positively charged positions with the serum
protein molecule.

In the precipitation of sulfadiazine in the

presence of serum at pH l+.O, an eleotrophoretio study revealed that
the P “, and y-globulins remained in the filtrate, and the albumin
and a-globulins were removed with the precipitate.
The crystals formed in a solution of serum and sodium sulfa­
diazine at pH 9.2 and placed at 4*C., contained serum protein, which
was found to be almost pure P-globulin as determined by elaotrophoretic analysis.
At pH 7.7 sulfadiazine reacted with 1 molar solutions of lysine,
arginine, n-butyl amine but did not react appreciably with acetic
acid, glutamic acid, urea, tyrosine, histidine, threonine, and
glycine.

The sulfadiazine was found to react with positively

charged areas in the amino aoids, such as the 6 -amino group of
lysine and the guanidinium group of arginine.

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-47-

ACKNOWLEDGMENT

The author wishes to express his appreoi&tlon to
Dr. I, Forest Huddleson for the opportunity to have been
guided by him in this work; to Dr. R. E. Sanders for the
instructions in the operation of the Tiseleus apparatus;
and to the National Institute of Health for the Fellowship
appointment•

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