Journal of Biochemical and Btophystcal Methods, 7 (1982) 7-13
Elsevier Biomedical Press
7
Inorganic phosphate assay with malachite green: An
improvement and evaluation *
Stephen G. Carter and Daniel W. Karl **
Department of Biochemistry and Btophystcs, lowa State Umverstty of Sctence and Technology, A rues,
1,4 50011, U.S.A.
(Received 28 April 1982)
(Accepted 1 July 1982)
Summary
An improvement over existing procedures for the determination of nanomole quantities of inorganic
phosphate (P,) is described. The protocol is simplified, and the effective concentration range of P, in
which the assay may be used is increased to 60 nmol/ml. Many of the substances commonly used in
association with P~ assays (i.e. phosphohydrolase studies) are shown not to interfere with the measurement
of P~ by this method. The effects of detergents and protein on the assay also were investigated, and
methods for avoiding interferences by them are described.
Key words: inorganic phosphate assay; malachite green.
Introduction
The large number and intrinsic importance of phosphohydrolases has created the
need for an inorganic phosphate (Pi) assay that is sensitive, specific for P~, accurate,
and simple to perform. The most sensitive spectrophotometric assays for P~ employ
the reaction of phosphomolybdate with the basic dye malachite green to form a
colored complex. Methods based on this reaction were developed independently by
Itaya and Ui [1] and by Altmann et al. [2]. Important refinements of this procedure
have been published by Bastiaanse and Meijers [3], Muszbek et al. [4] and by Hess
and Derr [5]. Muzbek et al. improved the specificity of the assay by adding H2SO4
after a brief period of color development. The H2SO4 reacts with the uncomplexed
malachite green [2], reducing the background color and eliminating the contribution
* Journal Paper No. J-10406 of the Iowa Agriculture and Home Economics Experiment Station, Ames.
Project No. 2243.
** Present address: Department of Biochemistry, Temple University, Philadelphia, Pa., U.S.A.
0165-022X/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
of PI produced from any subsequent nonenzymatic hydrolysis of the phosphate-containing substrate. This report presents a modification and simplification of the
method of Muszbek et al. [4] which permits a great reduction in the time required to
perform the assay. We have also studied a number of possible interfering substances,
typically used in phosphohydrolase studies, and have evaluated their effects on this
assay procedure.
Modified procedure
The following assay procedure is used routinely in our laboratory. Solution A is
made by mixing 4 vols. of 2N HCI and 3 vols. of 0.1 M Na2MoO 4. Solution B is a
0.042% (w/v) malachite green dye solution in 1% (w/v) polyvinyl alcohol. Solution
C is a 7.8% (v/v) H2SO4 solution. To a 1 ml sample, 0.7 ml of solution A is added,
followed immediately by 0.3 ml of solution B. Mix and allow to stand for 2 min for
color development between the malachite green dye and the phosphomolybdate
complex, then add 2 ml of solution C and mix thoroughly. This mixture is allowed to
stand at room temperature for 1 h to complete the color development, after which
the absorbances may be read at 625 nm with the color remaining stable for several
hours. The sensitivity of this assay is approximately 1 nanomole Pi per 0.02 A625n m
units.
This procedure contains two changes from that of Muszbek et al. [4]. First,
substitution of hydrochloric acid (HC1) for trichloroacetic acid (TCA) in the protein
denaturation step permits the combination of the acid and the Na2MoO 4 solutions,
simplifying sample handling. We have found that small, constant amounts of protein
( < 200/~g/ml) affect the assay in a predictable manner and consequently can be
corrected for conveniently, thereby eliminating the need to remove the protein from
the assay mixture. Second, increasing the concentration of malachite green dye
from 0.0132 mg/ml to 0.0315 mg/ml increased the usable concentration range of PI
three fold, from 20 to 60 nmol Pi/ml. Malachite green is still the limiting reagent at
high Pi concentrations, so a further extension of the linear range should be possible,
if necessary.
Evaluation of interferences
In addition to the procedural modifications described, we also have evaluated a
number of experimental conditions for their effect on the assay. As seen in Table 1,
many of the chemicals that are typically used in ATPase studies have no effect on
the phosphate standard curve, indicating the general applicability of this procedure.
However, dithiothreitol caused a significant (12%) depression of color development.
Of particular interest was the effect of the polyvinyl alcohol (PVA), which is
included in the malachite green reagent, on the absorbance values obtained in the
assay. Fig. 1 shows that, in the absence of PVA, the standard curve is nonlinear. This
is a result of the precipitation of the malachite green-phosphomolybdate complex.
9
TABLE 1
THE EFFECT OF VARIOUS ADDITIVES TO THE INCUBATION MIXTURE ON THE KHzPO4
STANDARD CURVE
Substance
Slope (A62sam/nmol Pi)
Conuol, no additions
5 mM fl-mercaptoethanol
5 mM dithiothreitol
100/~M Na~VO4
50 mM KC1
3 mM MgC12
50 mM KCI+ 3 mM MgCI2
3 mM ATP
3 mM ATP+3 mM MgCI2
200 #M adenosine
2 mM adenosine
0.0211
0.0209
0.0186
0.0211
0.021 i
0.0211
0.0210
0.0205
0.0205
0.0212
0.0212
T h e a d d i t i o n of 1% P V A (100% hydrolyzed, M r ----- 14000) f r o m A l d r i c h C h e m i c a l Co.
resulted in a linear s t a n d a r d curve.
P V A f r o m Sigma C h e m i c a l Co. (No. P-8136, lot 75C-0344) was n o t suitable as
obt ai n ed . It a p p e a r e d to c o m p e t e with the m a l a c h i t e green dye for b i n d i n g to
p h o s p h o m o l y b d a t e (Fig. 1). This interference m a y be d u e to the presence of residual
1.5
1.4
1,3
1,2
1.1
1.0
0.9
0.8
0,6
0.50.40.30.2-0.1-i0
20
30
40
50
60
70
80
nmoles KH2P%/ml
Fig. 1. The effect of polyvinyl alcohol (PVA) on the inorganic phosphate standard curve determination.
0.042% (w/v) malachite green dye was dissolved in: O, deionized H 2 0 ; A, 1% (W/V) Sigma PVA; O, 1%
(w/v) Aldrich PVA. With the above conditions for the malachite green reagent, an inorganic phosphate
standard curve was determined by the method described in the text.
10
acetyl groups on the polymer. If the Sigma PVA was subjected to mild hydrolysis
with either acid or base, the interference was eliminated.
Salts of carboxylic acids (e.g. potassium formate and glycine), at concentrations
above 0.2 M, also interfere with the asssay by forming a green color similar to that
formed from P,. This interference may be eliminated by adding additional HC1 to
compensate for the acid consumed in protonating the carboxylate anions. This
interference is not due solely to an alteration of the assay pH, since reducing the
H2SO 4 content of the assay did not produce an equivalent color.
Detergents which may be present in an enzyme assay mixture for solubilization,
stabilization or activation have a complex effect on the P, determination. Fig. 2
demonstrates the effect of increasing concentrations of Zwittergent 3,12 and sucrose
dilaurate on the color yield from 20 nmol P,. The different influences of these
detergents on a P, standard curve is shown in Fig. 3. The assay may be used in the
presence of detergents; however, since different detergents have different effects on
the assay, it is important that the proper controls are performed. The influence of
the detergent can be circumvented by including consistent amounts of the detergent
in the samples and the P, standards, since the detergent influences only the slope of
the standard curve and not the linearity.
The elimination of the protein precipitation step in the procedure described here
0.2--
I
L
I
[
,01
,02
.03
.04
.05
% (w/v) DETERGEI'IT
Fig. 2. The effect of increasing concentration of sucrose dilaurate (O) and Zwittergent 3,12 (A) on the
determination of 20/~M KH2PO4. The absorbance values for the samples containing sucrose dilaurate
were corrected for turbidity contribution. The procedure for this correction was to subtract the A750nm
from the A625nm,since the absorption spectrum for malachite green has a zero absorbance at 750 nm and
a maximum at 625 nm.
II
1.6
1.4-Y~
1.2
~,
1.0
0.8
0.6
0.4
0.2
[
J
[
h
L
I
i0
20
30
40
50
60
nmoles KH2PO4/ml
Fig. 3. The effect of detergents on the inorganic phosphate standard curve determination. 0.01% (w/v)
sucrose dilanrate (/x), no detergent control (0), and 0.01% (w/v) Zwittergent 3,12 ([:]) were added to
each of the phosphate standards tested, then the assay as described in the text was performed.
0.5I
0.4
A625NM
0.3
0,2
o,i~0
I
I
I
I
I
20
40
60
80
100
I
200
~G PROTEIN/ ML
Fig. 4. The effect of increasing concentrations of bovine serum albumin (F1), ovalbumin (O), wheat germ
acid phosphatase (O) and pyruvate kinase (/x) on the determination of 20 #M KH2PO 4 by the method
described in the text.
12
is a significant improvement over the procedure of Muszbek [4] with respect to the
ease and speed of sample processing. As shown in Fig. 4, the result of this
modification is a minimal interference by increasing protein concentrations which is
expressed as a small, linear decline in the absorbance values from the assay of 20
nmol P,. The consistent nature of this interference makes any necessary corrections
very easy to perform. Bovine serum albumin (BSA) is an exception. The phosphate
assay is inhibited by BSA to a greater degree than by the other proteins tested as
shown in Fig. 4. It is well known, however, that BSA has a number of binding sites
for hydrophobic and anionic materials and it is possible that it competes with
malachite green for binding of the phosphomolybdate. Because this is not a problem
with most proteins, elimination of the protein precipitation step is justified by the
increased convenience and rapidity of processing samples.
Fig. 5 demonstrates the effect of 100 # g / m l ovalbumin on the P1 standard curve.
In the presence of ovalbumin, the standard curve is straight with a 2% decrease in
the slope compared to the control curve. This small difference may be corrected for
easily and reinforces the general applicability of the procedure described here to
such uses as assay of column chromatography fractions and enzyme kinetics
measurements.
1,3
B
1,2
1,1
1,0
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
t
I
I
10
2o
30
i
40
50
6o
NMO~S
Fig. 5. The Pi standard curve, in the absence ( 0 ) and presence ( © ) of' 100 /~g/ml ovaibumin by the
method described in this text.
13
Conclusion
This r e p o r t describes a p r o c e d u r e for the assay o f Pi which modifies existing
m e t h o d s b y s i m p l i f y i n g the p r o t o c o l a n d increasing the effective c o n c e n t r a t i o n range
o f P~ which m a y be assayed. W e investigated a n u m b e r o f substances often
a s s o c i a t e d with P~ assays for effects on the assay. In general, very few of the
c o m p o u n d s tested h a d a n y effect, with the exception of detergents. Because the two
d e t e r g e n t s tested affected the assay differently, the e v a l u a t i o n of a n y new detergent
used in the assay m i x t u r e is necessary p r i o r to using this m e t h o d ,
W i t h the e x c e p t i o n of BSA, the p r o t e i n s tested, at 100 # g / m l , cause a 2 - 5 %
r e d u c t i o n in the slope o f the s t a n d a r d curve, with no effect on the linearity of the
curve. Thus, for m o s t a p p l i c a t i o n s , the presence of small a m o u n t s of p r o t e i n does
n o t interfere seriously with the assay.
Simplified description of the method and its applications
To a l ml sample, 0.7 ml of solution A (4 vols. of 2 N HCI and 3 vols. of 0.1 M Na2MoO4) is added,
followed immediately by 0.3 ml of solution B (0.042% malachite green dye solution in l~ polyvinyl
alcohol). Mix and allow to stand for 2 rain, then add 2 ml of 7.8% H2SO4 and mix thoroughly. This
mixture is allowed to stand at room temperature for 1 h, after which the absorbances at 625 nm are read.
The major advantage of this procedure over existing procedures for inorganic phosphate analysis is the
speed and ease that samples are processed, while maintaining a high level of sensitivity. This procedure
should be of value to enzyme kinetics and column chromatography measurements.
Acknowledgments
W e t h a n k Professor Carl L. T i p t o n , in whose l a b o r a t o r y this w o r k was p e r f o r m e d ,
for m a n y helpful discussions a n d careful review of the m a n u s c r i p t .
References
1
2
3
4
5
Itaya, K. and Ui, M. (1966) Clin. Chim. Acta 14, 361.
Altmann, H.J., Fiirstenau, E., Gielewski, A. and Scholz, L. (1971) Z. Anal. Chem. 256, 274-276.
Bastiaanse, A.J. and Meijers, C.A.M. (1968) Z. Klin. Chem. U. Klin. Biochem. 6, 48-51.
Muszbek, L., Szab6, T. and F6s~s, L. (1977) Anal. Biochem. 77, 286-288.
Hess, H.H. and Derr, J.E. 0975) Anal. Biochem. 63, 607-613.