396 SHORT COMMUNICATIONS VOL. 2 0 (z956) potassium D-3-phosphoglycerate, 5 ° (containing o.12 pM D-2,3-diphosphoglycerate); MgSO 4, io; tris(hydroxymethyl)aminomethane, pH 7.o, Ion; and 1.2 enzyme units of mutase-free enolase*. Up to o.25 enzyme units of mutase may be taken for assay. In the enolase assay nL-2-phosphoglycerate is substituted for the n-3-phosphoglycerate. Protein is estimated by a semimicro adaptation of a standard Biuret method. V. W. RODWELL Mcllvain Biochemical Cardiovascular Laboratories, J.C. TOWNE* * Department o/Medicine, University o/Kansas, Medical Center, S. GRISOLIA** * Kansas City, Kansas (U.S.A.) 1 p. OESPER, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. I; Academic Press, Inc., New York, I955, p. 423 • 2 R. W. COWGILL AND L. PIZER, Federation Proc., 14 (I955) I98. 3 0 . WARBURG AND W. CHRISTIAN, Biochem. Z., 31o (1941) 384 . Received January 23rd, 1956 • Prepared from Anheuser-Busch dried bottom yeast strain BSC by a modified procedure of the method of WARBURG AND CHRISTIAN 3. We are indebted to Dr. D. P. WALLACH Of this laboratory for aid in this preparation. • * Post Doctorate Fellow of the National Heart Institute, National Institutes of Health. • ** Established Investigator of the American Heart Association, Preparation of paper for quantitative chromatography of corticosteroids Quantitative chromatography of steroid mixtures is usually performed on columnsl,2, 3 despite the existence of paper methods of high resolving powerS,S; yet paper chromatography offers considerable advantages both in simplicity and reproducibility. The drawback to paper work has been the high blank value of the eluate with many methods of determination, including fluorimetry in sulphuric acid 6 and in potassium tert-butoxide ~, ultraviolet spectrophotometry and arsenomolybdate reduction s. Washing of papers in the Soxhlet apparatus has not been impressive in reducing blank values, but the following technique has been consistently satisfactory in over a year's use in this laboratory. Whatman No. 2 "Paper for Chromatography" is cut into 5° × 1. 5 cm strips. These are washed chromatographically for 24 hours with 2 N ethanolic sodium hydroxide, prepared from freshly redistilled 95 % ethanol. The papers are then washed with distilled water until the eluate is free from alkali, and finally with 95 % ethanol for three hours. After drying they may be stored indefinitely. SWEAT'S sulphuric acid-induced fluorescence has been employed by us in plasma cortico- steroid estimations involving paper chromatography. When 8 cm lengths of the prepared strips are eluted with absolute ethanol and compared fluorimetrically with i pg cortisol as standard, blank values never exceed lO% of the cortisol figure, and are usually in the range 3~°/o . The eluate from unwashed paper gives a value 1- 3 times greater than the standard. In absorptiometry at 24o rap, the paperblank reading is 5-1o% of that obtained with io pg cortisol. Limited experience with the potassium tert-butoxide method 9 indicates that blank values are even lower than with sulphuric acid. Department o/ Physiology, University o/Cape Town (South Africa) BARRY LExVIS x C. J. O. R. MORRIS AND D. C. WILLIAMS, Biochem. J., 54 (1953) .523. t M. L. SWEAT, Anal. Chem., 26 (1954) 1964. 3 T. E. WEICHSELBAUM AND H. W. MARGRAF, J. Clin. Endocrinol. and Metabolism, 15 (z955) 97 o. 4 I. E. BusH, Biochem. J., 5o (1952) 37 o. 5 R. B. BURTON, A. ZAFFARONI AND E. S. KEUTMAN, Science. i11 (i95o) 6. 6 M. L. SWEAT, Anal. Chem., 26 (1954) 773. 7 p. K. BoNnY, personal communication, (195.5). 8 V. SCHWARZ, Biochem. J., 53 (I953) 148. 9 D. ABELSON AND P. K. BoNnY, Arch. Biochem. Biophys., 57 (1955) 2o8. Received January *8th, I956

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Page 1: Preparation of paper for quantitative chromatography of corticosteroids

396 SHORT COMMUNICATIONS VOL. 20 (z956)

potass ium D-3-phosphoglycerate, 5 ° (containing o.12 p M D-2,3-diphosphoglycerate); MgSO 4, io; t r i s (hydroxymethyl )aminomethane , p H 7.o, Ion; and 1.2 enzyme units of mutase-free enolase*. Up to o.25 enzyme units of mutase may be taken for assay. In the enolase assay nL-2-phosphoglycerate is subst i tu ted for the n-3-phosphoglycerate. Protein is est imated by a semimicro adapta t ion of a s tandard Biuret method.

V. W. RODWELL Mcllvain Biochemical Cardiovascular Laboratories, J . C . TOWNE* *

Department o/Medicine, University o/Kansas, Medical Center, S. GRISOLIA** * Kansas City, Kansas (U.S.A.)

1 p. OESPER, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. I; Academic Press, Inc., New York, I955, p. 423 •

2 R. W. COWGILL AND L. PIZER, Federation Proc., 14 (I955) I98. 3 0 . WARBURG AND W. CHRISTIAN, Biochem. Z., 31o (1941) 384 .

Received January 23rd, 1956

• Prepared from Anheuser-Busch dried bo t tom yeast strain BSC by a modified procedure of the method of WARBURG AND CHRISTIAN 3. We are indebted to Dr. D. P. WALLACH Of this labora- t o r y for aid in this preparat ion.

• * Post Doctorate Fellow of the National Hear t Inst i tute , National Ins t i tu tes of Health. • ** Established Investigator of the American Hear t Association,

Preparation of paper for quantitative chromatography of corticosteroids

Quant i ta t ive chromatography of steroid mixtures is usually performed on columnsl,2, 3 despite the existence of paper methods of high resolving powerS,S; yet paper chromatography offers considerable advantages both in simplicity and reproducibility. The drawback to paper work has been the high blank value of the eluate with many methods of determination, including fluorimetry in sulphuric acid 6 and in potassium tert-butoxide ~, ultraviolet spec t rophotometry and arsenomolybdate reduction s. Washing of papers in the Soxhlet appara tus has not been impressive in reducing blank values, bu t the following technique has been consistently satisfactory in over a year ' s use in this laboratory.

W h a t m a n No. 2 "Pape r for Chromatography" is cut into 5 ° × 1. 5 cm strips. These are washed chromatographical ly for 24 hours with 2 N ethanolic sodium hydroxide, prepared from freshly redistilled 95 % ethanol. The papers are then washed with distilled water until the eluate is free from alkali, and finally with 95 % ethanol for three hours. After drying they may be stored indefinitely.

SWEAT'S sulphuric acid-induced fluorescence has been employed by us in plasma cortico- steroid est imations involving paper chromatography. When 8 cm lengths of the prepared strips are eluted with absolute ethanol and compared fluorimetrically with i pg cortisol as standard, b lank values never exceed lO% of the cortisol figure, and are usually in the range 3~° /o . The eluate from unwashed paper gives a value 1- 3 times greater than the s tandard.

In absorpt iometry at 24o rap, the paperblank reading is 5 -1o% of tha t obtained with io pg cortisol. Limited experience with the potass ium tert-butoxide method 9 indicates tha t blank values are even lower than with sulphuric acid.

Department o/ Physiology, University o/Cape Town (South Africa) BARRY LExVIS

x C. J. O. R. MORRIS AND D. C. WILLIAMS, Biochem. J., 54 (1953) .523. t M. L. SWEAT, Anal. Chem., 26 (1954) 1964. 3 T. E. WEICHSELBAUM AND H. W. MARGRAF, J. Clin. Endocrinol. and Metabolism, 15 (z955) 97 o. 4 I. E. BusH, Biochem. J., 5o (1952) 37 o. 5 R. B. BURTON, A. ZAFFARONI AND E. S . KEUTMAN, Science. i11 (i95o) 6. 6 M. L. SWEAT, Anal. Chem., 26 (1954) 773. 7 p. K. BoNnY, personal communicat ion, (195.5). 8 V. SCHWARZ, Biochem. J., 53 (I953) 148. 9 D. ABELSON AND P. K. BoNnY, Arch. Biochem. Biophys., 57 (1955) 2o8.

Received Janua ry *8th, I956