THE USE OF DYES FOR THE DETERMINATION OF ACID AND

BASIC GROUPS IN PROTEINS

BY HEINZ FRAENKEL-CONRAT AND MITZI COOPER
(From the Western Regional Research Laboratory,* Albany, California)

(Received for publication, March 4, 1944)

Only a few methods are available for the determination of acid and basic
groups of proteins. Titration curves have been most generally used for
this purpose. Their application is restricted, however, to proteins which
are either soluble over a wide pH range (1, 2) or completely insoluble
(3, 4) ; in addition, considerable amounts of material are needed. The

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usefulnessof titration curves is also limited by difficulties of interpretation.
Metaphosphoric acid has recently been suggested as a reagent for the
determination of basic protein groups with which it combines stoichio-
metrically in acid solution (5).
Acid and basic dyes are known to combine with protein groups of oppo-
site ionic charge. The pioneer work of Loeb (6) was carried further by
Chapman, Greenberg, and Schmidt (7-9) who measured the amounts of
acid and basic dyes bound by proteins at various pH values and correlated
their findings with those of titration studies. The present paper reports
analytical methods for the determination of the total acid and basic groups
of proteins based upon this ability to combine with dyes in buffered alkaline
or acid solutions. The technique is rapid and simple and is applicable to
both soluble and insoluble proteins; it is based on the photoelectric deter-
mination of the uncombined dye and therefore can be carried out with
protein samplesas small as the accuracy of weighing permits.

EXPERIMENTAL

Reagents-
Dye solutions; 0.1 per cent orange G (Coleman and Bell) and 0.2 per
cent safranine 0 (National Aniline) in water.
The orange G, while labeled as of 78 per cent dye content, was found to
be of constant chromogenic value and nitrogen and sulfur content after
fractionation and recrystallization. Air-dried samples of both dyes con-
tained approximately 10 per cent of water. Standard solutions were pre-
pared from material dried to constant weight at 70” regardless of the “dye
content” indicated on the labels.
* This is one of four regional research laboratories operated by the Bureau of
Agricultural and Industrial Chemistry, Agricultural Research Administration,
United States Department, of Agriculture.
239
246 ACID AND BASIC PROTEIN GROUPS

Buffers; pH 2.2, 980 ml. of 0.1 M citric acid and 20 ml. of 0.2 M disodium
phosphate (10). pH 11.5,250 ml. of 0.2 M disodium phosphate and 200 ml.
of 0.1 N sodium hydroxide, water to 1000 ml. (11).
Determination of Orange G Bound by Proteins (Basic Groups)-To each of
four 15 ml. test-tubes, suitable for use in an angle head centrifuge, are
added 5 mg. of the protein, 1 ml. of pH 2.2 buffer, increasing am0unt.s
(1, 2, 3, or 4 ml.) of 0.1 per cent orange G, and two glass beads. If the
protein dissolves in the buffer, it will be reprecipitated by the dye. The
suspensions are shaken metihanically for 20 to 24 hours. They are then
centrifuged, and aliquots of the supernatant solutions are diluted lOO-fold.
The color intensities are determined by means of a photoelectric calorimeter

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(Klett-Summerson), with a blue filter (Corning No. 038 + Pyrex NQ. 554).
The dye concentration is read from a standard curve prepared from data
obtained with appropriate dilutions of the orange G stock solution. This
curve is a straight line in the range of 0.5 to 10 mg. of dye per liter. The
readings are not affected by the presence of the acid buffer.
The dye bound by the protein is determined by subtracting the excess
found in the supernatant from the amount added. Saturation of tjhe
protein with dye is indicated when no more dye is bound by the samples
to which greater amounts of dye are added. For routine analyses a series
of three samples is regarded as sufficient if the results agree within 10 per
cent. If greater exactness is desired (as in all determinations listed in
Tables I and II), six to twelve samples are prepared. More dye is used if a
definite trend in the first series indicates that saturation has not been
reached. The average of all values for the maximal amount of dye bound,
in mg., multiplied by a conversion factor of 8.85’ yields the results in terms
of acid equivalents of dye bound per gm. of protein X 104.2
An alternate technique applicable to proteins soluble at pH 2.2 permits
the analysis of only about 1.5 mg. of protein. For example, 1 ml. of a 0.75
per cerit solution (in distilled water, dilute salt solution, or 0.01 N hydro-
chloric acid) is diluted with 1.5 ml. of the pH 2.2 buffer and four 0.5 ml.
aliquots of this mixture are treated with 0.5 to 2.0 ml. portions of dye solu-
tion. The resulting precipitate is centrifuged off the following day and
the supernatant treated as described above.
Determination of Xafranine Bound by Proteins (Acid Groups)-The pro-
cedure is the same as that for the basic groups except for the use of the
1 The factor represents the valence of the dye (2), X 10, divided by the molecular
weight of the dye (452) X the amount of protein used (0.005 gm.). The empirical
formula of orange G is C,eII1(IN20(503)2=Na2’.+.
2 Analyses on proteins containing few basic or acid groups are necessarily inexact
(MO per cent) since they are derived from the differences between large figures.
With proteins of typical composition, the averages of three or four samples can
generally be reproduced within 5 per cent.
 13. FRAENKEL-CONRAT AND M. COOPER 241

pH 11.5 buffer and the 0.2 per cent safranine solution. After 24 hours
of shaking and subsequent centrifugation, the solutions are diluted lOO- or
200-fold and read with the same blue filter used for orange G. The stand-
ard curve is a straight line for 0 to 10 mg. of dye per liter. The factor
for calculating the results in terms of moles (= base equivalents) of dye
bound per gm. of protein X lo4 is 5.62.” Saturation of the protein with
dye is again indicated by the lack of a progressive trend in the amounts of
dye bound by samples treated with increasing amounts of dye solution.
The results of at least three samples, agreeing within 10 per cent, are
averaged.2
Of proteins soluble at pH 11.5, samples of only 1.5 mg. are needed, with

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a technique corresponding to that described above.
Effect of Variations in Experimental Conditions on Amounts of Dye
Bound by Proteins. pH-For purposes of convenience and practicability,
the protein-dye combination was carried out in buffered solutions, thus
circumventing the need for pH measurements and adjustments. Buffers
of pH 2.2 and 11.5 were selected becausethe work of Chapman, Greenberg,
and Schmidt (7-9) had indicated that complete dissociation of protein
groups in the presence of dyes was approached at these points. It did not
seem advisable to use more strongly acid or alkaline buffers, since the ad-
vantages of more complete dissociation of protein groups might be over-
shadowed by the disadvantages of protein breakdown. Thus proteins
treated with safranine at pH 12.0 bound considerably larger amounts of
dye which increased with the excess added. This phenomenon which
may be due to protein breakdown or to physical adsorption of the dye under
these more extreme conditions was not further investigated.4
Concentration-When proteins were treated with an excess of orange G,
the amount bound was independent of protein or dye concentration within
the limits used (0.06 to 0.2 per cent protein and 0.05 to 0.1 per cent dye).
Of safranine, slightly smaller amounts were bound at lower than at higher
concentrations; thus the values obtained with solutions of 0.06 to 0.12 per
cent protein concentration were 5 to 10 per cent lower than those obtained
with 0.1 to 0.2 per cent protein concentration (dye concentration 0.12 to
0.16 per cent in both series).
When lessthan equivalent amounts of the dyes were added to dissolved
3 Safranine 0 is a mixture of homologous monoacid bases; the molecular weights
of the two main constituents, (C20Ht~N4)+Cl-and (C~IH~IN&U-, are 350.5 and 364.5.
Calculations were based on a value of 355.
4 Studies are in.progress aiming at differential determination of the most strongly
basic and acid groups by equilibrating proteins with the dyes in buffers less acid or
basic than pH 2.2 or 11.5. A method for the determination of the approximate
isoelectric point of insoluble proteins through measurement of the dyes bound at
various pH levels will be described elsewhere.
242 ACID AlUD BASIC PROTEIN GROUPS

proteins, the resulting protein-dye complexes were not completely pre-

cipitated. This resulted, paradoxically, in higher calorimetric readings in
the soluble phase than when sufficient dye for saturation of the protein
was added. This finding confirmed similar observations of Rawlins and
Schmidt (9).
Time, Shaking, State of Protein-For routine analyses, a 20 hour period
was found practical. That equilibration was completed during that time
was indicated by the fact that no greater amounts of dye were bound by
proteins after 48 hours of interaction.6 A shorter period may be sufficient
for many purposes, since egg albumin was found to bind within 10 minutes
90 and 86 per cent of the maximal amounts of orange G and safranine,

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respectively.
Efficient shaking was essential for equilibration when protein samples
were treated in test-tubes with more than 2 ml. of dye solution.
The state of dispersion of the protein did not greatly affect its capacity
for the dyes. Thus insoluble proteins (keratins) of 60 to 80 mesh part,icle
size bound only 10 to 20 per cent less dye than material which passed a
200 mesh screen. Also proteins which were insoluble in neutral solution
but soluble in the buffers used bound the same amount of dye when dis-
solved in the buffer before addition of the dye as when solution was pre-
vented by adding the dye before the buffer.
To investigate any possible effect due to incipient denaturation by the
acid or alkaline buffer, samples of egg albumin were denatured by heating
for 5 minutes to 70” at pH 2.5 or 11.7, and were subsequently analyzed for
their dye-binding capacity. This was found to be unaffected by such
treatment. That the heat treatment, but not the pH alone, led to appre-
ciable denaturation was evident upon neutralization of aliquot samples.
Signijkance of Dye-Binding Capacity of Proteins-The amounts of the
two dyes bound by proteins, expressed in terms of acid and base equivalents,
have been regarded as indicative of the number of basic and acid protein
groups dissociated under the conditions of the test at pH 2.2 and 11.5. To
ascertain which types of groups were thus determined, several proteins
were analyzed. Two of these, egg albumin and ,&lactoglobulin, can be
regarded as approximately pure proteins, inasmuch as the preparations
had been repeatedly recrystallized and had been electrodialyzed. Lyso-
zyme and insulin were crystalline preparations not electrodialyzed.6 The
others were commercial protein samples.
6 Wool, in contrast to other proteins studied including keratins and silk fibroin,
did not reach an equilibrium with the dye solution within 20 to 24 hours.
6 The preparations of crystalline egg albumin, ,%lactoglobulin, and lysozyme were
kindly placed at our disposal by Dr. F. E. Lindquist, Dr. E. F. Jansen, and Dr. H. L.
Fevold, respectively; crystalline zinc insulin was supplied by Eli Lilly and Company.
 H. FRAENKEL-CONRAT AND M. COOPER 243

A comparison of the number of basic groups which bind orange G with

that number as determined by the methods of the literature (1,2,5,12,13)
is summarized in Table I. The good agreement between corresponding
values for the well characterized proteins may be regarded as evidence for
the reliability of the proposed method. The approximate proportionality
of results obtained with the crude proteins contributes additional support.
It is concluded from these data that the number of basic groups binding
orange G at pH 2.2 represents the sum of the guanidyl, imidazole, and amino
(a- and E-) groups of proteins.

TABLE I

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Comparison of Basic Groups of Proteins As Determined bw Various Methods

Basic residues per gm. protein X 104

Protein’
Dye-binding Titration m&$$ Analysis or
capacityt (1, 2, 12, 13) isolation (12, 13)
capacity (5)

Egg albumin ...... 8.8$ 8.0- 8.7 7.3- 8.0 9.0

,%Laetoglobulin ... 11.61 11.5 11.5
Casein ............ 6.8 7.6- 9.0 8.0-9.48
Fibrin ............ 12.0 13.15
Gelatin ........... 6.0 8.9- 9.6 10.75
Gliadin. .......... 4.3$ 3.4 4.4#
Insulin. ........... 9.4t 10.1 10.3-11.0 9.5
Lysozyme. ........ 11.51 11.7-12.8 12.2
Zein. ............. 1.9 1.8- 2.1 1.5

* Egg albumin and ,%lactoglobulin were electrodialyzed; all proteins were cor-
rected for moisture content. Casein, gelatin, gliadin, and zein were commercial
preparations. Two casein preparations gave identical values. See foot-note 6
concerning the other proteins.
t Moles of orange G bound at pH 2.2, X2.
$ These analyses represent averages of data obtained with 5 and 1.5 mg. protein
samples, with protein concentrations ranging from 0.1 to 0.2 and from 0.06 to 0.15
per cent, respectively. Results of the two techniques agreed within 5 per cent.
8 Determined by nitrogen distribution.

A similar comparison of the groups binding safranine at pH 11.5 with

the carboxyl and with the total acid (i.e. carboxyl + phenol + thiol)
groups of several proteins is listed in Table II. The literature values given
for most proteins must be regarded as rough approximations, inasmuch as
they were calculated from incompletely confirmed determinations, by isola-
tion, titration, and calorimetry, of the content of dicarboxylic amino acids,
tyrosine, cysteine, and amide nitrogen. Only the data for egg albumin
and @-lactoglobulin appear reliable, since they were obtained by recent
improvements in analytical methods (14), supported by titration data
244 ACID AND BASIC PRO’l!EIN OflOUPS

(1, 2). These two proteins showed a capacity to bind safranine in an

amount which corresponded to their total acid groups, a finding which was
supported by the data on most of the other proteins studied. It thus
appears that the number of acid groups binding safranine 0 at pH 11.5
comprises the sum of the carboxyl, phenol, and thiol groups of proteins.’
Uses and Applications--The described micromethods for the routine
determination of acid and basic protein groups were developed primarily
for use in studies of protein derivatives. A considerable number of deriva-
tives (of keratins, gluten, egg albumin, /34actoglobulin, casein, etc.) has
recently been prepared in this Laboratory by treatment with epoxides,

II

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TABLE
Comparison of I’otal Acid and Carboxyl Groups of Proteins with’ Their Capacity
to Bind Sajranine
Protein* Dye boundt Total acid groupsS Carboxyl groupst

Egg albumin. .................. 13.58 13.8 10.4

@-Lactoglobulin ................. 17.65 17.5 14.5
Casein ......................... 19.4 16 13
Gelatin ......................... 12.7 10 10
Glisdin. ........................ 5.5 7 5
Insulin ......................... 17.5 13 6
Zein ............................ 5.5 6 3
* See the corresponding foot-note to Table I.
t Moles of safranine 0 bound at pH 11.5 by lo4 gm. of protein (range of protein
concentrations, 0.1 to 0.2 per cent).
$ Of lo4 gm. of protein; calculated from analyses for glutamic and aspartic acids,
amide N, tyrosine, and cysteine as summarized by Cohn and Edsall (12), Chibnall
et al. (14), and Brand and Kassell (15), and as amended for glutamic acid of insulin,
gelatin, and gliadin by Olcott (16). The unknown number of terminal carboxyl
groups of the polypeptide chains was disregarded.
5 In more dilute solution (protein concentrations 0.06 to 0.15 per cent), 12.4 and
16.5 moles of dye were bound by egg albumin and @-lactoglobulin, respectively.

aromatic isocyanates, anhydrides, aldehydes, nitrous acid, and combina-

tions of these reagents. Epoxides were found to combine with both the
acid and the primary amino groups of proteins (17). The esterification
was demonstrated by the decreasesin the number of acid groups of the
7 Before suitable conditions for the determination of total acid groups had been
recognized, a relative measure of the acidity of various proteins and derivatives ivas
obtained from their tendency to bind dyes when the amounts added were less than
those needed for saturation. For these studies both safranine and methylene blue
were used. The amounts of these dyes most readily bound yielded comparative data
on the acidity of proteins and derivatives which have since been confirmed by
determinations of their total acid groups.
 H. FRAENKEL-CONRAT-AND M. COOPER 245

derivatives, as estimated by dye methods, particularly with those of sub-

maximal combination which are believed to measure primarily the carboxyl
groups.’ On the other hand, the introduction of alkoxy residues in the
amino groups did not remove their basic nature. Actually a slight increase
in the basic groups was found in many epoxide-treated proteins, a phe-
nomenon which is not yet understood.
Intensive treatment of certain proteins with phenyl isocyanate led to
introduction of the reagent to the extent of up to 30 per cent (by weight)
of the protein.8 Dye methods have now yielded information which may
contribute to an accounting for this extent of interaction. A few typical
results obtained with cattle hoof powder and rennet casein are listed in

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TABLE III
Efects of Various Reagents on Basic and Acid Groups of Proteins
-
Protein and treatment* Basic Total acid
residuest residuest

Hoof powder, more than 200 mesh, untreated.. .......... 8.8 10.4
‘I “ 40-60 mesh, untreated ..................... 8.0 8.0
Phenyl isocyanate$. ................................... 0.0 2.1
‘Phthalic anhydride ................................... 3.6 13.3
Propylene oxide ...................................... 9.2 8.7
Same, followed by phenyl isocyanate. ................ 0.0 1.7
Formaldehyde ........................................ 7.1 11.2
Nitrous acid. ......................................... 3.8 13.0
Casein,untreated ...................................... 6.8 20.3
Phenyl isocyanate. ................................... 0.0 4.0
Propylene oxide ...................................... 6.4 7.2
- - -
* See foot-note 8 for the methods of preparation and treatment.
t Per gm. of protein X 104. Determinations based on the capacity to bind orange
G and safranine.0 at pH 2.2 and 11.5, respectively.
$ All hoof derivatives were powders which passed a 200 mesh screen.

Table III. Treatment with phenyl isocyanate was found to cause a loss of
almost all basic groups and of a considerable proportion of the acid groups
of the prot.eins. On the other hand, phthalic anhydride was found to react
only with part of the basic groups. In contrast to phenyl isocyanate,
phthalic anhydride increased the number of acid groups, as would be
expected from introduction of phthalic acid residues. With both reagents
the observed decreasein the basic groups corresponded to the lossin amino
nitrogen.8 The increase in the acid groups of the deaminated protein may
have been due to nitration of the phenol residues. Treatment with formal-
dehyde caused only minor changes in both acid and basic groups. This is
8 Fraenkel-Conrat, H., and Olcott. H. S., in preparation for press.
240 ACID AND BASIC PROTEIN QROUPS

in contrast to a marked decrease in the primary ammo nitrogen, indicating

that the N-methyl01 or N-methylene groups resulting from the interaction
of amino groups with aldehydes retain sufficient basicity to bind orange G
at pH 2.2.
SUMMARY

Microanalytical methods were developed for the estimation of the number

of acid and basic groups of proteins. These were based on the tendency of
the polar groups to bind dyes of the opposite charge, resulting in a precipi-
tation of the protein-dye complex. The acid dye, orange G, combined
stoichiometrically with basic protein groups in a buffer of pH 2.2. The
basic dye, safranine 0, reacted with acid groups at pH 11.5, but the extent

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of combination was in this case slightly affected by other factors, such as
protein concentration.
The number of protein groups binding these dyes corresponded well to
the total number of basic (guanidyl, imidazole, ammo) and acid (carboxyl,
phenol, thiol) groups of crystalline egg albumin and /34actoglobulin and,
approximately, to those of several crude proteins studied.
The proposed micromethods were applicable to both soluble and insoluble
proteins. They have proved useful tools in the interpretation of the action
of various chemical agents on proteins.

The valuable suggestions and criticisms of H. S. Olcott of this Laboratory

are gratefully acknowledged.
BIBLIOGRAPHY

1. Cannan, R. K., Kibrick, A. C., and Palmer, A. H., Ann. New York Acad. SC.,
41,243 (1941).
2. Cannan, R. K., Palmer, A. H., and Kibrick, A. C., J. BioZ. Chem., 142,803 (1942).
3. Steinhardt, J., and Harris, M., J. Res. Nat. Bur. Standards, 24,335 (1940).
4. Theis, E. R., and Jacoby, T. F., J. Biol. Chem., 146,163 (1942).
5. Perlmann, G. E., J. Biol. Chem., 137,707 (1941).
6. Loeb, J., Proteins and the theory of colloidal behavior, New York and London,
2nd edition, 36 (1924).
7. Chapman, L. M., Greenberg, D. M., and Schmidt, C. L. A., J. Biol. Chem., 72,
707 (1927).
8. C. Rawlins, L. M., and Schmidt, C. L. A., J. Biol. Chem., 82, 709 (1929).
9. C. Rawlins, L. M., and Schmidt, C. L. A., J. Biol. Chem., 88,271 (1930).
10. McIlvaine, T. C., J. Biol. Chem., 49, 183 (1921).
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12. Cohn, E. J., and Edsall, J. T., Proteins, amino acids and peptides, Amerioan
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13. Abraham, E. P., Biochem. J., 33, 622 (1939).
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16. Olcott, H. S., J. BioZ. Chem., 163, 71 (1944).
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