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