308 F. LIPMANN, L. C. TUTTLE VOL. 4 (1950) 



A survey and comparison of results obtained with the Hver and pancreas enzyme 

 appear in Fig. 4. Particularly the difference in the chain length optimum may be noted, 

 the optimum being found at octanoate for liver and at dodecanoate for pancreas lipase. 

 The previously mentioned inhibitory effect of free long-chain fatty acids surely affects 

 somewhat the situation of this optimum. In the experiments with solutions of the salts 

 of higher members of the fatty acid series, the solution was prepared by warming the 

 acid with equivalent amounts of sodium hydroxide. Such solutions jelled on cooling 

 and had to be re warmed for use in the experiment. 



DISCUSSION 



There are primarily two points that seem to deserve comment; one, the low energy 

 requirement of the hydroxamic acid condensation and tico, the apparent non-specificity 

 of this reaction for an esterase. Although no attempts were made here to determine 

 accurately the equilibrium point, it is quite obvious from the relatively low concentra- 

 tion of the reactants which are sufficient to support condensation on the catalyst that 

 the change of free energy with this condensation cannot be more than a few hundred 

 calories. It nevertheless is well known that spontaneous reaction between the free car- 

 boxyl group and hydroxylamine will not occur* and that therefore hydroxylamine re- 

 mains to be regarded a trapping reagent for activated carboxyl groups. It is true that 

 such activation need not mean the actual input of considerable energy by a creation of 

 an energy-rich link. However, the acetate^ or glutamate^^ activation by primary reaction 

 with ATP, so easily measured by use of the hydroxamic acid reaction, bears evidence how 

 valuable a tool hydroxylamine has become for a detection of this type of reaction. Never- 

 theless as rightly emphasized by Chantrenne^^, a judicious evaluation of the particular 

 experimental conditions is required and the use of lower concentration of hydroxylamine 

 may be recommended in cases where an activation of carboxyl by primary formation 

 of an energy-rich linkage is suspected. 



The "non-specificity" of the here described esterase activity appears of some 

 significance. The link formed here by esterase action may be considered rather a peptidic 

 link. It is thus tempting to look at this reaction as the reverse phenomenon to the 

 esterase activity of chymotrypsin, uncovered recently by Neurath and his group^^. 



SUMMARY 



A lipase-catalysed condensation of fatty acid and hydroxylamine is described. Reaction in liver 

 extracts follows the inhibition pattern of liver lipase, hexyl resorcinol and fluoride acting as powerful 

 inhibitors. On fractionation of hog liver extract, the esterase and condensation activities remain 

 associated. An analogous reaction is found with pancreatine. 



The condensation with hy roxylamine on lipase occurs only with relatively high concentrations 

 of hydroxylamine and the reaction is further enhanced by increase of the fatty acid concentration. 

 To obtain considerable hydroxamic acid formation, the concentration of 0.4 to 0.6 molar of hydroxyl- 

 amine is required. Witli liver esterase, the chain length optimum is found with octanoate, while 

 pancreas lipase reacts little with compounds containing below 8 carbons, and shows optimum activity 

 with dodecanoate. 



The observations indicate that a relatively small change of free energy occurs with condensation 

 of fatty acids with hydroxylamine to form hydroxamic acid. 



For the determination of the hydroxamic acid of long-chain fatty acids, a 50% alcoholic medium 

 is required because of the water insolubility of this compound. The hydroxamic acid determination 

 was modified for 50% ethanol-water. 



References p. jog. 



