An Experiment to QuantitateOrganically Bound Phosphate With Special Emphasis on Biochemical Molecules Richard E. Palmer Hampden-Sydney College, Hampden-Sydney. Virginia 23943 An excellent assay for inorganic phosphate was originally develoned bv Fiske and Subbarow? This colorimetric assav is baseh on the reduction of acid molyhdate by sodium bisuifite and elon and has meat simnlicitv and utilitv for the . rapid determination of inorganic phosphate from any source. Biochemists expanded the orieinal assav to include another class of compounds, the organic phosphates, which occupy a central role in metabolic pathways. By employing a variety of conditions to liberate phosphate from these organic compounds, the seven-minute and total-hydrolysis procedures were developed. Bonds characterized by a relatively high free energy of hydrolysis (25.0 kcal/mol) are degraded when treated with dilute acid at 100°C for 7 min. Since glucose-l-phosphate falls into this cateeorv. " . i t was chosen as the comnound of reference. In contrast, a bond with a lower free energy of hvdrolvsis ester - " and ereater stabilitv.-.such as the nhosnhate . . bond in glucose-6-phosphate, requires more drastic conditions for breakdown (see below). Therefore, one can differentiate between molecules which contain phosphate honds of different reactivities by carefully controlling the experimental conditions. By using models of various types of phosphate linkages (such as the ester bond in alucose-6-~hosphate, the aEeta~bond inglucose-l-phosphate, and the phosphoric acid anhydride honds in the two terminal honds of ATP), the student can compare the bonds in experimental compounds to the models, predict the category into which the honds in these comnounds will fall. and infer the exnerimental conditions toefiwt the hydrolysis. Thus, theroncept of reactivities of v h o s ~ h a t ehonds can he clarified and reinforced by incorioratkg this assay into the biochemistry laboratory. The m&ecules that I use in this experiment include the standards of a l u c o s e - l - ~ h o s ~ h aand t e alucose-6-vhos~hate for the seven-minute and total-phospgate d e t e r k i n a b s , respectively, and experimental compounds such as ATP, ADP, AMP, NAD+, NADP+, rihose-5-phosphate, phosphoenolpyruvate, and fructose diphosphate. Data from severa1 student laboratory groups are summarized in the table. The results are expressed in terms of the moles of phosphate released ner mole~ofcomnound assaved so that the niedict i ~ n and s results can he readily and clearly analyzed. Since each group of students uses a different set of compounds, a wide range of relevant biochemical molecules are studied and the cost of the experiment is kept relatively low. Discussion of experimental error and the use of unknowns is easily accomplished. Additional intellectual exercises can be included by requiring the students to use their knowledge of chemistry and biochemistry to devise alternate theoretical methods for measuring the ~eactionby focusing on the other products released. Emphasis is thus placed on proof and corroboration of experimental results. Finally, if used early in the semester, the experiment can serve as a review of Beer's Law and of spectrophotometer operation. In summary, these experiments are quick and easy to perform, yield quantitative information on a variety of levels, emphasize the concept of experimental controls, and
Quantltationof Phosphate Released from Varlous Compounds under Condltlons of Seven-Minute and Total Hvdrolvsis
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Journal of Chemical Education
Compound
Seven-Minme Hydrolysis Males P,/ Moles Compounde
Glucose-l-phosphate Glucose-8-phosphate AMP ATP NADt NADPt
Ribose-5-ohosohale
Total Hydrolysis Moles P,d Moles Compound
1.07f 0.11 (11) 1.05 i- 0.20 (23) 1.01 i- 0.125(28) 2.80 0.30 (29) 2.06 0.23 (27) 2.94 + 0.38 (27) 1.11 i-0.18 12%)
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D a t a are repried as mean standard deviation. Numbers in parenmeres ln61cate numbers d V ~ I Y B used B in me oalculation of me mean.
integrate the experimental with the theoretical using an extremely important class of compounds, the organic phosphates, as the experimental system. Experimental Reagents
1.0 mAf glucose-l-phosphate 1.0mAl ylucoie-6-pho*phalc 1.0 mM concentrations of various compounds 10.0%Mg(NO& in ethanol (wlv)
Procedure Inorganic Phosphate Determination. Prepare phosphate standards from 0.10 to 1.0 pmol in 3.0 ml of water. Add 1.0 ml of acid molybdate reagent, 1.0 ml of reducing reagent, and dilute to 10.0 ml with water. Thoroughly mix samples after each addition of reagent, and let stand for 20 min at room temperature. Read the absorbance at 660 nm. Determination of Organically Bound Phosphotes-Higher Energy Bands. Prepare samples of glucose-l-phosphateandlor experimental compounds from 0.10 to 1.0 pmol and add 1.0 ml of 1.0 M HCI. Heat in a boiling water bath for 7.0 min, cool, and neutralize with 1.0 M NaOH. Dilute t o 3.0 ml, and determine the amount of inorganic phosphate as in the first procedure, above. Determination of Organically Bound Phosphote-Lower Energy Bonds. Prepare samples of glucose-6-phosphateandlor experimental compounds from 0.10 to 1.0 pmol and add 6 drops of magnesium nitrate solution. In a hood cautiously boil tubes to dryness with a Bunsen burner until anash is visible. Coal tubes, add 1.0 mlof 1.0M HCI, and heat in a boiling water bath for 10min.Neutralize with 1.0 M NaOH, dilute to 3.0 ml with water, and determine the amount of inorganic phosphate as in procedure 1 above.
' Fiske. C. H . and Subbarow, Y.. J. Biol Chem.. 66, 375 (1925); Clark, J. M.. and Sw.tzer.R. ,. ' Experimental Biochemistiy, ' 2nded.. W. H. Freeman and Company. New York. 1977. pp 160-162.
