THE USE OF COPPER AND IRON SALTS FOR THE

DEPROTEINIZATION OF BLOOD

BY MICHAEL SOMOGYI
(From the Laboratory of the Jewish Hospital of St. Louis, St. Louis)

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(Received for publication, December 11, 1930)
As reported in a preliminary note (l), copper and iron salts were
found to be suitable for the precipitation of blood proteins by the
use of the simple technique followed in our method with zinc salts
(2). Below we briefly describe the procedures and results obtained
with these metals.
Copper
If to laked blood 1 volume of 10 per cent copper sulfate and 1
volume of 0.5 N sodium hydroxide are added, both proteins and
non-sugar reducing substances are precipitated, leaving sugar as
the sole reducing substance in the protein-free filtrate. Some
copper always passesinto the filtrate but this does not interfere
with the determination of sugar. In cases of hyperglycemia,
however, some sugar is lost in the process of precipitation in
spite of the fact that the hydrogen ion concentration of the
mixture (pH below 6) does not warrant the formation of insoluble
copper hydroxide-sugar complexes. The probable explanation
of the loss is the formation in the process of neutralization with
sodium hydroxide of transitory alkaline strata in which sugar is
precipitated with copper hydroxide and is not fully redissolved
even though the ultimate reaction of the mixture is distinctly acid.
In order to overcome this difficulty we sought for a very weakly
alkaline reagent to replace sodium hydroxide in the neutralization
of the copper sulfate. Sodium tungstate proved to serve the
purpose excellently. It precipitates copper at a lower pH than
does sodium hydroxide, never renders the alkalinity of the mixture
high enough to allow t,he formation of copper-sugar complexes,
and at the same time is a very efficacious buffer at the slight,ly acid
reaction optimal for the complete precipitation of blood proteins.
725
726 Deproteinization of Blood

The reagent.s for the deproteinization of whole blood are as

follows :
Solution I-7 per cent solution of CuS04.5HzO.
Solution II-10 per cent solution of Na2W04.2Hz0.
Procedure-To prepare blood filtrates in 1: 10 dilution, lake
1 volume of blood in 7 volumes of water, add 1 volume of Solution
I, and mix. Then introduce with continuous shaking 1 volume of
Solution II, stopper the flask, shake well, and filter through dry
filter paper after a few minutes. The filtrate is as a rule practically
colorless since the excess copper is precipitated in the form of

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tungstate.
For the deproteinization of corpuscles the reagents must be used
in concentrations twice as high as for whole blood. This does not
necessitate the preparation of separate solutions, instead the
corpuscles are laked in 5 volumes of water, and 2 volumes of each
of the above solutions are added. It is advisable to allow the
laked corpuscles to stand for a minute or 2 with the copper sulfate
before adding the tungstate. This permits the transformation of
hemoglobin into acid hematin, and as a consequence filtration is
accelerated and a better yield of filtrate is obtained.
For the precipitation of plasma or serum proteins copper is
superior to zinc. We made the observation in the tungstate
method of Folin and Wu that the optimal reaction for the complete
precipitation of the proteins of separated plasma or serum lies at a
slightly higher acidity than for the total proteins of whole blood.
The same holds true for precipitation by zinc or copper. If, how-
ever, the acidity required for a perfect precipitation of plasma
proteins is maintained in the zinc method, zinc salts pass into the
filtrate in quantities that interfere with the sugar determination.
This entails the added work of removing the zinc from the filtrate
by precipitation with alkali and a second filtration. The use of
copper instead of zinc obviates this operation, since the presence of
copper in the protein-free filtrate does not affect in the least the
determination of sugar with alkaline copper reagents. It also is
superfluous to neutralize the filtrates if the alkaline copper reagent
is an efficacious buffer solution as the modified Shaffer-Hartmann
reagent. Accordingly, we use for the deproteinization of plasma
or serum relatively less alkali (sodium tungstate) than for whole
blood. The reagents securing a perfect precipitation of plasma
proteins are as follows :
 IV. Somogyi 727

Xolution 1-5 per cent solution of CuSO4.5HzO.

Solution II-6 per cent solution of NasWOd. 2H,O.
The procedure is the same as for whole blood.
Protein-free blood filtrates, prepared by the copper method,
yield true sugar values. In Table I are presented examples of
comparative determinations in copper filtrates, zinc filtrates, and
tungstate filtrates of blood samples obtained from hospital
patients. As can be seen, there is good agreement between the
sugar values in copper and zinc filtrates, and both are well in line

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TABLE I
Comparative Sugar Determinations Showing That Blood Filtrates Prepared
by Deproteinization with Copper Yield True Sugar Values
Results are given per 100 cc. of blood, in terms of glucose.
- -7
Tungstate precipitation
-
Expkmt Copper Zinc Apparent Reducing
precipitation precipitation Bugair non-sugars True sugar
/[total reduc- (non- $mmmtable)
tion) f‘ermentable)
_- _-
ml. wg. mg. mg. mg.
1 74 70 98 27 71
2 214 215 238 23 215
3 204 210 231 19 212
4 288 289 319 26 293
5 92 93 110 16 94
6 230 231 264 26 238
7 260 259 285 25 260
8 67 62 82 22 60
9 154 147 171 22 149
10 143 144 167 35 142
-

with the true sugar values (apparent sugar minus reducing non-
sugars) in tungstate filtrates.
Comparative determinations of plasma sugar show in copper
filtrates 5 to 10 mg. per cent lower values than in tungstate filtrates.
The differences correspond to the amounts of non-sugar reducing
substances occurring in the tungstate filtrates of blood plasma.

Iron
Iron was introduced as a precipitant of blood proteins by
Michaelis and Rona about 20 years ago. In their method the iron
728 Deproteinization of Blood

is added to the laked blood in the form of colloidal ferric hydroxide.

We found that a complete precipitation is also effected by the more
convenient and simple technique of adding the iron in the form of
a ferric salt (preferably sulfate) and subsequent precipitation with
alkali, as in our technique with zinc or copper salts.
The reagents for the procedure are an approximately 15 per cent
solution of ferric sulfate (Merck’s C.P. reagent was used) and
$ N sodium hydroxide. The two reagents must be so balanced
that when 2 cc. of the ferric sulfate solution are titrated with the
alkali, 4.9 to 5.1 cc. are required to produce a permanent pink color

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with phenolphthalein. The ferric sulfate is diluted with about 50
cc. of water before titration. It is essential to run in the alkali
slowly with continuous shaking.
Procedure-To prepare filtrates in 1: 10 dilution, lake the blood
in 6 volumes of water, introduce 1 volume of the ferric sulfate
solution, mix, then add with continuous shaking 2 volumes of the
alkali, stopper the flask, and shake well. Filter through dry filter
paper a few minutes later.
The filtrate is clear, but has a faint yellow tinge due to the
presence of a slight quantity of iron. This is inevitable, since a
higher degree of alkalinity, which would suffice to remove the last
traces of iron, prevents the complete precipitation of proteins.
The iron, however, interferes with the sugar determination, in
that it lowers the results by 2 to 4 mg. per cent, and, therefore,
must be eliminated prior to the sugar determination. By the
addition of a few granules of sodium carbonate to the filtrate, the
iron is precipitated and may be removed either by filtration or by
centrifugation. The resulting colorless fluid yields true sugar
values well in line with those obtained upon precipitation by zinc
or copper. Notwithstanding the complication arising from the
presence of iron in the protein-free filtrate, the technique with
iron salts is simpler than precipitation by colloidal ferric hydroxide,
especially since the latter needs to be combined with heat coagula-
tion to give good results (3).

SUMMARY

Iron salts can be used instead of colloidal iron hydroxide for the
removal of blood proteins in the determination of true sugar.
 M. Somogyi 729

Copper salts are preferable to iron salts for this purpose, and
fully the equal of zinc salts in regard to speed and simplicity of
technique. For the precipitation of plasma or serum proteins
copper is superior to zinc.

BIBLIOGRAPHY

1. Somogyi, M., J. Bid. Chem., 87, p. xxxii (1930).

2. Somogyi, M., J. Biol. Chem., 86,655 (193q).
3. Shaffer, P. A., J. Biol. Chem., 19,285 (1914).

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THE USE OF COPPER AND IRON SALTS
FOR THE DEPROTEINIZATION OF
BLOOD
Michael Somogyi
J. Biol. Chem. 1931, 90:725-729.

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