212 E. RACKER VOL. 4 (195OJ 



removal of interfering absorbing substances, particularly proteins and nucleic acid. 

 Of the enzymes catalyzing the formation of unsaturated intermediates of meta- 

 bolism, fumarase, aconitase and aspartase were selected for study. 



PREPARATION OF ENZYMES 



a) Fumarase. Fumarase was prepared according to the method of Laki and Laki^ and fumarase 

 activity was measured at each stage of the purification^. It was found that the preparation at the 

 final stage still contained contaminating proteins. The crystalline precipitate obtained was found to 

 have lost most of the fumarase activity after four subsequent recrystallizations while the supernatant 

 retained the fumarase activity^. These findings confirm the report by Scott' who observed that the 

 crystalline fraction lost fumarase activity on recrj'stallization while the amorphous fraction had a 

 specific activity equal to that ascribed to the crystals by Laki and Laki^. Furthermore, the purified 

 fumarase preparations of Laki and Laki still contain considerable quantities of contaminating 

 proteins. Appreciable aconitase activity has been found in these preparations as will be described 

 below, as well as very active lactic acid dehydrogenase which represents about 20% of the protein 

 present^. 



b) Aconitase. Fumarase prepared by the method of Laki and Laki^, and kindly supplied by 

 Dr J. B. V. Salles, was found to contain an active aconitase as noted above. This preparation of 

 fumarase had been kept at 0° for several weeks and retained considerable aconitase activity. Because 

 of the known lability of purified aconitase, it was decided to investigate this preparation further. 



Fumarase was prepared, therefore, according to the method of Laki and Laki^ and fumarase 

 and aconitase activity were measured in all fractions^. A large proportion of the aconitase activity 

 was retained by the heart muscle pulp after thorough washing with water; the pulp was then ex- 

 tracted by the phosphate buffer treatment used for obtaining the fumarase activity ^. Both aconitase 

 and fumarase were purified. Aconitase showed a somewhat greater sensitivity to the acid pn used 

 in the course of the purification. On fractionation with ammonium sulphate, the fumarase precipitated 

 at lower salt concentrations, so that partial separation of the two enzymes was accomplished. 



An aconitase preparation was also made from Fleischmann's baker's yeast. Maceration juice 

 was obtained by extracting dried yeast with M/15 disodium phosphate for 3 hours at 37°. The macera- 

 tion juice was fractionated at -5° with acetone. An active fraction was obtained which precipitated 

 between 30 and 50% acetone concentration. This was dissolved in cold water and dialysed for two 

 hours against running tap water. Following centrifugation, the supernatant was further fractionated 

 by the addition of solid ammonium sulphate. The precipitate obtained at 50% saturation was col- 

 lected. Solid ammonium sulphate was added to the supernatant and the fractions precipitated up to 

 80% saturation were also collected. The aconitase activity of these fractions will be described later 

 in this paper. 



c) Aspartase. This enzyme was prepared from E. coli (strain B). The bacteria were grown in 

 neopeptone broth for 18 hours at 37° with vigorous aeration, then centrifuged and washed once 

 distilled water. They were then suspended in a small volume of distilled water and disintegrated by 

 sonic vibration^ for five minutes. After centrifugation for 20 minutes at 18000 rpm in a refrigerated 

 centrifuge, the supernatant was fractionated by means of ammonium sulphate. The precipitate ob- 

 tained at 50% saturation was dissolved and dialysed against distilled water at 0° for 24 hours. This 

 preparation of aspartase was used for the studies described in this paper and was found to be quite 

 stable if kept at 0°. 



SPECTROPHOTOMETRIC MEASUREMENTS 



a) Fumarase. The enzymatic activity of fumarase was determined in a Beckman DU 

 quartz spectrophotometer. The final volume was 3 ml including 0.05 M potassium- 

 phosphate buffer at Ph 74 and 0.05 M sodium L-malate. After addition of the enzyme, 

 the changes in absorption at 240 m/< were recorded at intervals of 15 seconds. The control 

 cell contained all the solutions except the substrate. The enzymatic reaction follows a 

 zero order course for several minutes and is measured during this period. One unit is 



defined as a change of log -5- of o.ooi per minute. The increments in optical density at 

 240 m/u. are proportional to the amount of enzyme added (Fig. 2). 



* Sonic oscillator manufactured by Ratheon Corp., Waltham, Massachusetts, U. S. A. 

 References p. 214. 



