VOL. 12 (1953) ENZYMIC OXIDATION OF j8-HYDROXYBUTYRATE 189 



itself an improvement of that of McKenzie^. The quinine salt, after one recrystalhzation from acetone 

 at 2° and five at about 16°, yielded a^-acid with the [aJD shown in Table I, c being determined by 

 titration with NaOH with exclusion of COa- The mother hquors from the first five recrystallizations 

 of the quinine dl-salt were evaporated in vacuo, and the resulting residue recrystalhzed three times 

 from water at 0-2°, the crystals being filtered in the cold room and washed. This material then yielded 

 /-acid with the [aJD shown in Table I, c being determined as before; [a]D was independent of con- 

 centration, in agreement with Magnus-Levy^, and decreased by approx. o.io per 1° rise of tempera- 

 ture. Allowing for temperature, the specific rotations of the d- and /-acids compared favorably with 

 the greatest values reported in the literature. The acids were converted into sodium salts, which were 

 recrystalhzed from absolute ethanol-dry ether. The specific rotations (Table I) were determined with 

 samples dried to constant weight over PgOg (o.i mm, room temperature). 



TABLE I 



specific rotations in water of d- AND /-^-HYDROXYBUTYRIC ACIDS AND THEIR SODIUM SALTS 



* After correction for 1.1% of moisture in dried salt. 

 Previous values for [a]D'- ^-Acid, +24.3 (10°; c, 2.2) (McKenzie^), /-Acid, — 24.2 (21°; c, 

 1.4-11.0) (Magnus-Levy*), — 24.8 (20°; c, 3.3) (McKenzie^), — 24.5 (25°; c, 5) (Levene and 

 HallerIS), /-Salt, —14.3 (18-20°; c, 2.4-12.6) (Magnus-Levy*,) —14.5 (15°; c, 8.5), —14.3 (I7^ 

 c, 3.4), —13.8 (17°, c, 1.4) (McKenzieS), — 14.5 (15°) (Lenoel1«). 



As /3-hydroxybutyric acid can form anhydro-derivatives^'^^-^* a sample of the /-acid was heated 

 with a measured excess of aqueous NaOH on a boiling water bath for 30 min and back-titrated 

 (phenolphthalein) with HCl. The alkah neutrahzed exceeded that required for direct titration of the 

 /-acid at room temperature by only 0.2%. A similar rest revealed no alkali-labile anhydro-compound 

 in the sodium salt of the ^-acid. 



Enzyme preparations. The easily sedimented particulate elements, consisting largely of mito- 

 chondria and nuclei, were obtained from homogenates of rat liver and kidney in two volumes of 

 0.14 M KCl - o.oi M phosphate, pH 7.5, by centrifugation as previously described in detail". The 

 residues were washed twice with cold KCl-phosphate and finally suspended in cold KCl-phosphate. 

 Such preparations were used for the experiments described in Tables II-IV. 



For the preparation of mitochondrial extracts acetone-dried powders of mitochondria formed 

 the starting material. Two types of preparation were employed. The mitochondrial fraction was ob- 

 tained from homogenates of rat liver in 8.5% sucrose as described by Schneider^^, washed three 

 times with cold 0.15 M KCl in the refrigerated centrifuge, and then suspended in about twenty 

 volumes of acetone at — 15° C with stirring. The suspension in cold acetone was repeated 4 times, 

 separating the solid material each time by means of the centrifuge. The final residue was freed of 

 acetone in vacuo, yielding a white powder which was stored at — 15° C in a desiccator. Since this 

 procedure was relatively laborious and required the use of large volumes of homogenate to obtain 

 any significant amounts of the dry powder, the procedure was abridged in the following method. 



The livers of 12 albino rats (147 g wet weight) were homogenized for exactly thirty seconds in a 

 Waring blender with 600 ml of cold 0.15 M KCl. The homogenate was strained through gauze and 

 centrifuged at 1500 g for 15 minutes in the cold. The supernatant was decanted and the residue re- 

 suspended in 600 ml fresh cold 0.15 M KCl and again centrifuged. This washing was repeated once 

 more. To the final washed liver residues (largely mitochondria and nuclei) was added 600 ml of acetone 

 at — 15° C and the mixture well dispersed. The solid material was washed twice more with acetone, 

 then freed of acetone in vacuo, yielding 9.1 g of a fluffy white powder. This was stable for at least <> 

 weeks in a desiccator at — 15° C. 



References p. 202. 



