A method for the concentration and for the colorimetric determination of nanomoles of inorganic pyrophosphate
Heinonen, J.K.; Honkasalo, S.H.; Kukko, E.I.
Analytical Biochemistry 117(2): 293-300
1981
ISSN/ISBN: 0003-2697 PMID: 6275737 Document Number: 168740
A generally applicable, inexpensive and sensitive method for the determination of PPi was developed. PPi was quantitatively separable from solution even in nanomolar concentrations by filtration through a membrane filter in the presence of CaCl2 and KF. The separated PPi was dissolved by immersing the filter in 0.5 NH2SO4. Pi was removed by precipitating it as a phosphomolybdate-triethylamine complex and the PPi was measured as a green pyrophosphomolybdate in the presence of 2-mercaptoethanol. Nucleotides and phosphate esters do not react. PPi can be accurately assayed even when there is a 104-fold excess of Pi. Trimetaphosphate, tripolyphosphate and tetrapolyphosphate also give this green color but the rate of the color formation is 50 times slower than that with PPi. Thus this interference of the polyphosphates can be eliminated or the polyphosphates can be assayed simultaneously with the PPi in the same sample.