VOL. 4 (1950) 



BIOCHIMICA ET BIOPHYSICA ACTA 



211 



SPECTROPHOTOMETRIC MEASUREMENTS OF THE ENZYMATIC 

 FORMATION OF FUMARIC AND C75-ACONITIC ACIDS 



by 



E. RACKER 



Department of Microbiology, New York University College of Medicine and College of Dentistry, 



New York {U.S.A.) 



Fumaric and czs-aconitic acids are intermediates in the main pathway of substances 

 oxidized through the tricarboxylic acid cycle. With the exception of the keto-acid 

 oxidases, the enzymes participating in the cycle have been obtained in solution and 

 after purification can be studied in isolated and defined systems. Compounds such as 

 fumaric and cw-aconitic acid with an unsaturated C = C linkage have a marked absorption 

 in the ultraviolet. This property can be utilized in a spectrophotometric test, measuring 

 appearance and disappearance of these substances in the course of enzymatic reactions. 



A rapid and convenient test for the ^i i.ooOr 

 enzymes catalysing the formation of fuma- 

 ric acid from malic acid or aspartic acid and 

 the formation of czs-aconitic acid from 

 citric acid or isocitric acid will be described 

 in this paper. 



0.800 



EXPERIMENTAL 



0.600 



0.400 



0.200 



Ultraviolet Absorption Spectrum of 



Fumaric Acid and Cis-Aconitic Acid 



The ultraviolet absorption spectrum 

 of the sodium salts of these two acids is re- 

 corded in Fig. I. The fumaric acid used in 

 this experiment was a recrystalHzed com- 

 mercial preparation; the czs-aconitic acid 

 was kindly supplied by Dr S. Ochoa. As 

 can be seen from Fig. i, the absorption of 

 these compounds shows a steady rise to- 

 ward the short wave lengths. Because pro- 

 teins and nucleic acids absorb considerable 

 amounts of ultraviolet light in this region, 

 enzymes used for spectrophotometric stu- 

 dies must have a fairly high turnover num- 

 ber so that activity measurements can be 



carried out at high enzyme dilutions. The activity of enzymes with a low turnover 

 number can be tested spectrophotometrically only after considerable purification, with 



References p. 214. 



Fig. 



Ultraviolet absorption spectrum of sodium 

 fumarate and sodium c/s-aconitate. 



