ARTICLE IN PRESS

ANALYTICAL
BIOCHEMISTRY
Analytical Biochemistry 323 (2003) 180187
www.elsevier.com/locate/yabio

A simplied method for inorganic phosphate determination

and its application for phosphate analysis in enzyme assays
Subhash D. Katewa*,1 and Surendra S. Katyare
Department of Biochemistry, Faculty of Science, M.S. University of Baroda, Vadodara 390 002, India
Received 14 June 2003

Abstract

A simplied method for inorganic phosphate determination has been developed. The method is sensitive, easy, economic, and
applicable for estimation of phosphate released in both enzymatic and nonenzymatic reactions. A mixture of hydrazine sulfate and
ascorbic acid was used as the reducing agent and the conditions for the development of the molybdenum blue color were optimized.
Thus in the 4.0 ml assay system, 0.4 ml of the reducing agent solution containing 20 mg each of hydrazine sulfate and ascorbic acid
per milliliter of 1.0 N H2 SO4 gave a rapid optimum color development with absorption maximum at 820 nm. Color development
showed a linear relationship up to 10 lg Pi concentration. Thus the method has a 2.5
higher range of Pi estimation than that of the
Bartlett method. The molar extinction coecient at 820 nm was higher than that obtained in the Bartlett procedure. Also the
molybdenum blue color formed was stable up to 24 h. Under the standard assay conditions, interference from acid-labile phosphate
as in the case of Na ,K ATPase was at the minimum. The applicability of the method for assay of microsomal Na ,K ATPase
and glucose-6-phosphatase was checked in microassays (nal volume 0.1 ml) in comparison to the conventional procedures which
use 34 times higher volumes. Likewise the applicability of the method for phospholipid analysis was compared with that of the
conventional Bartlett method. Under both test systems the results obtained by the micromethod were identical to those obtained by
the conventional methods. In general the method, which rapidly produces quantitatively molybdenum blue color, not only is rapid
economical, and convenient but also has wide applicability.
2003 Elsevier Inc. All rights reserved.

Importance of phosphorous in intermediary metab-
olism is well recognized and well documented and needs
no overemphasis [1,2]. Because of its pivotal role, a
sensitive procedure for the assay of phosphorous in
microquantities assumes importance [3]. The initial
gravimetric/nephelometric methods were changed to
colorimetric procedures which were based on the prop-
erty of orthophosphate to form a complex with molyb-
dic acid [3]. It was realized that phosphomolybdate
could be reduced to produce a deep-blue-colored com-
plex called molybdenum blue [3]. The rst quantitative
and reproducible method for colorimetric estimation of
phosphate was described by Fiske and Subba Row [4],
where 1-amino-2-naphthol-4-sulfonic acid in combina-
*
Corresponding author.
E-mail address: sdkatewa@bualo.edu (S.D. Katewa).
1
Present address: Department of Biochemistry, School of Medi-
cine and Biomedical Sciences, State University of New York at
Bualo, Bualo, NY 14214, USA.
tion with sodium sulte and sodium metabisulte was
used as the reducing agent. Use of several other reducing
agents such as hydroquinone, 2,4-diaminophenol,
ascorbic acid, thiosulfate, stannous chloride, hydrazine
sulfate, etc. has since been described [[3] and references
cited therein]. The basic problem encountered in these
methods is that the molybdenum blue color is unstable
after a duration of 510 min depending on the method
employed [3,5]. Also, the acid hydrolysis of labile
phosphorous poses a problem of interference and
quantitative estimation although alternate procedures
have been devised [3,6]. The sensitivity of the colori-
metric method for phosphate estimation was improved
by Bartlett [7]; simultaneously the problem of color in-
stability was also solved by this procedure [7]. The
Bartlett method requires that the reduction to molyb-
denum blue be carried out in a boiling water bath for
7 min; this produced stable molybdenum blue species [7].
However, because of the boiling step, obviously the
method is of little value for estimation of phosphate in

0003-2697/$ - see front matter 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2003.08.024
 ARTICLE IN PRESS
S.D. Katewa, S.S. Katyare / Analytical Biochemistry 323 (2003) 180187
enzyme assays especially if the substrate contains acid-
labile phosphorus, e.g., ATP. Hurst [5] tried to over-
come the problem by using a mixture of hydrazine sul-
fate and stannous chloride as the reducing agents.
Under this condition the color developed maximally in
3 min and was stable up to 30 min or so. Also, the nature
of molybdenum blue species produced was dierent
from that obtained with the Fiske and Subba Row or
Bartlett procedures [4,5,7]. The intensity of molybde-
num blue with the Fiske and Subba Row procedure can
be monitored over a wavelength range of 600700 nm
[4]. The molybdenum blue produced with the Bartlett
method showed absorption maximum at 830 nm
whereas the molybdenum blue produced with the Hurst
method showed an absorption maximum at 700 nm
[5,7]. Thus, obviously depending on the procedure em-
ployed, molybdenum blue species of variable absorption
characteristics and stability seems to be produced [4,5,7].
In the light of the above it was felt that it would be
desirable for a sensitive assay procedure which produced a
stable molybdenum blue color and at the same time ob-
viated the boiling step as in the case of the Bartlett pro-
cedure [5] to be devised so that the method can be utilized
for analysis of phosphate released in enzymatic assays and
of the phosphate released by chemical reactions; elimi-
nation of the boiling step would provide a time-saving and
less-cumbersome device for the color development.
From a literature survey it became apparent that the
stability of the reducing agents is of primary importance.
Ascorbic acid and hydrazine sulfate are stable at room
temperature. Hence we decided to use a combination of
these two reagents in dierent proportions to optimize
the conditions for molybdenum blue color development
and to get a stabilized color complex. The details of these
experiments for developing a sensitive method for
phosphorous estimation with an extended range of more
than 4 lg [7] and its application to estimation of phos-
phate released in enzymatic reactions, which use phos-
phate ester substrates containing acid-labile and acid-
stable phosphorus, e.g., ATP and G6P, respectively, are
described below. The method can be used most conve-
niently for inorganic phosphate determination without
the necessity of boiling of the samples for color devel-
opment, as is necessary with the Bartlett procedure [7].
The details of the same are also given below.
Materials and methods
Chemicals
Glucose 6-phosphate (G6P)2 and bovine serum al-
bumin fraction V (BSA) were purchased from Sigma
2
Abbreviations used: G6P, glucose-6-phosphate; BSA, bovine
serum albumin; G6Pase, glucose-6-phosphatase.
181
Chemical Co., USA. Sodium salt of ATP was obtained
from SRL, India. Silica gel G was from E. Merck,
Germany. Hydrazine sulfate and L -ascorbic acid were
purchased from Merck, India. All other chemicals were
purchased locally and were of analyticalreagent grade.
Reducing agents
A mixture of hydrazine sulfate and ascorbic acid in
varying proportions was prepared in 1.0 N H2 SO4 [5]
and 0.4 ml of the solution was used as reducing agent for
the development of the color. The contribution to the
nal normality of H2 SO4 in the 4.0 ml assay system by
the reducing reagents solution is 0.1 N. Under standard
assay conditions the reducing agent contained 20 mg
each of hydrazine sulfate and ascorbic acid in 1.0 ml of
0.1 N H2 SO4 .
Assay procedure
Aliquots of solution containing 110 lg of Pi were
taken and the volume was made up to 2.4 ml with dis-
tilled water. To this 0.8 ml of 3 N H2 SO4 was added
followed by 0.4 ml of 2.5% (w/v) ammonium molybdate
(prepared in 3 N H2 SO4 ). The contents were mixed
thoroughly and reduction of phosphomolybdic acid was
eected by adding 0.4 ml of reducing reagent solution.
Thus in the nal assay procedure the concentration of
H2 SO4 was 1.0 N.
The variations in the assay procedure for optimiza-
tion of the color development conditions with respect to
normality of H2 SO4 and the composition of reducing
agent mixture are detailed in the individual experiments.
In separate experiments the time course of color
development was determined under optimized assay
conditions.
Enzyme assays
The applicability of the procedure for the determi-
nation of phosphohydrolases activities was ascertained
using ATP and G6P as the substrates, respectively, for
Na ,K ATPase and glucose-6-phosphatase (G6Pase)
assays. Rat liver microsomes were used as the source of
the enzyme [8,9].
In separate experiments the susceptibility of ATP and
G6P to acid hydrolysis was assessed for up to 24 h; two
dierent concentrations of ATP and G6P were used in
these experiments (e.g., for details see Fig. 5).
Measurement of Na ,K ATPase activity was car-
ried out in a nal assay system (total volume 0.1 ml)
containing 50 mM TrisHCl buer, pH 7.4, 120 mM
NaCl, 10 mM KCl, and 5 mM MgCl2 [8,10]. For sub-
strate kinetics studies the concentration of ATP was
varied from 0.1 to 5 mM. After preincubating the en-
zyme (4050 lg of microsomal protein) for 23 min at
 ARTICLE IN PRESS
182 S.D. Katewa, S.S. Katyare / Analytical Biochemistry 323 (2003) 180187
37 C, the reaction was initiated by the addition of ATP
and allowed to proceed for 10 min. At the end of the
incubation period, the reaction was terminated by the
addition of 50 ll of 5% (w/v) sodium dodecyl sulfate
(SDS) solution. The liberated inorganic phosphate was
estimated by the method described above. Thus after
terminating the reaction, the volume was brought up to
2.4 ml with distilled water. This was followed by the
addition of 0.8 ml of 3 N H2 SO4 and 0.4 ml of 2.5% (w/v)
ammonium molybdate prepared in 3 N H2 SO4 . After
thoroughly mixing the contents 0.4 ml of the reducing
reagent solution was added. The optical density mea-
surements were carried out at the end of 30 min of color
development, where the extent of acid hydrolysis of the
substrate was minimum (Fig. 5) and about 80% of the
optimum color development occurred (Figs. 1C and F).
The corresponding slope (Figs. 1C and F) was used for
calculating the specic activities.
In a separate set of experiments the Na ,K ATPase
activity was determined by following conventional pro-
cedure routinely used in our laboratory [9] where the
assay volume was 0.4 ml. After terminating the reaction,
the entire sample was used for the phosphate estimation
according to the method of Fiske and Subba Row [4].
Measurement of G6Pase activity was carried out in
the assay system (total volume 0.1 ml) comprising
Fig. 1. Eect of varying the concentrations of hydrazine sulfate and
ascorbic acid on the course of color development. In the 4.0-ml assay
system the Pi concentrations were 5 lg (AC) and 10 lg (DF) and the
nal concentrations of H2 SO4 and ammonium molybdate were 1.0 N
and 0.25%, respectively. In A and D, the concentrations of hydrazine
sulfate and ascorbic acid were 10 and 3 mg, respectively, per milliliters
of 1.0 N H2 SO4 (j) and 20 and 3 mg, respectively per milliliter of
1.0 N H2 SO4 (D). In B and E the concentrations of hydrazine
sulfate and ascorbic acid were 10 mg each per milliliter of 1.0 N H2 SO4
(j) and 20 and 10 mg respectively, per milliliter of 1.0 N H2 SO4 (
D). In C and F the concentrations of hydrazine sulfate and ascorbic
acid were 20 mg each per milliliter of 1.0 N H2 SO4 (j). The optical
density was measured at 820 nm at various time intervals as indicated.
100 mM sodium acetate, 5 mM G6P, and ca. 5060 lg of
microsomal protein [9,11]. For substrate kinetics studies
the concentration of G6P was varied from 0.1 to 20 mM.
After preincubating the enzyme for 23 min at 37 C, the
reaction was initiated by the addition of the substrate
and was allowed to proceed for 10 min after which the
reaction was terminated by adding 50 ll of 5% (w/v)
SDS solution. After terminating the reaction, the vol-
ume was brought up to 2.4 ml with distilled water. The
rest of the procedure followed for the estimation of
liberated inorganic phosphate by employing the opti-
mized assay conditions described above for the mea-
surement of Na ,K ATPase activity. The optical
density measurements were carried out at the end of 1 h.
The corresponding slope (Figs. 1C and F) was used for
calculating the specic activities.
In separate set of experiments the G6Pase activity
was also determined by following conventional proce-
dure routinely used in our laboratory where the assay
volume is 0.3 ml [11]. After terminating the reaction, the
entire sample was used for the phosphate estimation
according to the method of Fiske and Subba Row [4].
In the conventional procedures in which the assay
volume was 34 times higher than that with the micro-
methods (e.g., vide supra), the inputs of components
including the substrate and the enzyme were increased
proportionately.
For the determination of Km and Vmax values the data
on substrate kinetics were analyzed by the Lineweaver
Burk, EadieHofstee and Eisenthal and CornishBow-
den methods [12]. The values of Km and Vmax obtained
by the three methods were in close agreement and were
averaged for the nal presentation of the data.
All the kinetics data were computer analyzed em-
ploying Sigma plot version 5.0 [10,13].
Inorganic phosphate determination
The suitability of the present procedure as a sub-
stitute for the Bartlett method was ascertained by car-
rying out analysis of mitochondrial/microsomal
phospholipids. Extraction of total phospholipids using a
freshly prepared chloroform:methanol mixture (2:1 v/v)
was essentially as described previously [9,10,1315].
Separation of phospholipid classes by thin-layer chro-
matography (TLC) was according to the procedure of
Skipsky et al. [16]. Suitable aliquots of total phospho-
lipids or individual phospholipid spots from TLC plates
were subjected to digestion with H2 SO4 and complete
oxidation was insured by treatment with perchloric acid
as described in detail previously [15]. Each sample was
processed in duplicate.
In those samples where phosphorous estimation was
carried out by the Bartlett method digestion was eected
using 0.5 ml of 10 N H2 SO4 and development of color
was by following the Bartlett procedure [7].
 ARTICLE IN PRESS
S.D. Katewa, S.S. Katyare / Analytical Biochemistry 323 (2003) 180187
In a second set of experiments where the present
procedure was used for phosphorous determination,
0.3 ml of 10 N H2 SO4 was used for sample digestion.
This precaution is necessary to maintain the normality
of H2 SO4 in the nal color development assay at 1.0 N.
After completion of digestion of the samples, the volume
was brought up to 2.4 ml with distilled water. The rest of
the procedure for color development followed the opti-
mized assay conditions described above.
Protein estimation was by the method of Lowry et al.
[17] with BSA used as the standard.
Results and discussion
In the rst set of experiments the eects of varying
proportions of hydrazine sulfate and ascorbic acid on
the time course of color development were evaluated
using 5- and 10-lg Pi samples. The results are shown in
Fig. 1. From Figs. 1A and D it can be noted that the
addition of reducing reagent solution containing hy-
drazine sulfate and ascorbic acid at the concentrations
of 10 and 3 mg, respectively, per milliliter of 1.0 N
H2 SO4 or 20 and 3 mg, respectively, per milliliter of
1.0 N H2 SO4 resulted in a slow rate of color develop-
ment. Thus by 1 h only about 50% of the color devel-
opment occurred; at the end of 2 h the color
development reached 7580% and optimum color de-
velopment was seen at 24 h. In the next set (Figs. 1B and
E) the concentration of hydrazine sulfate was the same
as that in A and D i.e., 20 mg/ml of 1.0 N H2 SO4 but the
concentration of ascorbic acid was raised to 10 mg/ml of
1.0 N H2 SO4 . With this combination the color devel-
oped more rapidly. Thus at the end of 1 h the color
development reached about 6065% of the optimum
value. At the end of 2 h 8590% of the optimum color
development occurred. Finally (Figs. 1C and F) hydra-
zine sulfate and ascorbic acid were used at the concen-
tration of 20 mg each/ml of 1.0 N H2 SO4 and, as is
evident, the color development was very rapid; within
10 min 5565% of the optimum color developed. By
30 min 7580% of the optimum color development had
occurred and at the end of 1 h 95% of the optimum color
had developed. Keeping this advantage of rapid color
development with the last combination in mind, in all
the experiments performed subsequently, the concen-
trations of hydrazine sulfate and ascorbic acid were kept
at 20 mg each/ml of 1 N H2 SO4 .
Bartlett [7] has shown that normality of H2 SO4 is
important for color development in both the Fiske and
Subba Row and the Bartlett procedures. Hurst [5] has
shown that in his assay procedure the color was stable
over the range of 0.81.1 N H2 SO4 . We therefore as-
certained the eect of the H2 SO4 concentration on the
color development. These data are shown in Fig. 2. As
can be noted there was hardly any color development in
183
Fig. 2. Eect of sulfuric acid concentration on the color development.
In the 4.0-ml assay system containing 5 lg Pi, the concentration of
H2 SO4 was varied as indicated from 0.4 to 1.0 N. Final concentration
of ammonium molybdate was 0.25% while concentrations of hydrazine
sulfate and ascorbic acid were the same as indicated for Figs. 1C and F
i.e., 20 mg each per milliliter of 1.0 N H2 SO4 .
0.4 N H2 SO4 . In fact, occasionally the blank gave higher
color density than the sample tubes. In 0.5 N H2 SO4 the
color development was only about 20% of the optimum
value, whereas over the range of 0.61.0 N H2 SO4 , al-
most a plateau region was seen.
We then ascertained the nature of molybdenum blue
formed in terms of its absorption characteristics. Si-
multaneously, for comparison, the spectra of molybde-
num blue formed using the Fiske and Subba Row and
the Bartlett methods [4,7] were also recorded using the
same concentration of Pi. These spectra are shown in
Fig. 3. As is evident the molybdenum blue formed using
the Fiske and Subba Row method (Fig. 3A) showed no
well dened peak, whereas the molybdenum blue
formed using the Bartlett (Fig. 3B) and the present
procedures (Fig. 3C) showed absorption maxima at
820 nm. Also the peak height was higher with the pres-
ent procedure than that with the Bartlett method.
It is of interest to note here that the molybdenum blue
species formed using the Fiske and Subba Row method
showed ill-dened absorption characteristics (Fig. 3A).
Optimum color development occurred by 910 min,
beyond which the color was unstable [4]. The molyb-
denum blue species formed using the Hurst procedure,
which absorbs at 700 nm, is stable up to 30 min [5],
whereas the molybdenum blue species produced by the
Bartlett procedure [7] and by our procedure, which ab-
sorb at 820 nm, were stable up to 24 h (e.g., see Fig. 1).
Taken together, the results suggest that the molybde-
num blue species with absorption maximum at 820 nm
represents the most stable species.
The standard curve for phosphorous estimation ob-
tained under optimized conditions by following the
present procedure is shown in Fig. 4. As can be noted,
not only is the color development linear with the
 ARTICLE IN PRESS
184 S.D. Katewa, S.S. Katyare / Analytical Biochemistry 323 (2003) 180187
Fig. 3. Spectra of molybdenum blue produced by dierent assay pro-
cedures. In all three methods, the nal volume for color development
was 4.0 ml and the concentration of Pi was 5 lg. Procedures for color
development as detailed under Materials and methods were followed.
The spectra were recorded in a Shimadzu Model UV-160A UV/VIS
spectrophotometer over the wavelength span of 500 to 900 nm. The
optical density measurements were carried out at the end of 10 min for
the Fiske and Subba Row method (A) and at the end of 24 h for the
Bartlett (B) and the present (C) methods.
Fig. 4. Standard curve for Pi determination by the present procedure.
In a 4.0-ml assay system the concentration of Pi was varied from 1 to
10 lg. Other experimental conditions were the same as described for
Figs. 1C and F. The measurement of optical density was carried out at
820 nm at the end of 24 h.
Table 1
Comparison of the sensitivity of the three procedures for the determination of phosphorous by the molybdenum blue method
Method Reducing agent
Fiske and Subba Row 1 Amino-2-naphthol-4-sulfonic acid
Bartlett 1 Amino-2-naphthol-4-sulfonic acid
Present Hydrazine sulfate and ascorbic acid
concentration of Pi but also that the range for estima-
tion is extended to 10 lg of Pi, which is 2.5 times higher
than that with the Bartlett procedure [7].
A comparison of the molar extinction coecients of
molybdenum blue produced by dierent procedures
(e.g., see Fig. 3) at 700 and 820 nm is shown in Table 1.
As can be noted, using the Fiske and Subba Row pro-
cedure, the values of the molar extinction coecient at
both wavelengths were the lowest and almost compa-
rable. As against this, the Bartlett and the present pro-
cedures produced signicantly higher values even at
700 nm which increased more than twofolds at 820 nm.
Also, the molar extinction coecient values obtained
with the present procedure were higher than those with
other two procedures, signifying improved sensitivity.
The applicability of the present procedure for enzy-
matic analysis was then evaluated. Prior to performing
the enzyme assays we ascertained the extent of hydro-
lysis of the two substrates namely ATP and G6P for
over 24 h under conditions simulating the phosphorus
assay procedure. The two substrates at two concentra-
tions (2 and 5 mM ATP and 2 and 20 mM G6P, nal
concentration) were incubated under phosphorous assay
conditions where the nal concentration of H2 SO4 was
1.0 N and the release of inorganic phosphate was mon-
itored over 24 h. The results are presented as percentage
hydrolysis of the substrate (Fig. 5). As can be seen, G6P
was completely stable, whereas progressive ATP hy-
drolysis was seen up to 2 h after which a steady-state
value was reached. Thus the extent of acid hydrolysis of
ATP at 10, 20, 30, and 60 min was 2, 3, 4.5, and 7% after
which the value reached a constant level of 12.5%.
Since it was noted earlier that under optimized con-
ditions about 80% of the optimum color development
occurred within 30 min (Figs. 1C and F) it was decided
to measure the ATPase activity by monitoring the color
developed at the end of 30 min. The rationale for se-
lecting this time interval was that 4.5% hydrolysis of
ATP would not add substantially to the background
above which the Pi released by enzymatic hydrolysis can
be easily picked up. Additionally, the microassay pro-
cedure employed would substantially reduce the sub-
strate blank.
In the preliminary studies, the activities of the
Na ,K ATPase and G6Pase by following the conven-
tional and the modied micromethod (as described
above) were compared. Thus, the activity of Na , K
Heating period Molar extinction coecient
700 nm 820 nm
None 3645 4650
7 min 8903 21452
None 11036 26139
 ARTICLE IN PRESS
S.D. Katewa, S.S. Katyare / Analytical Biochemistry 323 (2003) 180187 185

A B

Fig. 5. Time courses of acid hydrolysis of ATP (A) and G6P (B). ATP at the nal concentrations of 2 mM (j) and 5 mM (m) and G6P at the
nal concentrations of 2 mM (j) and 20 mM (m) were incubated with sulfuric acid under the Pi assay condition. The concentration of H2 SO4
was 1.0 N. Release of inorganic phosphate was monitored at dierent time intervals up to 24 h as indicated.

ATPase expressed as l mol of Pi/h/mg protein was
8.91  0.57 and 9.18  1.01, respectively, by the two
procedures. Similarly, the activity of G6Pase expressed
in the same units was 7.01  0.18 and 6.87  0.50 by the
two procedures. Having ascertained that identical re-
sults are obtained by both the conventional method and
the micromethod, in the next series of experiments the
kinetic properties of the two enzymes were examined.
The substrate saturation curves and Lineweaver
Burk, EadieHofstee, and Eisenthal and Cornish
Bowden plots for Na ,K ATPase by the conventional
procedure and by the present method were comparable
and identical (data not shown) and the enzyme activity
resolved in two components consistent with our earlier
observations [9]. The computed data on Km and Vmax
values of the two components of Na ,K ATPase, de-
rived from the analysis of the LineweaverBurk, Eadie
Hofstee and Eisenthal and CornishBowden plots are
given in Table 2. As can be noted the results by the two
methods were identical. Also, the values of Km and Vmax
by the two methods were consistent with previously re-
ported observations [9]. As discussed earlier, the two
components represent the modulation of the enzyme
activity in response to changes in the substrate concen-
tration [9].
The substrate saturation curves and the correspond-
ing LineweaverBurk, EadieHofstee and Eisenthal and
CornishBowden plots for G6Pase by the conventional
method and the micromethod were comparable (data
not shown). The corresponding Km and Vmax values
computed as described above are given in Table 2. Once
again the results by the two methods were identical.
The applicability of the present method for
phospholipid analysis was also checked. As can be noted
from the data in Table 3 the values for mitochondrial
and microsomal total phospholipids by the two methods
were in excellent agreement as were the data for mito-
chondrial phospholipid composition (Table 4).
Table 3
Total phospholipid (TPL) content of rat liver mitochondria and mi-
crosomes
TPL content (lg /mg protein)
Assay procedure Mitochondria Microsomes
Bartlett 177.3  6.50 424.5  31.41
Present 185.4  8.11 415.6  21.04
Experimental details are as given in the text. The results are given
as mean  SE of six independent observations.

Table 2
Kinetic attributes of the rat liver microsomal Na ,K ATPase and glucose-6-phosphatase
Component I Component II
Enzyme Assay procedure Km Vmax Km Vmax

Na ,K ATPase Conventional 0.94  0.08 5.90  0.34 5.46  0.37 14.62  0.57
Present 0.93  0.09 5.42  0.67 5.65  0.47 14.69  0.96
G6Pase Conventional 0.23  0.01 4.64  0.21 0.85  0.15 12.10  0.89
Present 0.26  0.02 5.08  0.49 0.93  0.14 12.23  0.67
Experimental details are as given in the text. The Km ( in mM) and Vmax (expressed as lmol of Pi liberated/h/mg protein) values were calculated by
three dierent methods of analysis as described in the text using Sigma Plot version 5.0 and were averaged. The results are given as mean  SE of six
independent observations.
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186 S.D. Katewa, S.S. Katyare / Analytical Biochemistry 323 (2003) 180187
Table 4
Phospholipid composition of rat liver mitochondria
Phospholipid class Phospholipid composition (% of total)
Bartlett procedure Present procedure
Lyso 4.45  0.52 4.51  0.61
SPM 5.42  0.33 4.41  0.58
PC 42.13  1.99 43.95  2.50
PI 3.15  0.64 3.35  0.43
PS 3.06  0.54 2.91  0.43
PE 28.58  1.31 27.01  0.98
DPG 13.25  1.03 13.99  0.90
Experimental details are as given in the text. The results are given
as mean  SE of six independent observations. DPG, diphosphat-
idylglycerol; Lyso, lysophospholipid; PC, phosphatidylcholine; PE,
phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphati-
dylserine; SPM, sphingomyelin.
Thus the method that we have developed seems to be
more sensitive (e.g., see Table 1) with extended concen-
tration range for Pi analysis as compared to that of the
Bartlett method [7] In addition to the enhanced sensi-
tivity, the method obviates the need for boiling the
sample for color development when one is carrying out
inorganic phosphate analysis, as in the case of
phospholipid determinations [7]. The present procedure
also produced a stable molybdenum blue color species.
The color development followed a rapid course and
about 95% of the optimum color development was seen
within 1 h. Even under this condition the extent of hy-
drolysis of acid-labile phosphate was negligible (Fig. 5).
Nevertheless, for application to phosphohydrolase ac-
tivity using substrates with acid-labile phosphate, 30 min
time seems to be a safe period for monitoring color de-
velopment (Figs. 1 and 5). We also found that the color
was stable up to 24 h which gives exibility in making
optical density measurements in assays such as G6Pase
without following a xed time limit as is required in the
case of the Fiske and Subba Row procedure [4]. An ad-
ditional advantage is that the volume of enzyme assay is
reduced by a factor of 34, thereby reducing the input of
materials and sample. Thus the method becomes not
only more sensitive but also economical.
Although the present procedure has been standard-
ized for a nal assay volume of 4 ml, in principle it should
be possible to cut down the sample volume to 1.0 ml,
thereby increasing the sensitivity further by 4 times, and
the range of estimation will become 0.252.5 lg.
Lanzetta et al. [6] described a method for estimation
of nanomole quantities of inorganic phosphate. The
problem of color development with time in reagent
blank and in samples containing ATP was solved by
using citrate buer. Under these conditions, the color
developed optimally in 30 min and was stable for 4 h.
Malachite green and Sterox were the special reagents
required for color development [6].
As against this, in our present procedure, the blanks
were stable up to 24 h and thus no buering was
necessary for suppressing the blank. Also under the
simulated conditions of color development, the extent of
hydrolysis of ATP at 30-min and 1 h intervals was
negligible. In addition, no special chemicals or reagents
were required [6] for color development; hydrazine sul-
fate and ascorbic acid are bench chemicals routinely
available in the laboratory.
The additional advantage of the present method is
that the reducing agents hydrazine sulfate and ascorbic
acid are stable at room temperature as is sulfuric acid.
This obviates the need for preparing the reducing re-
agent mixture in large quantity and taking care for its
storage. Routinely, the required amounts of hydrazine
sulfate and ascorbic acid can be weighed and mixed with
1 N H2 SO4 as and when the need arises and reagent can
be prepared fresh. Also depending on the requirements
of the particular enzyme assay, the optical density
readings can be taken at convenient intervals and the
corresponding slope can be used for nal calculations of
the data as pointed out above.
Thus, the method described presents a versatile pro-
cedure for the estimation of inorganic phosphorous re-
leased in enzymatic reactions using acid-labile and acid-
stable substrates. Since the boiling step is eliminated, the
method also becomes less cumbersome for quantica-
tion of inorganic phosphorous released in chemical re-
actions as in the case of phospholipid analysis [7].
In conclusion it is felt that the method described here
is a convenient procedure for inorganic phosphate de-
termination on a routine day to day basis and allows the
handling of a large number of samples simultaneously.
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