was recorded, the voltage at the maximum, and its distance from a spectral reference peak. After correlating the peaks from the several files, the program averages the correlated peaks and tabulates them in terms of H z from TMS. TO ensure that the pen voltage is faithfully monitored and that no time scale distortion occurred in the data conversion, the program has the option of plotting the raw data o n a Calcomp plotter. Each analog spectrum, therefore, can be visually compared with its digitized version. The program will also punch the correlated frequencies o n cards and in the format required by LAOCN3 (2). A Fortran IV listing of program E D R E M is available upon request. RESULTS AND DISCUSSION
A typical N M R spectrum of benzisoxazole is shown in Figure 1 (top). The spectrum is reproduced from the pen record of a single left to right sweep-;.e., a low field to high field sweep. The bottom part of Figure 1 shows a Calcomp playback of the same sweep, as recorded simultaneously o n our data acquisition system. T o obtain the peak frequencies of benzisoxazole, we recorded ten such sweeps, five in each direction, as input to EDREM. The parameters for these sweeps were: W = 100 Hz, P = 3 sec-I, T = 985 sec, integration time per data point = ‘ / e sec. The discrimination and “noise” voltages used were 5 mV. The correlated and averaged peak positions found by E D R E M from these ten sweeps are shown as vertical lines in the top part of the figure. There is sufficient difference in the apparent peak positions between one sweep and the next, especially between sweeps in opposite directions, to require averaging over several sweeps.
One reason for these measured differences is the ringing phenomenon evident o n the right hand side of the peaks shown in the figure. The ringing prevents the pen from following each resonance faithfully and thereby gives a false indication of the peak positions. The average position of a peak, however, in the limit of a large number of sweeps taken in alternate directions, approaches the true position. In the case of the ten spectra, five in each sweep direction, recorded in the benzisoxazole analysis, the positions of 38 peaks (see Figure 1) were correlated and averaged by EDREM, which punched the computed frequencies onto cards in the format required by LAOCN3 ( 2 ) . In the absence of a data acquisition system, the usual procedure had been t o generate several spectra (typically three in each direction), manually measure the position of each peak in each spectrum, and compute the average positions. With the ability to automatically digitize and record the pen voltage, it is now feasible to carry out the peak identification and position averaging rapidly and without error o n a digital computer. Eliminating the manual labor also allows for an arbitrarily large number of sweeps to be included in the averaging, up to the limit imposed by instrument drift. The only hand computation remaining is that required to average the occasional peaks missed by EDREM. The utility of o u r technique is limited only by the spectrometer ability to resolve a particular spectrum-whether it be that of a solution or a neat liquid. RECEIVED for review April 20, 1971. Accepted June 16, 1971.
3’,5’-Cyclic Adenosine Monophosphate Phosphodiesterase Assay Using High Speed Liquid Chromatography Sam N. Pennington Dirision of’kledicaf Sciences and Department of Chemistry, East Carolina Unicersity , Greenaifle, N . C. 27834
THEIMPACT of cyclic adenosine monophosphate (Cyclic-AMP) on biochemical research has been extremely large and research publications dealing with this compound now number in excess of 4000 ( I ) . One of the particular areas of interest in Cyclic-AMP is that of the enzyme 3’,5’-Cyclic-AMP phosphodiesterase (PDE). This enzyme catalyzes the following reaction:
More thari 300 publications have appeared between 1957 and 1969 dealing with this particular enzyme ( 1 ) . A number of assay met nods exist for P D E including potentiometric ( 2 ) , isotopic (3), and spectrophotometric (3, 4). Spectrochemical methods gerierally involve coupled enzymatic reactions to yield inorganic phosphate which is determined by the method of Fiske-SubbaRow ( 5 ) and have good sensitivity. The potentiometric method lacks the sensitivity necessary to d o tissue level assay and isotopic methods have the problem of expense and hazards associated with radioactivity. We desired a rapid method capable of doing tissue level (liver) assays and preferred to measure the reaction products directly for the obvious analytical reasons. (1) “Cyclic AMP 1957-1969,” N. S. Semenuk and H. Zimmerberg,
adenosine-5’monophosphate ’ (AMP) HO OH
Ed., E. R. Squibb and Sons, Inc., Research and Development, Science Information Department, New Brunswick, N. J. 08903, @ 1970. (2) W. Y. Chwng, Ami. Biochem., 28, 182 (1969). (3) P. S. Schonhofer, I. F. Skidmore, G . Krishna, and H. R. Bourne, 2. .4nal. Chem., 252 182 (1970). (4) F. Eckstein and Hans-Peter Bar, Biochim. Biophys. Acta, 191 316 (1969). (5) C. H. Fisk.e and Y. SubbaRow. J. B i d . Chem., 66 375 (1925).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
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0.m
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Figure 2. Graph relating the production of AMP from C-AMP to protein from liver homogenate
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EXPERIhlETTAL
10
12
lu 16
15
20
22
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26
Time (minutes)
Figure 1, A. Chromatogram for the separation of 2, AMP; 3, C-AMP; 4, IMP Peak 1 represents the injection point. The operating conditions are given in the text
B . Chromatogram for the separation of the reaction products from liver homogenate 1, injection point; 2, unknown; 3, AMP; 4, C-AMP. Conditions given in the text
The rapid separation of A M P and Cyclic-AMP (6) reported by high pressure anion exchange chromatography appeared to be a logical method t o assay for A M P content. Certain questions had t o be answered to determine if this method was applicable to PDE assay. These included the possible effect of other enzymes, e.g., deamineases. This enzyme catalyzes the following reaction and would lead to obvious errors. "2
The chromatography system employed was composed of a Varian ultraviolet detector and power supply (Varian Associates, Walnut Creek, Calif.) fitted with a 10-foot by 0.040inch (i.d.) stainless steel column pack with pellicular anion exchange resin (7). The stainless steel eluent vessel was pressurized with helium (800 psig) which gave a flow rate of approximately 10 ml per hour. The column was heated to 50 "C i 1.0 by a Verabath (Fisher Scientific, Pittsburgh, Pa.) using water as the bath material. The solvent used was 0.013M HCI. A Beckman 1-mV span, IO-inch recorder (0.1 inch per minute) was used to record the chromatograms. Cyclic-AMP. AMP, and I M P were supplied by Sigma Chemical Co., St. Louis, Mo.. and stored desiccated at -20 "C. Liver tissue (3 grams) from Holzman rats was prepared by homogenating in a glass-Teflon (Du Pont) mill using tris buffer (20 ml), 0.01M (pH 7.0) that contained 1.0 m M Mn2+ ion. After homogenation, the resulting slurry was centrifuged at 5000 X G for 10 minutes and the supernatant decanted, diluted 1 ml to 5 ml in tris buffer, and used as a source of enzyme. The incubation mixture contained 0.2 ml of enzyme, 200 pg of Cyclic-AMP (sodium salt), and tris buffer to make 1 .0ml. The mixture was incubated for 30 minutes at 37 "C and then the reaction was stopped by placing the tubes in boiling water for 2 minutes. The tubes were then centrifuged for 2 minutes and the resulting supernatant (1.0 pl) was injected into the chromatograph. The protein content was determined with biuret (8) reagent using bovine serum albumin as a standard.
0
II
HO-P--0
RESLLTS AND DISCUSSION
-7Y
OH
H HO OH
+ H b bH Inosine-5'-monophosphate IMP
( 6 ) G. Brookw, ANAL.CHEM., 42 1108 (1970). 1702
"
Separation of AMP, Cyclic-AMP, and I M P are shown in Figure 1A. The conditions are as follows: Sample size, 1 p1 containing 0.3 pg of each compound; column temperature, 75 " C ; flow rate, 10 ml,hour (800 psig); sensitivity, 0.16 absorbance unit f u l l scale; eluent, 0.010M HCI. A typical separation of a reaction mixture is given in Figure 1B under the following conditions: Solvent 0.013M HCI; temperature, 50 "C; all other conditions are the same as Part A . There appeared t o be no interference from deami-
( 7 ) C. G. Horvath. B. A. Preiss, and S. R . Lipsky, ibid., 39, 1422 (1967). (8) "Experimental Biochemistry," J. M. Clark, Ed., W. H. Freeman and Company, San Francisco, Calif., 1964, p 95.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
Effect of Boiling on AMP to C-AMP Ratio in Reaction Mixture Ratio (AMP/C-AMP) Time at 100 "C, min 2 0.222 5 0.224 10 0.224 15 0.221
Table I.
nase as I M P was not detectable in the incubation mixture at a sensitivity of 20 nanograms (0.02 absorbance unit full scale). A plot of Cyclic-AMP and A M P concentration cs. A under the conditions of Figure 1B yields a straight line between 0.1 pg to 1.0 pg of the nucleotides. This allows one t o measure both product and substrate concentrations in a single run. Figure 2 gives the relationship between protein concentration and yield of A M P for a typical run. Depending o n protein concentration, one may vary the incubation time; however, it was best t o use a slightly lower protein concentration and a longer incubation t o keep the tissue blank small. All tissue examined contained a small, reproducible amount of A M P (0.00-0.03 pg) which was determined in a blank solution without added Cyclic-AMP. Blanks using boiled enzyme gave only a Cyclic-AMP peak. To determine the possible effects of boiling on the conversion of Cyclic-AMP to A M P in the reaction mixture, samples were analyzed repeatedly during a 15-minute period in a boiling bath. Other than a slight concentration due t o evaporation, n o effect was observed as evidenced by a constant A M P to Cyclic-AMP ration (Table I). Two other variables were examined. These were the egect of time o n the reaction mixture after stopping the reaction and the effects of aging o n the tissue homogenate. There appeared t o be no aging effect o n the sample mixture after boiling as samples held 24 hours at room temperature yielded identical amounts of Cyclic-AMP and A M P as previously determined. Attempted assays of brain tissue prepared in the same manner as liver tissue revealed that this material contains significant levels of 5'-nucleotidase activity under the conditions of the incubation. This enzyme converts A M P t o adenosine and inorganic phosphate, i.e.,
3 0.021.0.
0
I
5
10
w
15
20
25
(minutes)
Figure 3. Chromatogram of reaction products using rat brain tissue 1, injection point; 2, adenosine; 3, AMP; 4, C-AMP. Conditions are given in the text
0
HO-P-
II
This reaction causes a n apparent error in the PDE activity measurement in brain tissue based only o n A M P ; however, by using A M P as the substrate, one may assay for 5'-nucleotidase (see Figure 3).
RECEIVED for review April 5 , 1971. Accepted June 11, 1971
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