302 



F. LIPMANN, L. C. TUTTLE 



VOL. 4 (1950) 



condensation. As previously described, the precipitate is eventually removed by filtration or centri- 

 fugation and the color determined in the supernatant. 



Determination in 50^^ alcoholic solution. — When it appeared desirable to follow the hydroxamic 

 acid formation with fatty acids of increasing chain length, it was observed that these hydroxamic 

 acids became increasingly insoluble in water and on removing the protein precipitate, considerable 



amounts were lost. It was found, however, 



2^0 i that these longer chain hydroxamic acids 



are easily soluble in 50% ethyl alcohol. 

 Therefore in the experiments dealing with 

 higher fatty acids, a revised procedure was 

 used where, after incubation, the medium 

 was brought to a concentration of appro- 

 ximately 50% in ethyl alcohol. 



Procedure of Hydroxamic Acid Deter- 

 mination in Alcoholic Solution. — To 0.5 ml 

 of enzyme-substrate-hydroxylamine mix- 

 ture, 3 ml of 95% ethanol are added and 

 well mixed. Then 



1. 1.5 ml are added of a mixture of 

 equal volumes of 28% hydroxylamine-HCl, 

 3.5 normal NaOH and a hydrochloric acid, 

 obtained by dilution of concentrated HCl 

 with 2 volumes of water, 



2. 0.5 ml of 24% trichloracetic acid 

 and finally, 



3. 0.5 ml of 10% ferric chloride in 0.2 

 normal HCl are added. The precipitate is 

 filtered or centrifuged off and the color 

 measured in the supernatant. The main 

 change of procedure is in the use of more 

 highly concentrated solutions in order to 

 keep the volume down and give space for 

 the addition of ethanol. 



Since the appearance of our original 

 method, an interesting application of the 

 hydroxamic acid-iron colour for colori- 

 metry of fatty acid esters appeared^. Esters were found to react quantitatively with hydroxylamine 

 in strongly alkaline solution and this reaction is used by Hill' for a determination of fatty acid 

 esters. An extensive and very instructive discussion of the reaction between hydroxylamine and 

 carboxyl derivatives may be found in the spot test analysis of Fritz Feigl*. 



0.5 1.0 15 2.0 



jjM hydroxamic acid in 6cc 



Fig. I. Standard curve for hydroxamic acid determi- 

 nation in 50% ethanol. Lithium acetyl phosphate 

 was used. 



ENZYME PREPARATIONS 



Pigeon and rat liver homogenate were prepared as described previously^ using 3 to 4 volumes 

 of 1% potassium chloride and 0.02 M sodium bicarbonate solution. 



Hog liver fractionation. — In this fractionation we followed roughly the procedure elaborated 

 for the purification of liver lipase by King and his collaborators®-^. Fresh hog liver was obtained 

 from the slaughterhouse and 100 grams were homogenized in a Waring blender with 200 ml of o. i 

 molar disodium hydrogen phosphate. The homogenate was frozen overnight and then centrifuged 

 for half an hour after thawing. 



Fraction L-i, obtained by removal of inactive protein by acidification. — 75 ml of the extract were 

 further diluted with 2 volumes of o.i molar secondary phosphate and recentrifuged. To the super- 

 natant 75 ml of water were added and the mixture was now acidified with 11. 5 ml of normal acetic 

 wherewith the pn was brought to 4.8. A voluminous precipitate formed and was centrifuged off and 

 discarded. 127 ml of strongly reddish, almost clear supernatant were collected. The extract was 

 neutralized with 5 ml of normal ammonia to pn 6.8. 10 ml were taken for analysis. 



Fraction L-2, obtained by removal of inactive protein by half saturation with ammonium sulphate. — • 

 122 ml of fraction L-i were mixed with an equal volume of saturated ammonium sulphate solution. 

 The mixture was shortly warmed to 30° and filtered. The filtrate was dialysed against distilled water. 



Fraction L-3, 50^^ ammonium sulphate precipitate. — The precipitate on the filter was squeezed 

 between filter paper layers and dried as far as possible. The precipitate was dissolved in about 10 ml 

 of water and dialysed in cellophane against 4 liter of distilled water overnight in the cold room. Next 

 morning the globulin precipitate formed on dialysis was centrifuged and once washed with water. 



References p. 309. 



