\Ye wish to report the use of partition coluriir~s~:~ssaysin wliich starting materials with assay values in the isolation of sheep hormone preparations of ol 80 to 40 u./mg. gave Fractions I1 with 200 to high biological activities (200 to 400 u./rng.) without 400p./mg.Y When 1.1 ing. of the solids of Fraction prior peptic digestion. I1 were rechromatograrned, Fig. 1 (solid circlesj, Three to seven mg. of material purified by the a symmetrical peak containing virtually all of the oxycellulose method2b and containing 30 to 40 material (96%) emerged close to the expected posiu./mg. were placed on a column 1.2 cm. in diameter tion (I? value of 0.72). Thus the material compriscontaining 12 g. of kieselguhr6 and 10 ml. of 0.2 N ing Fraction I1 showed no gross inhomogeneity by HCl saturated with isobutyric acid7 as the station- the criterion of partition chromatography which ary phase. The moving phase was composed of iso- was applied. butyric acid saturated with 0.2 N HCI. The colThe authors are greatly indebted to Professor umn was run at a constant temperature of 24' and C. H. Li for the starting materials and for furnisha t a flow rate not greater than 6 ml. per hour. ing the facilities for the biological assays reported After the fraction containing the biological activity in this paper. had emerged, the remainder of the material was ((0 Based on weight as determined by the colorimetric procedure.& eluted with 6 N HC1. r)EPARl'hlENT O F BIOCHEMISTRY The results of a typical experiinent in which ilia- UNIVERSITY OF CALIFORNIA G E O R G E P. IIESS terial containing 0.820 mg. of nitrogen was placed RISRKELEY 4 CALIFORNIA 1'REDERICK 11. C A R P E N T E R on the column are shown in Fig. 1 ( o p w circles). IZECEIVEDSEP~EMBER 2, 1932 The amount of substance in each tube was deterAs judged mined by the method of Lowry, et by this colorimetric procedure, the starting material INCORPORATION OF LABELED CARBON DIOXIDE was recovered completely from the column. FracINTO PYRUVATE AND a-KETOGLUTARATE' tion I (11% of the nitrogen and 5% of the activity) Sir: started to emerge from the column after the first The pyruvic oxidase of pigeon breast muscle? 17 ml. and was contained in the following 14 ml. catalyzes the incorporation of C1*02into pyruvate The material in the next 17 i d . , Fraction 11, con- (Table I). No addition of cofactors is required for of the activity. tained 10% of the nitrogen and reaction but with added cocarboxylase the rate The material eluted by 6 iL'HCl, Fraction 111 con- this of incorporation is increased 3 to 4 fold. However, tained i'4yOof the nitrogen and no activity without added Mg++ or cocarboxylase there is no detectable decarboxylation of pyruvate to acetoin. Similarly the enzymatic oxidation of pyruvate by ferricyanide requires Mg++ and cocarboxylase. Thus the only activity exhibited by pyruvic oxidase without any additions is the incorporation of COS, presumably by an exchange reaction as follows I)yru\,ttc
g?
(,iccll.tltleh)rde-eii~yiiie) -t- CO1
'1 lie only I\tiowii cofactor presciit in pyruvic oxitlasc. is potogen or thioctic acid,' wliich according to (>urisalus,et ~ l . is, part ~ of a iiiore coniplex coeii~)-inc tentatively ideritified by Reed, et d.,5as
1i~)oic~l-thiainiiie pyrophosphate. TABLE I LABELED CARBON D I O X I D E I N r O PYRUVATE The components of the system were pyruvate (35 pmoles), KHC140a(2.1 X 106 cts./min.) and pyruvic oxidase (250 units) in a total volume of 1.3 ml a t PH 7.0; incubated in nitrogen at 37'. IUCORPORATION O F
16
24
32
40
48 86
94
ELUATE
102
110
I18
126
134
142
I50
VOLUME, m i
Fig. 1.-Chromatogram of oxycellulose purified sheep ACTH preparation (open circles); rechromatogram of Fraction I1 (closed circles).
In many experiments on different preparations of starting material similar results were obtained. The material in Fraction I1 had approximately the same R valuesa (0.GO to 0.70) from experiment to experiment and represented on a nitrogen basis an 8- to 12-fold purification of the starting material. This purification was also reflected in the biological ( 5 ) (a) A. J. P. Martin and R. L. M. Synge, Biochcm. J . . 35, 1358 (1941); (b) A. J. P. Martin and R. R.Porter, ibid., 49, 215 (1952). fa) Hyflo Super-cel, Johns-Manville Company. (7) F. H . Carpenter, C. T.Hess and C. H. 1.i. J . B i d Chent , 197, 7 (1 952). (8) 0. H. Lowry. N. J . Roscbr
