VOL. 4 (1950) BIOCHI MICA ET BIOPHYSICA ACTA 3OI 



LIPASE- CATALYSED 

 CONDENSATION OF FATTY ACIDS WITH HYDROXYLAMINE 



by 



FRITZ LIPMANN* and L. CONSTANCE TUTTLE** 



Biochemical Research Laboratory, 



Massachusetts General Hospital and Department of Biological Chemistry, 



Harvard Medical School, Boston, Massachusetts [U.S.A.) 



Some time ago we reported preliminarily on two different types of enz5miatic 

 reactions leading to a condensation with hydroxylamine^. Acetate when incubated with 

 adenosine triphosphate and hydroxylamine was found to yield acet-hydroxamic acid 

 in fresh pigeon liver extracts. This reaction is specific for acetate, depends strictly on 

 ATP, and occurs only in fresh liver extract of the pigeon but not of rat, rabbit or hog. 

 The reaction is lost with aging but is regenerated on addition of coenzyme A and thus 

 belongs in a class with the coenzyme A dependent acetyl transfer reaction. The charac- 

 teristics of this type of hydroxamic acid acid formation will be reported on elsewhere in 

 more detail. 



The second reaction was of an entirely different type. It occurred only with higher 

 concentrations of hydroxylamine and was fully independent of ATP. In the meantime 

 we studied this reaction extensively and are reporting here the results obtained. It is 

 found to occur only weakly with acetate but increasingly with the lengthening of the 

 fatty acid chain, up to an optimum at octanoate. It is present in comparable strength 

 in all liver extracts studied so far. It does not diminish appreciably on aging or dialysis. 

 In contrast to the acetate reaction with ATP, it was strongly inhibited by fluoride. 

 This and other observations eventually led to the conclusion that we were dealing here 

 with a lipase-catalysed condensation of the fatty acid carboxyl with hydroxylamine. 



METHODS AND ENZYME PREPARATIONS 



Hydroxamic Acid determination. — The previously described method^ was designed for a deter- 

 mination of acyl phosphate formed during enzymatic incubation. Hydroxylamine was added at the 

 end of incubation to react non-enzymatically with pre-formed acyl phosphate at a pH of slightly 

 above 6. Subsequently, after deproteinization with trichloracetic acid, the color was developed with 

 acid ferric chloride. In contrast to this earlier set-up, the hydroxylamine now is part of the reaction 

 system and is present during incubation ; the method is modified to determine the enzymatically 

 formed hydroxamic acid. The experiment is generally terminated by addition of a mixture of trich- 

 loracetic acid, hydrochloric acid and additional hydroxylamine. Finally ferric chloride is added. The 

 addition of hydroxylamine serves only to stabilize the color but does not participate in primary 



* I am happy for the opportunity to express with this contribution my gratitude and increas- 

 ingly realized indebtedness to Professor Otto Meyerhof and his laboratory for what I imbibed 

 there during my apprenticeship from 192 7- 1930. 



** Present address: Department of Chemistry, University of Nebraska. 



References p. jog. 



