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phosphatebondlab

phosphatebondlab - Chemistry 365 Biochemistry Laboratory...

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Chemistry 365 Biochemistry Laboratory Unit #4 Analysis of Organic Phosphate Bonds References : Fiske, C.H.; Subbarow, Y. J. Biol. Chem. 1925 , 66 , 375. Palmer, R.E. J. Chem. Ed. 1985 , 62 (10), 898. Introduction The phosphomolybdate method is one of the more common methods for determining inorganic phosphate in samples. Molybdic acid (generated by addition of sulfuric acid to ammonium molybdate) is added to the solution containing inorganic phosphate, and the colorless phosphomolybdic acid complex is formed. A reducing agent is then added which converts the complex to the intensely blue phosphomolybdous acid complex. This complex is quite stable and color generation follows Beer's Law. The method will not detect organically bound phosphate, so it can be used to determine inorganic phosphate, even in the presence of organic phosphates. Organic phosphates may then be hydrolyzed to yield inorganic phosphate, and this method used to quantitate organic phosphate. Organic phosphates have a central role as important intermediates in many metabolic pathways. Release of inorganic phosphate from these compounds under gentle hydrolysis conditions, allows one to characterize different forms of organic phosphate. Phosphate bonds with a free energy of hydrolysis
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