A simplified method for inorganic phosphate ...

ARTICLE IN PRESS ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 323 (2003) 180–187 www.elsevier.com/locate/yabio

A simplified 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 simplified 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 coefficient 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 Naþ ,Kþ ATPase was at the minimum. The applicability of the method for assay of microsomal Naþ ,Kþ ATPase and glucose-6-phosphatase was checked in microassays (final volume 0.1 ml) in comparison to the conventional procedures which use 3–4 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. Ó 2003 Elsevier Inc. All rights reserved.

Importance of phosphorous in intermediary metabolism 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 property of orthophosphate to form a complex with molybdic acid [3]. It was realized that phosphomolybdate could be reduced to produce a deep-blue-colored complex called molybdenum blue [3]. The first 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@buffalo.edu (S.D. Katewa). 1 Present address: Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14214, USA. 0003-2697/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2003.08.024

tion with sodium sulfite and sodium metabisulfite 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 5–10 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 colorimetric method for phosphate estimation was improved by Bartlett [7]; simultaneously the problem of color instability was also solved by this procedure [7]. The Bartlett method requires that the reduction to molybdenum 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

ARTICLE IN PRESS S.D. Katewa, S.S. Katyare / Analytical Biochemistry 323 (2003) 180–187

enzyme assays especially if the substrate contains acidlabile phosphorus, e.g., ATP. Hurst [5] tried to overcome the problem by using a mixture of hydrazine sulfate 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 different from that obtained with the Fiske and Subba Row or Bartlett procedures [4,5,7]. The intensity of molybdenum blue with the Fiske and Subba Row procedure can be monitored over a wavelength range of 600–700 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 employed, 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 obviated the boiling step as in the case of the Bartlett procedure [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; elimination 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 different 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 phosphate released in enzymatic reactions, which use phosphate ester substrates containing acid-labile and acidstable phosphorus, e.g., ATP and G6P, respectively, are described below. The method can be used most conveniently for inorganic phosphate determination without the necessity of boiling of the samples for color development, 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 albumin fraction V (BSA) were purchased from Sigma 2 Abbreviations used: G6P, glucose-6-phosphate; BSA, bovine serum albumin; G6Pase, glucose-6-phosphatase.

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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 analytical–reagent 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 final 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 1–10 lg of Pi were taken and the volume was made up to 2.4 ml with distilled 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 effected by adding 0.4 ml of reducing reagent solution. Thus in the final assay procedure the concentration of H2 SO4 was 1.0 N. The variations in the assay procedure for optimization 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 determination of phosphohydrolases activities was ascertained using ATP and G6P as the substrates, respectively, for Naþ ,Kþ 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 different concentrations of ATP and G6P were used in these experiments (e.g., for details see Fig. 5). Measurement of Naþ ,Kþ ATPase activity was carried out in a final assay system (total volume 0.1 ml) containing 50 mM Tris–HCl buffer, pH 7.4, 120 mM NaCl, 10 mM KCl, and 5 mM MgCl2 [8,10]. For substrate kinetics studies the concentration of ATP was varied from 0.1 to 5 mM. After preincubating the enzyme (40–50 lg of microsomal protein) for 2–3 min at

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S.D. Katewa, S.S. Katyare / Analytical Biochemistry 323 (2003) 180–187

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 measurements 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 specific activities. In a separate set of experiments the Naþ ,Kþ ATPase activity was determined by following conventional procedure 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

100 mM sodium acetate, 5 mM G6P, and ca. 50–60 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 2–3 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 volume 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 optimized assay conditions described above for the measurement of Naþ ,Kþ 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 specific activities. In separate set of experiments the G6Pase activity was also determined by following conventional procedure 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 3–4 times higher than that with the micromethods (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, Eadie–Hofstee and Eisenthal and Cornish–Bowden methods [12]. The values of Km and Vmax obtained by the three methods were in close agreement and were averaged for the final presentation of the data. All the kinetics data were computer analyzed employing Sigma plot version 5.0 [10,13]. Inorganic phosphate determination

Fig. 1. Effect 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 (A–C) and 10 lg (D–F) and the final 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.

The suitability of the present procedure as a substitute for the Bartlett method was ascertained by carrying 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 [9,10,13–15]. Separation of phospholipid classes by thin-layer chromatography (TLC) was according to the procedure of Skipsky et al. [16]. Suitable aliquots of total phospholipids 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 effected using 0.5 ml of 10 N H2 SO4 and development of color was by following the Bartlett procedure [7].


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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 final 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 optimized 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 first set of experiments the effects 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 hydrazine 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 development. Thus by 1 h only about 50% of the color development occurred; at the end of 2 h the color development reached 75–80% and optimum color development 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 developed more rapidly. Thus at the end of 1 h the color development reached about 60–65% of the optimum value. At the end of 2 h 85–90% of the optimum color development occurred. Finally (Figs. 1C and F) hydrazine sulfate and ascorbic acid were used at the concentration of 20 mg each/ml of 1.0 N H2 SO4 and, as is evident, the color development was very rapid; within 10 min 55–65% of the optimum color developed. By 30 min 75–80% 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 concentrations 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 0.8–1.1 N H2 SO4 . We therefore ascertained the effect 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

Fig. 2. Effect 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 0.6–1.0 N H2 SO4 , almost a plateau region was seen. We then ascertained the nature of molybdenum blue formed in terms of its absorption characteristics. Simultaneously, for comparison, the spectra of molybdenum 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 defined 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 present 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-defined absorption characteristics (Fig. 3A). Optimum color development occurred by 9–10 min, beyond which the color was unstable [4]. The molybdenum 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 absorb at 820 nm, were stable up to 24 h (e.g., see Fig. 1). Taken together, the results suggest that the molybdenum blue species with absorption maximum at 820 nm represents the most stable species. The standard curve for phosphorous estimation obtained 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



Fig. 3. Spectra of molybdenum blue produced by different assay procedures. In all three methods, the final 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.

concentration of Pi but also that the range for estimation 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 coefficients of molybdenum blue produced by different 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 procedure, the values of the molar extinction coefficient at both wavelengths were the lowest and almost comparable. As against this, the Bartlett and the present procedures produced significantly higher values even at 700 nm which increased more than twofolds at 820 nm. Also, the molar extinction coefficient values obtained with the present procedure were higher than those with other two procedures, signifying improved sensitivity. The applicability of the present procedure for enzymatic analysis was then evaluated. Prior to performing the enzyme assays we ascertained the extent of hydrolysis of the two substrates namely ATP and G6P for over 24 h under conditions simulating the phosphorus assay procedure. The two substrates at two concentrations (2 and 5 mM ATP and 2 and 20 mM G6P, final concentration) were incubated under phosphorous assay conditions where the final concentration of H2 SO4 was 1.0 N and the release of inorganic phosphate was monitored 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 hydrolysis 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 conditions 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 selecting 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 procedure employed would substantially reduce the substrate blank. In the preliminary studies, the activities of the Naþ ,Kþ ATPase and G6Pase by following the conventional and the modified micromethod (as described above) were compared. Thus, the activity of Naþ , Kþ

Table 1 Comparison of the sensitivity of the three procedures for the determination of phosphorous by the molybdenum blue method Method

Fiske and Subba Row Bartlett Present

Reducing agent

1 Amino-2-naphthol-4-sulfonic acid 1 Amino-2-naphthol-4-sulfonic acid Hydrazine sulfate and ascorbic acid

Heating period

None 7 min None

Molar extinction coefficient 700 nm

820 nm

3645 8903 11036

4650 21452 26139


A

185

B

Fig. 5. Time courses of acid hydrolysis of ATP (A) and G6P (B). ATP at the final concentrations of 2 mM (—j—) and 5 mM (—m—) and G6P at the final 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 different 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 results 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, Eadie–Hofstee, and Eisenthal and Cornish– Bowden plots for Naþ ,Kþ 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 Naþ ,Kþ ATPase, derived from the analysis of the Lineweaver–Burk, Eadie– Hofstee and Eisenthal and Cornish–Bowden 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 reported observations [9]. As discussed earlier, the two components represent the modulation of the enzyme activity in response to changes in the substrate concentration [9].

The substrate saturation curves and the corresponding Lineweaver–Burk, Eadie–Hofstee and Eisenthal and Cornish–Bowden 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 mitochondrial phospholipid composition (Table 4).

Table 3 Total phospholipid (TPL) content of rat liver mitochondria and microsomes TPL content (lg /mg protein) Assay procedure

Mitochondria

Microsomes

Bartlett Present

177.3 6.50 185.4 8.11

424.5 31.41 415.6 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 Naþ ,Kþ ATPase and glucose-6-phosphatase Component I Enzyme

Component II

Assay procedure

Km

Vmax

Km

Vmax

Na ,K ATPase

Conventional Present

0.94 0.08 0.93 0.09

5.90 0.34 5.42 0.67

5.46 0.37 5.65 0.47

14.62 0.57 14.69 0.96

G6Pase

Conventional Present

0.23 0.01 0.26 0.02

4.64 0.21 5.08 0.49

0.85 0.15 0.93 0.14

12.10 0.89 12.23 0.67

þ

þ

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 different 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.



Table 4 Phospholipid composition of rat liver mitochondria Phospholipid class

Lyso SPM PC PI PS PE DPG

Phospholipid composition (% of total) Bartlett procedure

Present procedure

4.45 0.52 5.42 0.33 42.13 1.99 3.15 0.64 3.06 0.54 28.58 1.31 13.25 1.03

4.51 0.61 4.41 0.58 43.95 2.50 3.35 0.43 2.91 0.43 27.01 0.98 13.99 0.90

Experimental details are as given in the text. The results are given as mean SE of six independent observations. DPG, diphosphatidylglycerol; Lyso, lysophospholipid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; SPM, sphingomyelin.

Thus the method that we have developed seems to be more sensitive (e.g., see Table 1) with extended concentration range for Pi analysis as compared to that of the Bartlett method [7] In addition to the enhanced sensitivity, 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 hydrolysis of acid-labile phosphate was negligible (Fig. 5). Nevertheless, for application to phosphohydrolase activity using substrates with acid-labile phosphate, 30 min time seems to be a safe period for monitoring color development (Figs. 1 and 5). We also found that the color was stable up to 24 h which gives flexibility in making optical density measurements in assays such as G6Pase without following a fixed time limit as is required in the case of the Fiske and Subba Row procedure [4]. An additional advantage is that the volume of enzyme assay is reduced by a factor of 3–4, 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 standardized for a final 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 0.25–2.5 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 buffer. 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 buffering 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 sulfate 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 reagent 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 final calculations of the data as pointed out above. Thus, the method described presents a versatile procedure for the estimation of inorganic phosphorous released in enzymatic reactions using acid-labile and acidstable substrates. Since the boiling step is eliminated, the method also becomes less cumbersome for quantification of inorganic phosphorous released in chemical reactions 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 determination on a routine day to day basis and allows the handling of a large number of samples simultaneously.

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