VOL. 12 (1953) 



ACETYL COENZYME A SYNTHESIS 



143 



acceptor enzyme is minimized by the use of higher concentrations of hydroxylamine ^^. Thehydroxamic 

 acid assay has occasionally been checked using a procedure which measures arylamine acetylation, 

 namely: by combining the A-60 fraction of Chou with the yeast enzyme. The combined assay has the 

 advantage of greater sensitivity. As shown in Fig. ib, it is linear over a wider concentration range than 

 is the hydroxamic acid assay (Fig. la) which, at higher enzyme concentration, becomes less reliable. 



0.5 

 ml 



2.0 



xiooo 



2.5 



W l£ 2.0 05 W 15 

 enzyme * 100 ml enzyme 



Fig. la. lb. Comparison of hydroxamic acid and mixed enzyme assay. 



The combined assay system was worked out by Bessman^^, who will report on this procedure 

 separately. This assay is similar to the CoA assay described by Handschum.^cher et al'^^, using 

 however, instead of their aminoazobenzene, aminoazobenzene sulfonate as acetyl acceptor. This 

 compound has the advantage of greater solubility. 



Hydroxamic acid assay'^^. One ml assay mixture contains: 25 units CoA, 10 j^iM ATP*, 10 fiM 

 potassium acetate, 100 jiM potassium phosphate buffer, 200 /iM hydroxylamine, (neutralized with 

 KOH to pH 7.4), 50 1.1M KF, 10 jxM MgClj, and 10 /{M glutathione. The tubes are incubated at 

 37° for 20 minutes, at which time are added 1.5 ml of a ferric chloride reagent which contains 10% 

 FeCl3 and 3.3% trichloroacetic acid in 0.66 N HCl. The acethydroxamate formed is measured in the 

 Klett-Summerson colorimeter with a No. 54 filter using i ml of HgO and 1.5 ml of the FeC^ reagent 

 to set the zero point. In all cases a blank tube which contains no CoA is incubated along with the. 

 tube containing CoA. The blank reading is subtracted from the reading obtained when CoA is present 

 With increasing purification the blank reading becomes negligible. One unit enzyme activity is 

 defined as that amount of enzyme which gives a reading of 100 (0.4 j^iM of hydroxamic acid) under 

 these conditions. 



Mixed assay. One ml of assay mixture contains: 100 /<M potassium phosphate, 50 /tM KF, 

 10 [xM potassium acetate, 25 units CoA, 10 /<M ATP, 10 units A-60 enzyme^'', 10 /<M glutathione, 

 10 fj.M MgClg, and 0.6 /<M aminoazobenzene sulfonate. The pH of the phosphate-acetate-fiuoride 

 mixture is set at 7.4. The tubes are incubated 40 minutes at 37°. At this time 0.2 ml of the incubation 

 mixture is added to 3.0 ml of 10% TCA in 50% ethanol and centrifuged. The amount of dye acetylated 

 is measured by the reduction in colour observed in the Klett-Summerson colorimeter with a No. 50 

 filter. The zero time reading is about 290. The colour difference should be less than 150 Klett units. 



THE COMPONENTS OF THE ENZYMIC SYSTEM 



Chou had already found that magnesium was a component of the ATP-acetate 

 reaction^^. Magnesium dependence is shown in Fig. 2. It may be mentioned here that 

 we routinely used all salts in the form of potassium salts in view of the general experience 

 that this type of reaction seems to go better in a potassium-containing medium. We have, 

 however, not studied the effect of monovalent ions in detail. 



In Fig. 3, a pH-activity curve is shown, indicating an optimum between 7.2 and 7.8. 



CoA reduction. When hydroxylamine was used to trap the acetyl CoA formed, 

 glutathione was used to reduce the coenzyme. In stoichiometric balance experiments 

 hydrogen sulfide was generally used except when a sulfhydryl balance was desirable. 

 In this instance the CoA was reduced with potassium borohydride (Metal Hydrides, Inc.) 



* The ATP used was purchased as the disodium salt from the Pabst Brewing Company. For a 

 majority of the experiments, the CoA preparations were prepared from Stveptomyces fradiae cultures 

 by Dr. J. D. Gregory. These products were 65 to 80% pure. For some of the experiments, the CoA 

 was purchased from the Pabst Brewing Company. This product is about 75% pure. 



References p. 149. 



