CLIN.
CHEM.
19/6,
621-623
(1973)
Evaluation of EDTA as the Chelator in the Biuret Reagent
Harold Van KIey and Carol S. Claywell
Use of ethylenediaminetetraacetate
(EDTA) as the
chelating agent in the biuret reaction (in place of
tartrateor citrate)was investigated.We found that
this suggested modification of the reagent results in
lower color yields and prolongs the time for color
development. The EDTA reagent is more stable if
color development is hastened by heating, but conditions are described under which the more customary
reagent may be so used.
Bovine serum albumin (BSA) was dried for at least two
days under reduced pressure over P205; reference and
stock solutions were prepared with physiological saline.
For rapid color development,
samples were heated in a
boiling water bath for the desired time, then quickly
cooled to room temperature
in ice, filtered through Whatman No. 1 paper to remove any precipitated
Cu20 or protein, and the color was measured as quickly as possible.
Gasbarro et al. (1) recently suggested that ethylenediaminetetraacetate(EDTA) be used as the chelating agent
in place of potassium
sodium tartrate
to stabilize
the
basic cupric ion, Cu(H), in the biuret reagent.
They
claimed that this resulted in a more stable reagent, lower
blank values, a perfect Beer-Lambert
law relationship
up
to 140 g of protein per liter, and the possibility of rapid
color development
at 100#{176}C
instead of at room temperature. Tris(hydroxymethylamino)methane
has a binding
constantof 3-4 (2) for its complexes with Cu(ll) and interfereswith development of the biuret color (3, 4), so
EDTA, with a binding constant of 18 (5), might be expected to cause even greater interference.
Indeed, the
transferof Cu(H) from chelation with a peptide to EDTA
has been studied (6). However, any suggested improvement in this commonly used assay for protein deserves
further investigation.
Gasbarro et al. (1) did not specify the formula for the
biuretreagentthey tested.We have investigated
two formulations
with tartrate
as the complexing
agent (7, 8),
and one with citrate (9).
The blank with the EDTA biuret reagents was indeed
lower (Table 1); however, the advantage
of this may be
questioned,
because spectrophotometers
may be readily
set to zero with the usual reagent. The absorption peak of
the biuret color is very broad; decreased color intensity of
the blank will not greatly increase the sensitivity
of the
assay.
A comparison of standard curves for bovine serum albumin with the EDTA biuret reagents and the normal formulations is shown in Figure 1. When EDTA was substituted for tartrate, color development
was almost completely abolishedwith the Weichselbaum reagent (Figure 1A),
whereas the other formulations
showed a decreased slope
of the standard curve after EDTA was substituted
(Figure
lB and 1C). After the mixture has stood at room temperature for 2.5 h and was then heated for 3 mm, no further
color developed in the EDTA samples of Figure l.A. Linearity of the assay extended to 10-12.5 mg of protein per
milliliter,
except for the Henry assay, in which linearity
extended to 80 mg of BSA per milliliter with the normal
formula and to at least 100 mg/ml with EDTA. Different
ratios of protein and Cu(ll) and different
total volumes
are used in each procedure, so the absorbance
per milligram of protein is expected to be different.
Development
of color with the Gornall reagents at room
temperature
was studied by repeated scans every 5 mm
from 450 to 650 nm. Essentially
no increase in absorbance
Materials and Methods
Alkalinebiuretreagentswere prepared as describedby
Weichselbaum
(7), Gornall
et al. (8), the “Diagnostest”
(10) modificationof the method
of Henry et al. (9), and
the correspondingreagents with disodium
EDTA substituted in equimolar quantities
for tartrate
(7, 8) or citrate
(9, 10).
Samples were read at 555 or 540 nm in 1-cm cuvets in a
ZeissPMQII spectrophotometer.Repeated scans from 450
to 650 nm, to follow development
of color with time, were
made on a Perkin-Elmer Model 202 spectrophotometer.
Results and Discussion
Table 1. Comparison of Blank Absorbance
Values vs. Water for Traditional and
EDTA-Modified Bluret Reagents
Reagent
From the Laboratory
for Biochemical
Research, St. Mary’s
Health Center, 6420 Clayton Rd., St. Louis, Mo. 63117; and the
Department
of Biochemistry,
St. Louis University School of Medicine, St. Louis, Mo.
Received Feb. 28, 1973; accepted April 6, 1973.
Standard
EDTA
A
Weichselbaum (7)
Gornalletal.(8)
“Diagnostest”
(10)
0.222
0.059
0.093
0.198
0.008
0.004
CLINICAL CHEMISTRY, Vol. 19, No. 6, 1973
621
‘0
#{149}0
‘0
0
#{149}0
0
1)
10
g CuSO4
Fig. 2. Relationship of copper
velopment of the biuret color
mg BSA/ml
Fig. 1. Standard
uret reagents
curves
for usual
and EDTA-modified
bi-
0--- 0,
79/liter
O---O, normal formulation;
S
S. EDTA. Reagents
are: A,
Welchselbaum (7); 8. Gornall et al. (8); C, “Dlagnostest”
(10) modificatiOn of Henry et al. (9)
usual formulation,
5H20/liter
complex
#{149}
#{149},
EDTA.
and protein
Protein
to de-
concentration,
V.”
0
0
__8---#{176}
9.99.8O
was found after 10 mm with the normal formulation,but
the EDTA-modified reagent did not reach constant ab0
sorbance until25-30 mm. Thus development of color is
delayed as well as decreased.Maximum
absorptionwas
found at 560 nm rather than 540 nm recommended by
Gornall etal.(8).
As shown in Figure 2, an increase in the ratio of Cu(U)
0)
and chelator to protein caused a slight decrease in color
0
10
20
30
development
at higher concentrations
with the tartrate
Gornall reagent; maximum
color was observed in the
MINUTES OF HEATING
range of copper recommended for this assay. With the
Fig. 3. Development
of biuret color at boiling water temEDTA formulation,
the color was only about two-thirds as
perature.
All points are the difference
compared
to a
intense as with the traditional
reagents and color yield deblank similarly heated
creased more at higher concentrations.
If the chelator and
O---O,
usual formulation;
#{149}
#{149},
EDTA. Protein concentration,
protein concentrations
were held constant and the concen10 9/liter
tration of Cu(fl) was increased, there was a sharp increase
in color yield at lower concentrations,
which became constant
at approximately
15 g of CuSO4-5H20
per liter.
reagent after 15 mm of heating, but none in the EDTA
The shape of the curve was as expected for competition
blanks.
between two species.
These results are consistent with a report by Ward and
Gasbarro et al. (1) reported that the EDTA reagent perFastiggi
(11),
which appeared
after
the completion
of
mits rapid development
of color at 100#{176}C
instead
of room
temperature.
Color development
was more rapid at 100#{176}Cthese studies: EDTA caused general reduction in color development in the Gornall assay (8) but no shift in the abwith both the tartrate and EDTA formulations
of the GOrsorption spectrum.
nall assay
(Figure
3). Color development
was relWe conclude that the modification
of the biuret reagent
atively diminished
at all times with the EDTA formulaas suggested by Gasbarro et al. (1) does provide a more
tion. Color increased
throughout
the time period studied, which may reflect some partial hydrolysis of peptide
stablereagent,but at a costof greatly reduced sensitivity.
The amount of reactiveCu(ll)in solutionisdecreasedbebonds and structural
rearrangement
in the hot alkaline
cause of the strong Cu(ll)-EDTA
complex. This is shown
solution. Carefully defined conditions of time and temperin color development
only half to two-thirds
that found
ature would be needed if the biuret color were developed
with tartrate
or citrate as the complexing
reagent. The
at elevated temperature,
but the usual Gornall formulation may be so treated and gives better sensitivity than
smaller slope in the curve with the EDTA reagents condoes the EDTA formulation.
The increased stability of the
tributes to loss of precision (but not necessarily of accuraEDTA reagent
was manifested
in this study in that
cy) in the assay. We have found no problems of stability
precipitates
of Cu20 formed in the blanks of the tartrate
of the tartrate reagent containing KI.
I’)
622
CLINICAL
CHEMISTRY,
Vol. 19,
No. 6,
1973
If total protein is to be measured in serum, the EDTA
formulationcan be used with a lesserdilutionbut gives no
real advantage. In biological fluids that have a smaller
protein concentration-such
as urine, pancreatic juice, or
cerebrospinal
fluid-the
decreased sensitivity
could be i
major handicap.
The biuretcolordevelopsrapidlyat elevatedtemperatures, but another potential source of error is introduced if
progressive color development
with time of heating (Figure 3) is not uniform
for all proteins. At room temperature
a plateau is reached and time is not a crucial factor for a
valid assay in manual methods. There may be some advantages for automated
biuret assays but, again, the normal biuret formulations
have more favorable characteristics than do the EDTA-containing
reagents.
References
1. Gasbarro, L., Bandinelli, R., and Tomassini, G., A new unified
biuret reagent stabilized with EDTA. Clin. Chim. Acta 36, 275
(1972).
2. Bai,
K. S., and
Martell,
A. E., The
(hydroxymethyl)-1,3-propanediol
ions. J. Inorg.
Nuci.
Chem.
interaction
of 2-amino-2-
with copper (II) and nickel (II)
31, 1697(1969).
3. Robson, R. M., Goll, D. E., and Temple, M. J., Determination
of proteins in “Tris” buffer by the biuret reaction. Anal. Biochem. 22, 339 (1968).
4. Stewart, L. E., Thomas, J. W., and Hull, G. E., Effect of Tris
buffer
on the biuret reaction. Anal. Chim. Acta 44, 453 (1969).
5. Schwarzenbach,
G., and Freitag, E., Komplexone
XX. Stabilitat.skonstanten
von
Schwermetall
Komplexen
der
Athylendi-
amin-tetraessigs#{227}ure.HeW. Chim. Acta 34, 1503 (1951).
6. Pagendorf, G. K., and Margerum, D. W., Mechanism for the
proton-transfer
reactions of a peptide hydrogen
in copper (11) tnglycine. J. Amer. Chem. Soc. 90,6963(1968).
7. Weichselbaum,
T. E., An accurate and rapid method for the
determination
of proteins in small amounts of blood serum and
J. Clin. Pathol. 16,40(1946).
8. Gornall, A. G., Bardawill, C. J., and David, M. M., Determination of serum proteins by means of the biuret reaction. J. Biol.
Chem. 177, 751 (1949).
9. Henry, R. J., Sobel, C., and Beckman, S., Interferences
with
biuret
methods
for serum proteins.
Use of Benedict’s qualitative
glucose reagent as a biuret reagent. Anal. Chem. 29, 1491 (1957).
10. DiagnostestT
reagent
set. for determination
of serum protein,
The Dow Chemical Co., Diagnostic Products, Indianapolis,
md.
11. Ward, W. W., and Fastiggi, R. J., Binding of EDTA to
DEAE-cellulose
and its interference
with protein
determinations.
Anal. Biochem. 50, 154 (1972).
plasma.Amer.
CLIN.CHEM. 19/6,623-625 (1973)
Evaluation of an Automated Creatininase Creatinine Procedure
Monty H. McLean,
Jerry Gallwas, and Mark Hendrixson
We have evaluated an automated
method
for determining serum creatinine
by use of the enzyme
creatininase. The method has been adapted
to the Beckman DSA-560 and measures
the Jaff#{233}-positive chromogens
present
in serum before and after enzymatic
hydrolysis
of creatinine.
The precision
and linearity
of the method are acceptable,
and the results correlate well with those obtained
by other accepted
techniques.
The method
offers the advantages
of automation,
enhanced
specificity,
micro-scale
sample
volume, and direct serum analysis.
source of enzyme material,
until recently, has rendered
the technique rather impractical.
An automated
creatinine method has been evaluated in
which the enzyme creatininase,
isolated as a crude preparation from a microbial source, is used.
The features of the instrument
required for this type of
assay include an incubated
transport
system, micro-scale
sample delivery, and a dual-beam
colorimeter
for measurement
of the Jaffe-positive
chromogens
present with
and without the enzymatic
hydrolysis of creatinine.
The
results are printed
out in digital form on the Teletype in
concentration
units.
The feasibility of an enzymatic
procedure for determination of serum creatinine was first demonstrated
in 1937
by Dubos and Miller (1). A bacterial species was isolated
from soilthat can specifically
decompose creatinine.
They
coupledthiswith the classical Jaff#{233}
reaction for determination of creatinine before and after incubation with the, enzyme preparation.
The lackofa commercial or otherready
Materials and Methods
From
Applications
Research,
Clinical
Instruments
Division,
Beckman Instruments,
Inc., 2500 Harbor Blvd., Fullerton, Calif.
92634.
Presented in part at the 24th National
Meeting
of the AACC,
Cincinnati, Ohio, August 24, 1972.
Received Feb. 1, 1973; accepted Mar. 16, 1973.
Apparatus
The procedure was evaluated
with a DSA-560
Model 33 Teletype (Beckman
Instruments,
Inc.,
ton, Calif. 92634) and the standard AutoAnalyzer
nicon Instruments
Corp., Tarrytown, N. Y. 10591).
with a
Fuller(Tech-
Reagents
Lyophilized
creatininase
preparation
680021,
Microbics
Operations,
Beckman
Inc., Fullerton,
Calif. 92634). Reconstitute
distilled water immediately
after opening,
(Catalog
No.
Instruments,
with 10 ml of
because the en-
CLINICAL CHEMISTRY. Vol. 19. No. 6. 1973
623