. Cell chemistry; a collection of papers dedicated to Otto Warburg on the occasion of his 70th birthday. Warburg, Otto Heinrich, 1883-; Biochemistry. ^•OL. 12 (1953) ACETYL COENZYME A SYNTHESIS 145 Identification of the reaction products. Chou had frequently observed that the amount of phosphate Hberated was far below the amount of hydroxamic acid formed. Recently these studies were resumed, particularly in view of Lynen's identification of acetyl Co A as the mercaptoester of CoA^^. Various ob- servations then indicated that inorganic pyrophosphate rather than orthophosphate was being liberate
. Cell chemistry; a collection of papers dedicated to Otto Warburg on the occasion of his 70th birthday. Warburg, Otto Heinrich, 1883-; Biochemistry. ^•OL. 12 (1953) ACETYL COENZYME A SYNTHESIS 145 Identification of the reaction products. Chou had frequently observed that the amount of phosphate Hberated was far below the amount of hydroxamic acid formed. Recently these studies were resumed, particularly in view of Lynen's identification of acetyl Co A as the mercaptoester of CoA^^. Various ob- servations then indicated that inorganic pyrophosphate rather than orthophosphate was being liberated. It was found that the liberation of inorganic phosphate could be almost completely suppressed by the addition of fluoride. Under these conditions the easily hydrolyzable phosphate did not change, but a substance appeared which gave, with the Fiske-Subbarow molybdate reagent, a slowly developing colour similar to that observed earlier by Sacks and Davenport with inor- ganic pyrophosphate^^. In Table II an experi- ment is presented with Co A as acetyl acceptor. If fluoride is added, approximately equiva- lent amounts of pyrophosphate appear, as determined by the manganese precipitation method of Kornberg^^ and also by a colori- metric method utilizing the above-mentioned colour increase with the molybdate reagent. This colour reaction has been developed into the convenient colorimetric method for determination of inorganic pyrophosphate, which will be described ;o ?0 30 40 50 60 70 80 units CoA added Fig. 4. CoA-concentration curve. Each tube contained, in 1 ml: 25 units CoA; 10 ^M ATP; 10/^Mpotassiumacetate; 50 juM KF; 100 potassium phos- phate buffer (pH ); 200 hydroxylamine neutralized with KOH to pH ; 10 /xM glutathione; ml (i unit) of yeast fraction i. Tubes were incubated at 37° for 20 minutes at which time ml of the 10% FeClg reagent were added. TABLE II EFFECT OF FLUORIDE ON PYROPHOSPHATE FORMATION Fluoride added /iM per ml CoA '
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