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A microtiter plate assay for inorganic phosphate
Journal of Biochemical and Biophysical Methods, 19 (1989) 249-252
249
Elsevier JBBM 00760
Short Note
A microtiter plate assay for inorganic phosphate Margarethe Hoenig, R. John Lee and Duncan C. Ferguson Department of Physiology and Pharmacology, Collegeof Veterinary Medicine, The University of Georgia, Athens, GA 30602, U.S.A.
(Received 5 December 1988) (Accepted 2 April 1989)
Summary A microtiter assay for the detection of picomolar quantities of inorganic phosphate has been described. The assay, linear between 50 and 1000 pmol of inorganic phosphate, is simple and rapid, with results obtainable in several minutes. Results from 5'-nucleotidase and (Ca 2+ +Mg 2+ )ATPase assays using this method were compared with conventional phosphate assays and showed a high degree of correlation. The high sensitivity of this assay and the small sample size needed allows its widespread use in biochemical studies involving the generation of inorganic phosphate. Key words: Inorganic analysis; Phosphate assay; Phosphatase; Phosphate compound; Enzyme mechanism
The detection of s u b n a n o m o l a r quantities of inorganic p h o s p h a t e is i m p o r t a n t in the measurement of phosphatases in subcellular preparations where the q u a n t i t y of protein is a limiting factor or where it is desirable to avoid the use of 32p labelled substrate compounds. This report describes a simple and rapid microassay for inorganic phosphate measurement which provides high sensitivity compatible to that of assays using radiolabelled c o m p o u n d s without involving the hazard of such compounds. The microtiter plate assay was a modification of the m e t h o d described b y P e n n e y [1]. Reactions were carried out on Microtest I I I assay plates (96 U - b o t t o m wells, Becton Dickinson a n d C o m p a n y , Oxnard, CA). Five to twenty/~1 of sample were placed into the well and distilled water was added to give a final volume of 70 ~1. Blanks contained 70 /~1 of distilled water. Sodium m o l y b d a t e (2.6 g / 1 0 0 ml) was
Correspondence address: M. Hoenig, Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, U.S.A. This study was supported in part by PHS Grant R23DK39185 (MH) and R23DK38814 (DCF).
0165-022X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
250 0.3
O.2
E3 O 0.1
0.0
0.0
0.=
0.,
01e
01,
110
1.
nmol inorganic phospate
Fig. 1. The standard curve for Pi determination.KH 2PO4 was used as the phosphate standard.
mixed with an equal volume of 2.5 N HC1 prior to analysis. The Malachite green concentration was 0.126 g/100 ml. Malachite green was purchased from Fisher Scientific, Pittsburgh, PA, color index No. 4200. Malachite green was added to the mixture of sodium molybdate (Sigma, St. Louis, MO) and HCI in a ratio of 1" 36. One hundred and eighty/~1 of this solution were then added to the well. After 120 s, absorption readings were made with a BIO-TEK model EL308 microplate reader (Burlington, VT) using a 620 nm filter. Usually 18 wells were analyzed at one time in 5 s intervals. Results from this assay using a 5-20/~1 sample were compared to the nonisotopic phosphate assay of 500 #1 by the Fiske-Subbarow method [2] of membranal 5' nucleotide activity [3] and for a radioisotopic measurement of (Ca 2÷ + Mg 2÷)ATPase
[41. The major advantage of the described assay is the extreme sensitivity, which allows detection of inorganic phosphate in the subnanomolar range. As is shown in Fig. 1 the analysis of phosphate standards was linear between 0.05 and I nmol inorganic phosphate. This allows the use of this analysis for the detection of enzyme activity in subcellular fractions for which the small amounts of protein are often a limiting factor. We compared results of the assay using malachite green with the Fiske-Subbarow method for the detection of inorganic P liberation in a membrane preparation from an insulinoma [5]. When the plasma membrane marker 5'nucleotidase was measured with the Fiske-Subbarow method [2] and compared to this assay, very similar results were obtained. Table 1 shows the concentrations of 5'-nucleotidase in homogenate, a crude membrane fraction and a purified plasma membrane from the insulinoma, measured with both assays simultaneously in 6 different tumor preparations. Results measuring the activity of (Ca2+ + MgE÷)ATPase in a purified plasma membrane fraction from an insulinoma were also similar. When the enzyme activity was measured in an assay using [V-32p]ATP it was 78.8-_t-_10.2 n m o l / m g / m i n (n = 5), while it was 69.5 _ 15.3 n m o l / m g / m i n in the microassay. The assay has the
251 TABLE 1 COMPARISON OF 5'-NUCLEOTIDE ACTIVITIES (nmol/mg/min, _x-4-SEM, n = 6) IN AN INSULINOMA PREPARATION USING A CONVENTIONAL ASSAY METHOD (A) AND THE MICROTITER PLATE ASSAY (B) Fraction
A [2]
B
Homogenate Crude membrane Plasma membrane
62.3-4- 6.2 124.5 + 19.2 400.9 + 45.8
80.3 -4-17.6 a 132.4_ 26.2 b 365.9 + 39.6 c
Correlation coefficient R: ~ 0.974; b 0.927; c 0.915
l i m i t a t i o n that c o n c e n t r a t i o n s of A T P > 2 m M interfere with the m e a s u r e m e n t of i n o r g a n i c p h o s p h a t e liberation. I n summary, a microtiter plate assay for the d e t e r m i n a t i o n of i n o r g a n i c phosp h a t e is described which is r a p i d a n d highly sensitive. Because the s a m p l e size n e e d e d is small a n d the assay has great sensitivity, it c a n b e applied i n b i o c h e m i c a l studies which thus far h a d to rely o n m e t h o d s u s i n g r a d i o l a b e l e d c o m p o u n d s .
References 1 Penney, C.L. (1976) A simple micro-assay for inorganic phosphate. Anal. Biochem. 75, 201-210. 2 Hwang, K.J. (1976) Interference of ATP and acidity in the determination of inorganic phosphate by the Fiske and Subbarow method. Anal. Biochem. 75, 40-44. 3 Michel, R.H. and Hawthorne, J.M. (1965) The site of diphosphoinositide synthesis in rat liver. Biochem. Biophys. Res. Comm. 21, 333-338. 4 Kotagal, N., Colca, J.R., Buscetto, D. and McDaniel, M.L. (1985) Effect of insulin secretagogues and potential modulators of secretion on a plasma membrane (Ca2+ + Mg 2+ )-ATPase activity in islets of Langerhas. Arch. Biochem. Biophys. 238, 161-169. 5 Hoenig, M., Ferguson, D.C. and Matschinsky, F.M. (1984) Fuel induced insulin release in vitro from insulinomas transplanted into the rat kidneys. Diabetes 33, 1-7.
249
Elsevier JBBM 00760
Short Note
A microtiter plate assay for inorganic phosphate Margarethe Hoenig, R. John Lee and Duncan C. Ferguson Department of Physiology and Pharmacology, Collegeof Veterinary Medicine, The University of Georgia, Athens, GA 30602, U.S.A.
(Received 5 December 1988) (Accepted 2 April 1989)
Summary A microtiter assay for the detection of picomolar quantities of inorganic phosphate has been described. The assay, linear between 50 and 1000 pmol of inorganic phosphate, is simple and rapid, with results obtainable in several minutes. Results from 5'-nucleotidase and (Ca 2+ +Mg 2+ )ATPase assays using this method were compared with conventional phosphate assays and showed a high degree of correlation. The high sensitivity of this assay and the small sample size needed allows its widespread use in biochemical studies involving the generation of inorganic phosphate. Key words: Inorganic analysis; Phosphate assay; Phosphatase; Phosphate compound; Enzyme mechanism
The detection of s u b n a n o m o l a r quantities of inorganic p h o s p h a t e is i m p o r t a n t in the measurement of phosphatases in subcellular preparations where the q u a n t i t y of protein is a limiting factor or where it is desirable to avoid the use of 32p labelled substrate compounds. This report describes a simple and rapid microassay for inorganic phosphate measurement which provides high sensitivity compatible to that of assays using radiolabelled c o m p o u n d s without involving the hazard of such compounds. The microtiter plate assay was a modification of the m e t h o d described b y P e n n e y [1]. Reactions were carried out on Microtest I I I assay plates (96 U - b o t t o m wells, Becton Dickinson a n d C o m p a n y , Oxnard, CA). Five to twenty/~1 of sample were placed into the well and distilled water was added to give a final volume of 70 ~1. Blanks contained 70 /~1 of distilled water. Sodium m o l y b d a t e (2.6 g / 1 0 0 ml) was
Correspondence address: M. Hoenig, Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, U.S.A. This study was supported in part by PHS Grant R23DK39185 (MH) and R23DK38814 (DCF).
0165-022X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
250 0.3
O.2
E3 O 0.1
0.0
0.0
0.=
0.,
01e
01,
110
1.
nmol inorganic phospate
Fig. 1. The standard curve for Pi determination.KH 2PO4 was used as the phosphate standard.
mixed with an equal volume of 2.5 N HC1 prior to analysis. The Malachite green concentration was 0.126 g/100 ml. Malachite green was purchased from Fisher Scientific, Pittsburgh, PA, color index No. 4200. Malachite green was added to the mixture of sodium molybdate (Sigma, St. Louis, MO) and HCI in a ratio of 1" 36. One hundred and eighty/~1 of this solution were then added to the well. After 120 s, absorption readings were made with a BIO-TEK model EL308 microplate reader (Burlington, VT) using a 620 nm filter. Usually 18 wells were analyzed at one time in 5 s intervals. Results from this assay using a 5-20/~1 sample were compared to the nonisotopic phosphate assay of 500 #1 by the Fiske-Subbarow method [2] of membranal 5' nucleotide activity [3] and for a radioisotopic measurement of (Ca 2÷ + Mg 2÷)ATPase
[41. The major advantage of the described assay is the extreme sensitivity, which allows detection of inorganic phosphate in the subnanomolar range. As is shown in Fig. 1 the analysis of phosphate standards was linear between 0.05 and I nmol inorganic phosphate. This allows the use of this analysis for the detection of enzyme activity in subcellular fractions for which the small amounts of protein are often a limiting factor. We compared results of the assay using malachite green with the Fiske-Subbarow method for the detection of inorganic P liberation in a membrane preparation from an insulinoma [5]. When the plasma membrane marker 5'nucleotidase was measured with the Fiske-Subbarow method [2] and compared to this assay, very similar results were obtained. Table 1 shows the concentrations of 5'-nucleotidase in homogenate, a crude membrane fraction and a purified plasma membrane from the insulinoma, measured with both assays simultaneously in 6 different tumor preparations. Results measuring the activity of (Ca2+ + MgE÷)ATPase in a purified plasma membrane fraction from an insulinoma were also similar. When the enzyme activity was measured in an assay using [V-32p]ATP it was 78.8-_t-_10.2 n m o l / m g / m i n (n = 5), while it was 69.5 _ 15.3 n m o l / m g / m i n in the microassay. The assay has the
251 TABLE 1 COMPARISON OF 5'-NUCLEOTIDE ACTIVITIES (nmol/mg/min, _x-4-SEM, n = 6) IN AN INSULINOMA PREPARATION USING A CONVENTIONAL ASSAY METHOD (A) AND THE MICROTITER PLATE ASSAY (B) Fraction
A [2]
B
Homogenate Crude membrane Plasma membrane
62.3-4- 6.2 124.5 + 19.2 400.9 + 45.8
80.3 -4-17.6 a 132.4_ 26.2 b 365.9 + 39.6 c
Correlation coefficient R: ~ 0.974; b 0.927; c 0.915
l i m i t a t i o n that c o n c e n t r a t i o n s of A T P > 2 m M interfere with the m e a s u r e m e n t of i n o r g a n i c p h o s p h a t e liberation. I n summary, a microtiter plate assay for the d e t e r m i n a t i o n of i n o r g a n i c phosp h a t e is described which is r a p i d a n d highly sensitive. Because the s a m p l e size n e e d e d is small a n d the assay has great sensitivity, it c a n b e applied i n b i o c h e m i c a l studies which thus far h a d to rely o n m e t h o d s u s i n g r a d i o l a b e l e d c o m p o u n d s .
References 1 Penney, C.L. (1976) A simple micro-assay for inorganic phosphate. Anal. Biochem. 75, 201-210. 2 Hwang, K.J. (1976) Interference of ATP and acidity in the determination of inorganic phosphate by the Fiske and Subbarow method. Anal. Biochem. 75, 40-44. 3 Michel, R.H. and Hawthorne, J.M. (1965) The site of diphosphoinositide synthesis in rat liver. Biochem. Biophys. Res. Comm. 21, 333-338. 4 Kotagal, N., Colca, J.R., Buscetto, D. and McDaniel, M.L. (1985) Effect of insulin secretagogues and potential modulators of secretion on a plasma membrane (Ca2+ + Mg 2+ )-ATPase activity in islets of Langerhas. Arch. Biochem. Biophys. 238, 161-169. 5 Hoenig, M., Ferguson, D.C. and Matschinsky, F.M. (1984) Fuel induced insulin release in vitro from insulinomas transplanted into the rat kidneys. Diabetes 33, 1-7.