By Nathan P. Kaplan, Nathan P. Colowick, Donald B. McCormick, Lemuel D. Wright
The seriously acclaimed laboratory general, Methods in Enzymology, is among the so much hugely revered guides within the box of biochemistry. on the grounds that 1955, every one quantity has been eagerly awaited, usually consulted, and praised by means of researchers and reviewers alike. The sequence includes a lot fabric nonetheless suitable this day - actually a necessary booklet for researchers in all fields of existence sciences
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Extra resources for Vitamins and Coenzymes Part F
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Ohta and W. S. Beck, Arch. Biochem. Biophys. 174, 713 (1976). ~aM. Goulian and W. S. Beck, J. Biol. Chem. 241, 4233 (1966). 14R. H. Abeles, C. Myers, and T. A. Smith, Anal. Biochem. 15, 192 (1966). 4 ml. Tubes are placed in ice, evacuated, and flushed with nitrogen. Potassium borohydride, 1 mg, is added to each tube under a stream of nitrogen, followed by enzyme-containing samples. Tubes are again evacuated, flushed with nitrogen, and kept chilled until reduction of vitamin B12a is complete. The tube is incubated for 45 min in the dark at 37°; then cooled to 0°; a 10- to 50-/zl aliquot is assayed for AdoCbl.
Obshch. Khim. 42, 1202 (1972). 4 D. L e x a and J. M. Saveant, J. Am. Chem. Soc. 98, 2652 (1976). 5 I. Ya. Levitin, I. P. R u d a k o v a , A. L. Sigan, T. A. Pospelova, A. M. Yurkevich, and M. E. Vol'pin, Zh. Obshch. Khim. 45, 1879 (1975). 6 E. Knobloch, this series, Vol. 18B, p. 305. 7 B. Breyer a n d H. H. Bauer, " A l t e r n a t i n g Current Polarography and T e n s a m m e t r y . " Wiley (Interscience), N e w York, 1963. METHODS IN ENZYMOLOGY,VOL. 67 Copyright© 1980by Academic Press, Inc.
A typical standard curve (A) is shown in Fig. 1. The percentage bound o f the unknowns, determined by dividing the counts bound to the cellulose by the counts present in the 4-ml extract, is then read from the standard curve. Corrections are made for percentage r e c o v e r y , exogenous B,2 added, and the amount of tissue originally employed. The final concentration is then expressed as nanograms of B,2 per gram of tissue. Calculations cellulose counts % Bound of unknowns = extraction filtrate counts Measured pg B,2 = % bound of unknowns reading from standard curve (1) (2) Total pg Bx2 per amount = m e a s u r e d pg B12-[57Co]-B12 added (100 pg) (3) of tissue assayed % recovery B~2 ng per g tissue = total pg B12 per amount of tissue assayed corrected to 1 g and converted to ng (4) C o m p o s i t e Curve.