VOL. 4 (1950) 



ENZYMATIC CONDENSATIONS WITH NH2OH 



303 



The precipitate was dissolved with Krebs-Ringer containing o.oi molar ammoniuni hydroxide in 

 a 10 ml of Krebs-Ringer containing o.oi ammonium hydroxide. Most of it went into solution and a 

 little undissolved was discarded. This fraction L-3 was practically inactive. 



Fraction L-4 obtained by full saturation with ammonium sulphate. — This is the most active 

 fraction. To the half saturated ammonium sulphate solution (L-2) 37 grams per 100 ml of solid 

 ammonium sulphate were added. The total volume of 250 ml obtained. This was warmed to 30-35° 

 and filtered overnight in the cold room. The almost colourless filtrate was discarded. The precipitate 

 was dissolved in 15 ml water; it dissolved very completely to a dark red fluid. It was dialysed again.st 

 distilled water with agitation at room temperature for T,y2 hours. The volume increased to 32 ml and 

 very little precipitate was formed, which we centrifuged off and discarded. This is fraction L-4. 



Pancreas Lipase 



Pancreatine Parke-D.wis as obtainable on the market was used. Some fractionation of this 

 product is described later on in the text.. 



RESULTS 



In the first two tables, the Upase-catalysed hydroxamic acid formation is compared 

 with the acetate + ATP reaction. In Table I, the inactivity of ATP with octanoate is 

 contrasted with its action on acet-hydroxamic acid formation. It appears that the op- 

 timum concentration of hydroxylamine with ATP and acetate is 0.02 molar and that 

 at 0.05 molar already an inhibition is observed. Table II shows the effect of increased 

 concentrations of hydroxylamine on the condensation with octanoate. The strong 

 dependence of this reaction on the high concentration of hydroxylamine will be noted 

 as well as its independence on the presence of ATP. In the further study generally 

 an 0.4-0.6 molar concentration of hydroxylamine was used. 



TABLE I 



HYDROXAMIC ACID FORM.\TION WITH ACETATE -|- ATP AT VARIOUS CONCENTRATIONS OF HYDROXYLAMINE 



All tubes contained 0.5 ml of 10% fresh acetone pigeon liver extract in a total volume of i.i ml, 

 PH 7-3. temperature 37°, 60 minutes incubation. 



TABLE II 



HYDROXAMIC ACID FORMATION FROM OCTANOATE AT HIGHER CONCENTRATION OF HYDROXYLAMINE 



Each tube contained 0.5 ml rat liver homogenate (i :3 in 1% KCl, frozen for 4 days) in a total volume 

 of 1.4 ml, adjusted to pH 7-3, 37°, 60 minute incubation in air. 



Parallel experiment in a Warburg vessel with nitrogen in the gas phase. 



References p. jog. 



