CLIN. CHEM. 20/10, 1362-1363(1974)
Re-evaluation of EDTA-Chelated Biuret Reagent
V. Chromy, J. Fischer, and V. Kulh#{226}nek
We studied the optimal composition of EDTA-chelated
biuret reagent for the determination of protein in serum.
A reagent containing 18 mmol of EDTA, 15 mmol of
Cu2, and 1 mol of NaOH per liter exhibits a very low
blank but about the same sensitivity as tartrate-chelated
reagents. Maximum color intensity is attained within 25
mm at room temperature, and adheres to Beer’s law up
to 120 g of protein per liter.
Determination
of protein
in biological
fluids
is an impor-
tent diagnostic
method,
requiring
rapid and reliable technique. One of the most convenient such techniques is the
biuret method
of this method
(1 ). Because of the numerous
modifications
(cf. 2), the detailed
study of Gornall et al.
(3) dealing with a reagent stabilized
usual significance.
An important
reagent
composition
was made
by tartrate
modification
by Rosenthal
is of Un-
of the biuret
and Cundiff
Let stand for 25 mm at room temperature
sorbance
of the
sample
color of the protein
against
the
blank
and read the abat 545
nm.
The
complex is stable for at least 1 h.
Results and Discussion
The sensitivity
of all biuret reagents
toward the proteins
depends generally on the concentrations
of Cu2+, hydroxide or other alkali, and a chelator (3). These differ among
various recommended
reagents. The concentration
of cupric sulfate in the final biuret reaction mixtures varied
from 1 to 2 g/liter. The importance of sodium hydroxide in
the biuret reaction is well known and was studied in some
detail by Rising and Johnson (8 ). Before the development
of stabilized reagents, most biuret reaction
mixtures
contamed about 30 g of alkali per liter. It was necessary
to remove a precipitate
of cupric hydroxide before color cornparisons could be made or to enhance the concentration
of
alkali to avoid the precipitation,
especially
on standing
(9).
With the introduction
of chelators,
the alkali content
of
various formulations
varies from 4 to 40 g/liter (10, 11 ). Although
the sensitivity
and velocity
of color development
(4), who used ethylenediaminetetraacetate
(EDTA) as a
chelating and stabilizing agent. This reagent was “rediscovered” in 1972 (5), but it was rejected by Van Kley and
Claywell (6) because, with it, color yields were lower and
time for color development was prolonged.
However, in these modifications
(4-6) the optimal con-
with proteins increase with increasing amounts of copper
and alkali (3), the sensitivity of the reaction decreases with
centrations
increasing chelator concentration.
of Cu2+,
not been specified.
scribed
EDTA,
and
sodium
The EDTA-chelated
here obviates
these
hydroxide
have
biuret reagent
de-
Biuret reagent. In a 1-liter volumetric
flask, dissolve
3.8
CuSO4 . 5H20 and 6.7 g of Na2EDTA
in 700 ml of distilled water. Prepare a solution of 40 g of sodium hydroxide
in 200 ml of distilled
water, and add this solution slowly
and with constant stirring
into the volumetric
flask. Preliminary experiments
were made with reagents with vanous concentration
of individual components
(see legends to
g of
Figures 1-3).
Standard
solution.
We used a lyophilized
(Versatol
and Hyland),
the protein
content
tein nitrogen
press nitrogen
Kjeldahl
procedure
(7). The usual
as protein.
from
factor
total
control serum
of which was
and
of 6.25 was used
nonpro-
to ex-
Photometer.
We used a Unicam SP 800 Spectrophotometer, with 1-cm cuvettes.
Procedure.
In a test tube, mix 0. 1 ml of protein sample
(standard solution or serum) with 5.0 ml of biuret reagent.
Research
Institute
of Pure
Chemicals,
Lachema,
N. C., 621 33
Brno; and Institute of Medical Chemistry, School of Medicine,
E. Punkyn#{232}
University, Brno, Czechoslovakia.
Received
1362
April
CLINICAL
1 1, 1974; accepted
CHEMISTRY,
July
Thus it is desirable
with as low a chelator
Figure 1 shows the quantitative
tration of Cu2+ and absorbance
of
agent contained
1 mol of NaOH
ratio of EDTA to Cu2 was always
Reagents and Instrumentation
by the
a reagent
concentration
to
as
possible.
shortcomings.
Materials and Methods
assessed
prepare
12, 1974.
Vol. 20, No. 10, 1974
J.
relation
between
concen-
protein complex. The reper liter and the molar
kept equal to 1.05. After
further
experiments,
we selected
a Cu2+ concentration
of
15 mmol per liter. With cupric ion, EDTA forms a stable,
slightly colored 1:1 chelate. To keep Cu2 in solution
even
in an alkaline
medium,
at least an equimolar
amount
of
EDTA should be used. Such a reagent
is also the most sen-
sitive for proteins;
either increasing
or decreasing
the concentration
of EDTA decreases
the sensitivity
of the biuret
reaction (Figure 2). The reagent with an excess of unchelated Cu2+ ions tends to form a slight flocculent
coagulum,
but totally complexed
for several weeks. To
of the reagent,
to 1.2.
alkaline copper solutions
sufficient complexing
we increased
the molar
ratio
stable
capacity
are
assure
of EDTA
to Cu
It is evident from critical evaluation of EDTA-chelated
biuret reagents (6) that in those modifications
in which a
low concentration
of alkali was used (the Weichselbaum
reagent
as modified
by adding
EDTA)
the sensitivity
and color develops more slowly. Substitution
an equimolar amount of sodium-potassium
lowers
the effective
concentration
of alkali
is less
of EDTA for
tartrate even
originally
pres-
ent in a reagent.
For example, if tartrate in the Weichselbaum formulation
(11) is substituted
for Na2EDTA,
the
.
#{149}
I
I
I
#{149}
A
A
0.4
0.4
0.2
0.2
,
I
I
#{149}
a006
I
#{149}
0.5
0.018
0.012
Fig. 1 Relationship between Cu2 concentration
and absorbance of protein complex
1.0
1.5
(mol/liter)
.
NaOH, 1 mol/liter; molar ratio of EDTA to Cu, 1.05. The assays were carried
out as described under ‘Procedure,”
with a standard so’ution of protein (75
9/liter)
2.0
NaOH:
mol!Iiter
Fig. 3. Relationship between NaOH concentration and absorbance of the protein complex
Cu.2’ 15 mrnol/liter, NaEDTA,
,
18 mmol/liter
I
I
I
500
550
600
A
1.1’’’
I
0.6
A
0.4
-------.
0.4
0.2
02
I
0.01
#{149}
I
0.02
#{149}
I
0.03
#{149}
I
004
#{149}
I
0.05
Fig. 2. Relationship between EDTA concentration
and absorbance of protein complex
450
(mol/liter)
1. Protein
(1 12 9/liter) Vs. reagent
complex
final
References
chelator.
The relation
says. Clin. Chem.
reagent solution contains only 70% as much hydroxide
because pant is consumed to neutralize the acidity of the
between alkali concentration
and sensitivity
of the biuret reaction is shown in Figure 3. The sensitivity
of the reaction increases with increasing concentration
of
sodium hydroxide.
Maximum
sensitivity
is reached at
about 1 mol of NaOH/liter;
further addition of alkali has
influence
but affects
on the absorbance
the stability
of the protein
of the reagent,
complex,
which deteriorates
within
a few days. The reagent containing
1 mol of sodium
hydroxide can be used for weeks.
With the EDTA-chelated
reagent suggested, color develops rapidly,
even at room temperature,
maximum
color
yield is attained
within 25 mm, and the color that develops
is stable for at least 1 h. The protein complex has a broad
absorption
maximum
at 545 nm (Figure
4). The EDTAchelated reagent has a low blank (Figure 4, curve 2) but
about
(11 ).
the same sensitivity as reagent chelated by tartrate
Moreover, with EDTA-chelated
reagent, the color ad-
heres
to Beer’s
law up to 120 g of protein
per liter.
In some previous modifications
of the biuret method, a
precipitate
or turbidity
have been observed in the reagent
as well as in reagent
mixtures
with
tates may be inorganic
or organic.
proteins.
Precipitate
nn,
Fig. 4. Absorption spectra
Cu.2 15 mmol/llter: NSOH. 1 mol/liter
little
650
The precipiin the re-
agents is mainly insoluble
copper
hydroxides
or other
basic
copper salts. In body fluids, calcium and magnesium
appear to be the most likely cause of turbidity under the alkaline conditions
of the biuret reaction
(4 ). Organic turbidity
in the biuret
reaction
is reported
(12, 13), and its phospholipid nature was elucidated
by Levin et a!. (12). In the presence of EDTA, both the formation of precipitate
in the reagent and the turbidity
in the biuret
reaction
are suppressed.
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2. Kingsley,
blank: 2. Reagent
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of total protein
as-
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& A. Churchill, Ltd., London, 1964, 4th ad., p 140.
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C. A., The biuret reaction
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acid type.
I. The
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9.
for the determination
and visual colorime-
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T. E., An accurate and rapid method for the
ethylene
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Amer.
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CLINICAL
CHEMISTRY,
Vol. 20, No. 10, 1974
1363