employed techniques. Some typical advantages are : standardization of the bromine reagent is eliminated, the unsaturated concentration can be determined directly in a hydrocarbon media without prior dissolution in a suitable solvent, and the limits of detection are lower than any previously reported. The ready availability of commercially produced S2Brand the simplicity and sensitivity of this method merits careful consideration as to its applicability to routine quantitative determinations of olefinic unsaturation in various nonpolar solvents. By varying the volumes of the added bromine reagent and sample and, to a lesser degree, varying specific activity of the added bromine, the method could easily be scaled up or down. Thus, samples of widely varying size and total olefin content could be conveniently analyzed. However, the usefulness of this method is somewhat limited for routine determinations by the relatively short half-life (35.3 hr) of the s2Brisotope.
One of the more important results of this study is the demonstration of the inherent sensitivity and simplicity of a radiometric halogen addition for the determination of olefinic unsaturation. It should be feasible to extend the applicability of this method for routine determinations by the utilization of a halogen compound labeled with a longer half-lived halogen, such as l3lI (tl/? 8.05 d), in the form of IC1 or IBr. In addition, several of the previously established methods, such as those of Wijs and Hanus, and the numerous modifications thereof (2), could be readily adapted to a radiometric procedure with a resultant increase in sensitivity and simplicity. RECEIVED for review April 24, 1970. Accepted June 5, 1970. This research was supported by the US. Atomic Energy Commission under Contract No. AT(11-1)-1617. This is AEC Document COO-1617-22.
Determination of Picomole Amounts of Enzymatically Formed Adenosine 3’,5’-Cyclic Monophosphate by High Pressure Anion Exchange Chromatography Gary Brooker Departments of Medicine and Biochemistry, University of Southern California, School of Medicine, Los Angeles, C a l f . 90033
THEDETERMINATION of the nucleotide adenosine 3 ’,5 ’-cyclic monophosphate (cyclic AMP) formed by conversion of adenosine triphosphate (ATP) to cyclic AMP by the enzyme adenyl cyclase is important in biological chemistry, since cyclic AMP now appears to be an important regulator of the cells activities. Hormones appear to act in part by regulating the intracellular concentration of this nucleotide ( I ) . Current methods used to measure adenyl cyclase rely upon measurement of cyclic AMP formed from ATP by activation of the glycogen phosphorylase system (2) or by incubation of the enzyme with radioactive ATP (3) and subsequently counting a fraction purified by cation exchange chromatography and Zn-Ba precipitation. The first method, when proper care is taken, has been successful but is a complex method requiring much time. The isotope method, although rapid, is not completely specific since radioactivity not due to cyclic AMP has been found in the cyclic AMP fraction (4). Another disadvantage of the presently existing methods is their inherent lack of precision. The method reported here is based upon the high pressure anion exchange liquid chromatographic separation and quantitation of cyclic AMP formed by incubation of the enzyme with ATP. The chromatographic effluent is detected with a sensitive small volume ultraviolet flow cell. This method is rapid and has a high degree of reproducibility. (1) G. A. Robison, R. W. Butcher, and E. W. Sutherland, Ann. Reu. Biochem., 37, 149 (1968). (2) . , E. W. Sutherland, T. W. Rall, and T. Menon, J . Biol. Chem., 237, 1220 (1962). (3) G. Krishna, B. Weiss, and B. B. Brodie, J . Pharm. Expt. Therap., 163, 379 (1968). (4) H. P. Bar and 0. Hechter, A m / . Biochem., 29, 476 (1969).
1108
EXPERIMENTAL
Apparatus. High pressure anion exchange chromatography was performed using a 3-meter capillary “pellicular” anion exchange column as originally described by Horvath et at. (5). A Varian-Aerograph LCS 1000 liquid chromatograph with a sensitive, 8-111, 1-cm pathlength, 254-mp ultraviolet flow cell was used. The resin was converted to the chloride form by washing the column with 0.1N HCl, and then to neutrality with water. A Varian-Aerograph 1-mV full scale strip chart recorder was used. Full scale deflection equalled 0.008 optical density unit. Reagents. All chemicals used in this study were ACS reagent grade. Cyclic AMP and other nucleotides were obtained in their highest purity from Calbiochem, Los Angeles, or Sigma Chemical Company, St. Louis. Water used was either distilled-deionized or >18 megohm/cm resistance deionized water. Procedure, A rat brain was homogenized for 30 seconds at 0 “C in 10 volumes 0.25M sucrose/gram in a Teflon (Du Pont) pestle glass homogenizer. The brain homogenate (0-0.5 mg brain) was incubated at 30 “C in a total volume of 55 pl containing 2mM Na2ATP, 4mM MgC12, 10mM aminophylline, lOmM NaF, and lOOmM Tris-HC1 pH 8.0. The samples were incubated for times as indicated in the text and stopped by the addition of 50 pl of 5 ZnS04, 50 p1 of 0.3N Ba(OH)2 at 0 OC, and centrifuged for 3 min in a Spinco benchtop centrifuge. Fifty microliters of the supernatant was removed and 10 pl of 5x ZnS04 and 10 p1 of 0.3N Ba(OH)2 added and the sample was centrifuged again. The supernatant was injected. The column temperature was 80 OC and the flow rate 12 ml/hr of pH 2.20 HCl. The HCl was calibrated with a
x
(5) C . G. Horvath, B. A. Preiss, and S. R. Lipsky, ANAL.CHEM., 39, 1422 (1967).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 9, AUGUST 1970
