Wednesday, February 3, 2010

Quantitative Phosphate Determinations

Determination of Inorganic Phosphate
in Biologic Samples

A 1N perchloric acid Solutions/Reagents
B 5mM sodium molybdate (Na2MoO4·2H2O, Mr 241.95)
Important! Do not use ammonium molybdate!
C isopropylacetate
Standard 10mM KH2PO4 in 0.5Mperchloric acid
Put 1.5 ml of Soln. B and 2.0 ml of Soln. C into phosphate-free test
tubes. The sample,which should contain notmore than 100 nmoles
phosphate, is mixed with an equal volume of Soln. A. Give 0.5 ml of
this mixture to the above mixture of B and C. Shake the obtained
mixture vigorously for 30 s and then spin in a centrifuge for a short
period to separate the phases. To avoid the decomposition of labile
organic phosphates, the extraction should be done at 0 ◦C or below.
The molybdatophosphate complex remains in the organic
phase, which is removed and read at 725 nm against a blank.
Standards are made in the range of 5–100 nmoles phosphate
per 0.5 ml.
References
Wahler BE,Wollenberger A (1958) Biochem Z 329:508
Important! Use phosphate-free test tubes (cleaned with hot diluted
HCl) or plastic disposables.
This procedure is simpler andmore reliable than that by Fiske and
Subbarow.
Solutions/Reagents A 6N HCl
B 2.5% ammonium molybdate (w/v) in ddH2O
C 10% ascorbic acid (w/v) in ddH2O
D 2% urea (w/v) in ddH2O
E Reagent after ashing: Soln. B, C, and ddH2O are mixed in
a ratio of 1:1:8
E Reagent without previous ashing: Soln. A, B, C, and ddH2O
are mixed in a ratio of 1:1:1:7
E and E are stable only for 1 day.
Standard 10mM KH2PO4
conc. sulfuric acid
conc. nitric acid
Ashing
Ashing must be done if phosphate is at least partially covalently
bound as, for example, in nucleic acids, nucleotides, or phosphoproteins.
Give 0.2 ml of conc. sulfuric acid to 1–2 ml of aqueous sample.
Concentrate the liquid carefully in a hood at about 130 ◦C and then
heat to 280 ◦C until white fog appears. After cooling, add one to
two drops of conc. nitric acid and heat again until nitrous gases
are visible. After cooling, add 2 ml of Soln. D, boil the solution for
a short period, and fill up to 5.0 ml with ddH2O.
Determination
Mix 2.0 ml of the sample solution
Determination
Mix 2.0 ml of the sample solution after digestion or sample in
Soln. A with Soln. E and E . Close the test tubes and incubate in
the dark at 37 ◦C for 1.5–2 h. After that, read samples, blank, and
standards at 750 nm.
The standard curve is made in the range of 50–350 nmoles
phosphate.
100 nmoles phosphate = 9.497 μg PO4 = 3.097 μg P
References
Chen PS, Toribara TY (1956) Anal Chem 28:1756
1.3.3 Phospholipid Determination
Solutions/Reagents A chloroform/methanol 1:2 (v/v)
chloroform
Wet the lyophilized sample with 80 μl ddH2O. After that, add
300 μl of Soln. A and homogenize the sample with a glass-Teflon
homogenisator. After addition of 100 μl chloroform, centrifuge the
mixture for phase separation. This extraction is repeated three to
four times.
Combine the organic phases and vaporize the organic solvents
a nitrogen stream. The remaining solvent is removed in vacuum.
Use the dry residue for digestion and phosphate determination
described in Protocol 1.3.2.
1 nmoles phosphate ≈ 85 ng phospholipid; 1 μg phosphate ≈ 8.3 μg phospholipid with an average Mr of 800.
References
Blight EG, DyerW(1959) Can J Biochem Physiol 37:911
1.4 Monosaccharide Determination
The quantitative determinations of the monosaccharides ribose
and deoxyribose are given in Protocols 1.2.2 and 1.2.3, respectively.
The following protocol is useful for all monosaccharides.
80% phenol (w/v) in ddH2O (phenol must be colorless)
conc. sulfuric acid
Give 1.0 ml of the monosaccharide-containing solution (10–70 μg
of saccharide) into a centrifuge tube and mix with 20 μl of Soln. A.
Add 2.5 ml of conc. sulfuric acid onto the surface of the liquid
(caution, corrosive!), then cool for 10 min and keep at 25–30 ◦C for
10–20 min. After an additional 30 min at RT, read the absorption
(hexoses) at 490 nm, pentoses at 480 nm.
The standard curve is made fromthe appropriatemonosaccharide
dissolved in water.
Important! Because phenol is used, the waste has to be disposed
according to the local regulations.
Commercially available kits (e.g., Roche Glycan Quantitation Kit)
are based on the oxidation of the carbohydrates with periodic acid
and subsequent coupling of the formed aldehyde with a hydrazide
(e.g., digoxigenin hydrazide). The formed conjugate is estimated Immunochemical
immunochemically by ELISA.








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