Rapid and Simplified Method for Liquid Scintillation Counting of Radioactive Proteins Utilizing Aquasol Marvin A. Friedman, Gail Millar, Arthur McEvoy, and Samuel S. Epstein Children's Cancer Research Foundation, Inc., and Department of Pathology, Haroard Medical School, Boston, Mass., and New England Nuclear Corp., Boston, Mass.
LIQUIDSCINTILLATION COUNTING of radioactive protein samples is a common technique in a wide variety of analytical procedures. Generally, sample preparation involves acid precipitation of protein, removal of nucleic acids by hot acid extraction, and organic solvent extraction of lipids ( I ) . Although originally such extraction procedures involved removal of precipitated proteins by multiple centrifugations, more recent techniques have utilized filtration (2). A major difficulty in the determination of protein radioactivity is that acid precipitated protein is insoluble in most reagents used in liquid scintillation counting and must be solubilized prior to counting ; basic solubilizers used for such purposes, however, produce chemiluminescence at room temperature (3). We report here a simplified method for preparation of purified radioactive proteins and a simple efficient method for their counting by liquid scintillation.
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(1) E. Farber, J. McConomy, B. Franzen, F. Marroquin, G. A. Stewart, and P. N. Magee, Cancer Res., 27,1761 (1967). (2) F. J. Bollum, Adoan. Enzymol., 12(B), 169 (1968). (3) Technical Data Sheet, Protosol, New England Nuclear Corp., Boston, Mass., 1970. 780
ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
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MATERIALS AND METHODS Treatment of Animals. Male CD-1 mice weighing between 23 and 28 grams or male Wistar rats weighing about 220 grams were used in these studies. Mice were injected intraperitoneally with 64 mg/kg cycloheximide 2 hr prior to sacrifice and rats were injected intraperitoneally with 20 mg/kg dimethylnitrosamine 6 hr prior to sacrifice. Mice and rats were injected intraperitoneally with 1 or 5 pCi of L-leucine4,5-aH (New England Nuclear Corp.), respectively, 30 minutes before killing. Mice and rats were killed by cervical dislocation and by decapitation and exsanguination, respectively. Preparation of Liver Protein Samples for Counting. Individual livers were homogenized in 2 volumes of ice cold distilled water. Aliquots of 0.2 ml, unless otherwise specified, were added to 7 ml of 7 % trichloroacetic acid (TCA) containing 4 mg/ml celite (Celite Analytical Filter-aid, JohnsManville) and 0.1M leucine. Following heating at 95 "C for 30 minutes, the samples were applied to millipore filters (SCWP 0 25 00, Millipore, Inc.) under vacuum. The filter cake was then washed twice with 10 ml of 5 TCA and twice with 5 ml of 70 % ethanol. The filter cake was then placed in a 3-dram scintillation vial (Rochester Scientific, Rochester, N.Y.) along with 2 ml of distilled water. To this was added 6.75 ml of Aquasol (New England Nuclear, Corp., Boston, Mass.) and the vials were shaken well. These samples were then counted for 10 min each in a Beckman Scintillation Counter LS 250. Instrument counting efficiency was determined by internal standardization of duplicate samples with a calibrated tritiated water standard. In some experiments, a toluene/Triton X-100 (2: 1) scintillation solution containing 333 ml Triton X-100, 667 ml toluene, 5.5 grams PPO, and 0.1 gram POPOP per liter was used. Under normal operating conditions, an experiment with 20 animals could be completed and ready for counting 2 hr after killing the animals.
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VOLUME OF HOMOGENATE ASSAYED, ml
Figure 1. Linearity of observed radioactivity in liver protein with respect to volume of homogenate assayed
Table I. Comparison of Aquasol with Toluene/Triton X-100 Scintillation Solution Counting Radioefficiency, activitya Scintillation solutions 620 14 Aquasol Water Toluene/Triton X-100 (2 : 1) 330 31 Toluene/Triton X-100 (2:l) 440 18 2 ml water a Expressed as cpm/66 mg wet weight liver.
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RESULTS Linearity with Respect to Protein Concentration. The relationship between observed radioactivity and protein concentration was linear (Figure 1). At very high protein concentrations, self-absorption limited efficiency. Following 15 replicate determinations, mean activity from 0.2 ml of homogenate was 620 cpm with a standard deviation of 57 cpm. Since 20 cpm of this standard deviation were due to counting error, there was a 27 cpm or 4.3 deviation due to replicates. Comparison to Toluene/Triton X-100 Scintillation Solution. The results of analogous experiments using a toluene/Triton X-100 ( 2 : l ) scintillation solution are shown in Table I. When the millipore filters were placed in toluene/Triton X-100 ( 2 : l ) scintillation solution, only 50z as many counts were observed as with Aquasol-water. Following addition of 2
z
ml of water to the toluene/Triton X-100 scintillation solution, observed radioactivity was increased to 70% of the Aquasol group. The lowered counts in toluene/Triton X-100 scintillation solution were not due to lower efficiency. Inhibition of Protein Synthesis by Dimethylnitrosamine and Cycloheximide. The effects of two compounds known to inhibit protein synthesis, cycloheximide and dimethylnitrosamine, were measured by the technique described here. As can be seen, incorporation of aH-leucine following treatment with either inhibitor was clearly depressed (Table 11). DISCUSSION
Data have been presented here clearly indicating the advantages of Aquasol for determining radioactivity of proteins. Replicates prepared in Aquasol are highly reproducible; additionally, the observed radioactivity with Aquasol is higher than with a toluene/Triton X-100 (2 : 1) scintillation solution (Table I). Samples prepared in Aquasol accurately indicate the actual protein activity, as shown by a linear response to protein concentration (Figure l), and by a decrease in radioactivity of liver protein following cycloheximide and dimethylnitrosamine. Aquasol has the unique property of forming gels when mixed with water. This gel may keep the protein in
Table 11. Effects of Inhibition of Protein Synthesis of Observed Radioactivity Leucine incorporation,a Treatment Animals (No.) (mean k S.E.) Rat (4) 490 k 49 Control Rat (4) 167 + 19 Dimethylnitrosamine, 20 mg/kg Mouse (3) 438 =IC9 Control Cycloheximide, Mouse (3) 59 + 11 64 m d k g a Expressed as cpm per 66 mg wet weight liver.
solution. On the other hand, samples prepared in toluene/ Triton X-100 (2 : 1) scintillation solution settle on the bottom of the vial where self-absorption becomes an important factor. RECEIVED for review December 1, 1970. Accepted February 1, 1971. Work supported by N.I.H. Grants C-6516 and FR05526.
Luminescence Characteristics of n=Alkylcarbazoles A. W. Perry, P. Tidwell, J. J. Cetorelli, and J. D. Winefordner Department of Chemistry, University of Florida, Gainesville, Fla. 32601 IN THIS REPORT, the fluorescence and phosphorescence characteristics of several n-alkylcarbazoles are given; n-alkylcarbazoles have recently been found in cigarette smoke condensate ( I ) . These species have been shown to have high carcinogenic activity, (1-3) but have not been found in the human respiratory environment (4-6). Sensitive, selective methods of measurement of n-alkylcarbazoles are necessary to measure these species in cigarette smoke condensate ; the present study indicates that fluorimetry and phosphorimetry may have some use in these studies. EXPERIMENTAL
Apparatus. All fluorescence measurements were made with a modified Aminco-Bowman spectrophotofluorometer (No. 4-8202, American Instrument Co., Inc., Silver Spring, Md.). The standard source housing was replaced with an elliptical source condensing system (No. 4-8208, American Instrument Co.) to increase sensitivity. The xenon lamp was powered by a Harrison constant current power supply (No. 6280 A, Hewlett Packard, Palo Alto, Calif.) operated at 7.5 ampere to enhance the signal-to-noise ratio. The starter (1) D. Hoffmann, G. Rathkamp, and S. Nesnow, ANAL.CHEM.,
41,1256(1969). (2) D. Hoffmann and E. L. Wynder, Proc. Amer. Ass. Cancer Res., 7,32 (1966). ( 3 ) E. L. Wynder and D. Hoffmann, Science, 162,862 (1968). (4) R. L. Stedman, Cliem. Rec., 68,153 (1968). (5) E. L. Wynder and D. Hoffmann, “Tobacco and Tobacco Smoke, Studies in Experimental Carcinogenesis,”Academic Press, New York, N. Y . ,1967. (6) D. Hoffmann and E. L. Wynder, Chapter 20 in “Air Pollution,” Vol. 11, A. C. Stern, Ed., Academic Press, New York, N. Y . ,1968.
circuit for the lamp was described by Zweidinger and Winefordner (7). Spectra (and phosphorescence lifetimes) were recorded with the Aminco microphotometer and an Aminco x-y recorder (No. 1620-827). Intensity measurements for preparation of analytical curves were made using a low noise nanoammeter with a galvanometric recorder (8). The slit arrangement was adjusted for maximum sensitivity when making detection limit measurements and for maximum allowable resolution when recording spectra. All phosphorescence measurements were made with the modified Aminco-Bowman spectrophotofluorometer, the Aminco phosphoroscope attachment and the spinning sample cell assembly (7, 9). The same slit arrangement as described above was used for phosphorescence studies. Reagents. All carbazoles were purchased (Aldrich Chemical Co., Inc., Milwaukee, Wis.; K and K Labs, Inc., Plainview, N. Y.;Matheson, Coleman and Bell, Norwood, Ohio); the solvents, ethanol (Union Carbide Corp.) and cyclohexane (Matheson, Coleman and Bell), were prepared as follows. Ninety-five per cent ethanol was vacuum-distilled at 113 mm of Hg, resulting in ethanol of greater than 99 purity and no detectable background; cyclohexane was redistilled at atmospheric pressure in a five-foot vacuum-jacketed column, resulting in a background decrease by approximately one order of magnitude from the original starting solution. Stock solutions (0.1 mg/ml) of each carbazole were prepared. Solutions of lower concentrations were prepared by successive dilutions. (7) R. A. Zweidinger and J. D. Winefordner, ANAL.CHEM., 42, 639 (1970). (8) H. C. Hollifield and J. D. Winefordner, ibid.,40, 1759 (1968). (9) T. C. O’Haver and J. D. Winefordner, J. Cliem. Educ., 46, 241 ( 1969). ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
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