An improved scintillation cocktail of high-solubilizing power

ANALYTICAL

BIOCHEMISTRY

51, 173-179 (1973)

An Improved Scintillation Cocktail of High-Solubilizing Power LARRY Department

E. ANDERSON’ of

Biochemistry,

Received

May

AND

University

WILLIAM of

Illinois,

17, 1972; accepted August

0. McCLURE2 Urbana,

Illinois

61801

11, 1972

A scintillation cocktail containing 25% Triton X-114 in xylene is considered for a broad range of scintillation counting applications. The cocktail gives good counting efficiencies for “H (47%) and 14C (93%). It will accept up to 30% (v/v) aqueous sample. The scintillation fluid is also used effectively with samples which are difficult to solubilize, such as the degradation products from the solubilization of polyacrylamide gels. The cocktail can be formulated for less than $2.00 per gallon.

Samples which are insoluble or which produce nonhomogeneous solutions in scintillation cocktails present a difficult problem in liquid scintillation counting. The use of polyacrylamide gels for the separation and assay of radioactive proteins typically has been hampered by such difficulty. Although several techniques have been employed to overcome the counting drawbacks associated with the use of polyacrylamide gels, most of them suffer from one or a combination of the following: low counting efficiency (especially for tritium) ; time-consuming solubilization procedures ; or poor recovery of radioactivity (l-5). Anker has recently reported the use of N,N’-diallyltartardiamide (DATDA) as a cross-linking agent in polyacrylamide gels (6). His method employs the use of periodic acid for the digestion of the gel, and has the advantage of short solubilization time at room temperature. The result’ing solutions of linear polyacrylamide chains are quite soluble in water, but are insoluble in standard scintillation mixtures, such as Bray’s (7) and Kinard’s (8). A toluene-based scintillation system has been described employing Triton X-100, a nonionic detergent, as an emulsifier of aqueous samples (9-11). This paper describes a scintillation fluid of Triton X-114 and xylene which effectively overcomes the problems associated with ‘Taken from a thesis submitted to the Graduate College of the University Illinois in partial fulfillment of the requirements of the degree of Doctor Philosophy. ‘Alfred P. Sloan Fellow in Neurosciences, 1972-1974. 173 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

of of

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handling samples containing polyacrylamide gels, particularly gels in which DATDA is used as the cross-linking agent. The performance of the cocktail was also assessed for use in other applications. An often-encountered problem is the need for an inexpensive scintillation fluid which can accept a large volume of aqueous sample without sacrificing counting efficiency. A volume of 5 ml of Tritonxylene cocktail will accept up to 2 ml of water while retaining good counting efficiencies for both tritium and carbon-14. A linear relationship exists between efficiency and external standard ratio over a wide range of quenching. In addition, the cockt#ail works well with tissue samples hydrolyzed with tissue-solubilizing agents. By using components which are commercially available in bulk quantities, the cocktail can be produced for approximately $1.50 per gallon. MATERIALS

Scintillation

Cocktails

Rohm and Haas, Philadelphia, PA, supplied the four Triton detergents which were examined: X-100, N-101, X-102, X-114. Organic solvents used were reagent grade. All of the scintillation mixtures were based on 2,5 diphenyloxazole (PPO) obtained from Internat’ional Chemical and Nuclear Corp., Waltham, MA. PPO was used at 3 g/liter in all cocktails. The exact composition of each scintillation fluid mixture is given in Methods and Results. As a result of the testing described in Methods and Results, the recommended scintillant is of the composition 0.3% (w/v) PPO and 25% (v/v) Triton X-114 in xylene. If desired, 1,4-bis- [2- (5-phenyloxazolyl) ] benzene (POPOP, 0.2 g/liter) can be added. Detewnination

of Radioactivity

Absolute efficiency curves were prepared by using [3H] or [‘“C-l toluene obtained from New England Nuclear Corp., Boston, MA. Tritiated water and [3H] or [%-lleucine, supplied by New England Nuclear Corp., were used to assess the quenching behavior and counting efficiency of polar samples in the various scintillation mixtures. “Minivials” from Nuclear Associates Inc., Westbury, NY, holding 5.0 ml of scintillation fluid per sample were used. Samples were counted on a Beckman LS-230 liquid scintillation spectrometer until a two sigma statistical counting error of less than + 1% was obtained.

IMPROVED

Polyacrylamide

SCINTILLATION

175

COCKTAIL

Gels

Eastman Kodak Company supplied the allylamine and diethyl-ntartrate from which the cross-linking agent, DATDA, was synthesized (6). Acrylamide was also obt,ained from Eastman. Gel composition was 7.0% (w/v) acrylamide and 0.27% ( w / v ) cross-linking agent. Coomassie brilliant blue, used for staining, was supplied by Colab Laboratories, Inc., Chicago, IL. The other chemicals used were of reagent grade. Tissue Solubilixa tion Packard Instrument Company, Downers Grove, IL, was the supplier of Soluene-100, which was used in tissue solubilization. METHODS

AND

RESULTS

Four different Triton detergents vverc examined for their effect on the counting efficiency in a liquid scintillation system. This was done by adding appropriate volumes of nonradioactive diluents (detergent and toluene) to a stock solution of a tritiated polar sample and PPO in toluene. The resulting scintillation cocktails each contained 5 X lo5 dpm per 5.0 ml and either 25% or 50% (v/v) solution of Triton in toluene. A similar series of sample-s was formulated in which xylene replaced toluene. The counting efficiencies of the mixtures were determined (Table 1).

TABLE Absolute

1 Solubilizing Various Triton

Counting Efficiency and Cockt’ails

Containing

Counting Organic solvent Toluene

Xylene

25% Triton

Triton

x-100 N-101 x-102 x-114

39 35 39 47 46 37 39 46

x-100

N-101 x-102 x-114 a Absolute counting [3H]toluene of known b Solubilizing power

Power of Scintillation Detergents

efficiency

(pl)a 50% Triton 26 34 30 26 30 33

efficiencies of 3H were determined activity. is rated subjectively on a scale from

by

Solubilizing

poweld

25% Triton

50% Triton

4 3 5 1 3 3 5 2

4 3 5 2 3 3 4 2

reference

1 (very

good)

to

samples of

to 5 (poor).

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Subsequently, an assessment was made of the relative solubilizing power of the scintillation fluids containing the various Tritons. Of particular interest was the solubilizing ability of the different cocktails with respect to a specific polyacrylamide gel solution. This gel solution was obtained by preparing polyacrylamide gels following the method of Davis (12), except that equal molar amounts of DATDA were substituted for N,N’-methylenebisacrylamide (6). After the gels polymerized, they were sectioned transversely to obtain slices having a diameter of 5.0 mm and a thickness of 2.0 mm. The slices were then incubated at room temperature in 0.5 ml of 2% periodic acid for 2 hr to effect oxidation of the cross links. After dissolution of the gel, a few seconds of vigorous agitation on a Vortex mixer provided for good solubilization of the gel solution in the cocktail. An aliquot of 0.5 ml of this oxidized gel solution was added to 5.0 ml of each cocktail mixture, and the solubilizing ability was assessed on a scale from 1 (very good) to 5 (poor). As shown in Table 1, mixtures of Triton X-114 in toluene or xylene were superior to the other cocktail mixtures. To determine the optimal composition, toluene-based scintillation mixtures in which the amount of Triton X-114 varied from 10 to 50% were prepared as described above. Samples of known amounts of L- [3H] leucine were added, after which the cocktails were counted both initially and after successive cumulative additions of 0.1 ml vol of water. The highest counting efficiency was obtained within the range of 20-30s detergent in toluene. Further studies were carried out using a Triton X-114 concentration of 25%. The relationship between absolute efficiency and external standard ratios (ESR) of samples in scintillants made up of 25% Triton X-114 in toluene was determined as follows. [3H]Toluene (7.55 X lo4 dpm) or [Wltoluene (4.26 X lo4 dpm) was added to 5.0 ml of the scintillation mixture. Subsequently, counting and external standardization of the samples were performed before each addition of cumulative O.l-ml vol of water. External standard ratios obtained were plotted as a function of the corresponding absolute efficiencies. Counting efficiency curves were also obtained using 3H,0 or L-[~H] leucine as the radioactive sample. The curves generated for Triton-toluene mixtures containing either polar or apolar radioactive molecules revealed nonlinear quenching behavior. By replacing the toluene with xylene, a linear relationship was obtained with tritium over a range of absolute activities from 45 to 10% (Fig. 1). As can be seen in the figure, radioactivity from both polar (3H,0, [3H] leucine) and apolar ( [3H] toluene) solutes exhibited linear quenching behavior. In the same cocktail, solutes containing 14C

IMPROVED

0

0.1

SCINTILLATION

0.2

03

177

COCKTAIL

0.4

i 0.5

ESR

FIG. 1. The variation of absolute counting efficiency with external standard ratio for tritium (0, 0, 0) and C (A, A). ESR: External standard ratio. The isotopes were added to scintillants containing 0.3% PPO and 25% Triton X-114 in xylene and were counted to +l% error at ambient temperature in a Beckman LS-230 liquid scintillation spectrometer. Labeled compounds tested included laHltoluene (O), A3Hlleucine (O), 3H,0 (a), WZltoluene (A), and rA’%lleucine (A). Samples were quenched by successive additions of water. Data obtained at representative additions of water are indicated, per volume of 5 ml of scintillant, by the vertical lines.

yielded data which fell upon two intersecting straight lines. For example, efficiencies for [14C-]toluene ranged from 93 to 62% with an intersection point at 82%. The intersection point corresponded to the development of a stable white emulsion in the counting vial. Data obtained using L- [“Cl leucine exhibited similar behavior, with slightly different absolute values of efficiency. When the scintillation fluid was used to assay radioactivit’y in polyacrylamide gels, an estimation was needed of the recovery of counts. To determine the recovery, samples of 3H-labeled protein, taken from experiments involving the transport of material through axons of nerve cells, were prepared for gel electrophoresis. The discontinuous electrophoresis procedure of Davis (12) was then employed with the modification that DATDA was used as the cross-linking agent. After electrophoresis, gels were stained and destained according to Vesterberg (1.3)

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using 0.1% Coomassie brilliant blue. Slices from the entire length of a gel were solubilized in individual vials with periodic acid and counted after addition of the Triton-xylene scintillation cocktail. The percentage recovery of isotope was computed by dividing the total radioactivity found in all slices by the radioactivity added init’ially to the gel. The recovery of isotope was 92 + 5% (n = 5). The cocktail was also investigated to determine its utility with hydrolyzed tissue samples. Sections of up to 200 mg of tissue from nerve, muscle, or brain were heated at 90°C for 15 min in 0.5 ml of Soluene-100. The hydrolyzed tissues were readily dissolved in 5.0 ml of Triton-xylene scintillation fluid and yielded a counting efficiency of 30% (tritium). Chemiluminescence was sometimes a problem with these samples. This difficulty could be overcome by the addition to the scintillation fluid of a sufficient amount of benzoic acid to neutralize the Soluene. Such neutralized samples could be counted without difficulty after a 4 to 5hr delay. While most of the observed chemiluminescence is due to the action of tissue solubilizing agents, a small amount, which varies with the batch of the detergent, is contributed by the cocktail itself. The effects of other variables on the counting behavior of the new scintillation mixture were also examined. Cooling the samples had no effect upon either the ESR or the absolute efficiency as long as the amount of water in the system does not exceed a critical level of 0.6 ml of water in 5.0 ml of cocktail. Samples in which this amount of water was reached or exceeded form an opalescent gel, with a concomitant reduction of 3-4s in the absolute efficiency for tritium. Warming the gelled samples to room temperature caused liquefaction and a restoration of the loss in counting efficiency. Similar discontinuities have been reported for scintillants employing Triton X-100 (10). The effect of secondary scintillators was tested. Adding POPOP to the cocktail at a level of 0.2% resulted in a 3-4s increase in the absolute efficiency of counting tritiated water. Samples containing other agents were examined. The addition to the scintillation fluid of a given volume of 8 M urea or 1% sodium dodecyl sulfate had the same effect on absolute counting efficiency as the addition of a corresponding volume of water, but resulted in a more dramatic decrease in ESR. Certain hydrophilic samples, such as amino acids, require the presence of small amounts (l-2%) of water in the cocktail for complete solubilization. The reproducibility of counting efficiencies from batch to batch of xylene was tested. No significant variation in efficiency of counting was seen when comparing either xylene from different suppliers, or different batches of xylene from the same supplier.

IMPROVED

SCINTILLATION

COCKTAIL

179

DISCUSSION

The Triton-xylene scintillation mixture described will accept samples which are not solubilieed in other cocktails. In addition, the efficiencies of counting in this new cocktail are high. The best composition found so far is 25% Triton X-114 in xylene, which yields linear quench curves for tritium. Since both the basic reagents can be purchased for reasonable prices in bulk quantities, this scintillant may prove a valuable alternative to existing cocktails, most of which are either more difficult or more expensive to formulate. ACKNOWLEDGMENTS We thank This research (232-13RD), Institutes of

the Rohm and Haas Co. for their generous donation of detergents. was supported by the State of Illinois Department of Mental Health the Research Board of the University of Illinois, and the National Health (KS 09082). REFERENCES

1. FAIRBANKS, 2. 3. 4.

5. 6. 7. 8.

9. 10. 11.

12. 13.

G., LEVINTHAL, C., AND REEDER, R. H. (1965) Biochem. Res. Commun. 20, 393. YOUNG, R. W., AND FULHORST, H. W. (1965) Anal. Biochem. 11, 389. CIIOULES, G. L., AND ZIMM, B. H. (1965) Anal. Biochem. 13, 336. MAIZEL, J. V. (1966) Science 151, 988. PAUS, P. N. (1971) Anal. Biochem. 42, 372. ANKER, H. S. (1970) Fed. Eur. Biochem. Sot. Lett. 7, 293. BRAY, G. A. (1960) Anal. Biochem. 1, 279. KINARD, F. E. (1957) Rev. Sci. Z&rum. 28, 293. PATTERSOX, M. S., AND GREENE, R. C. (1965) Anal. Chem. 37, 854. BENSON, R. H. (1966) Anal. Chem. 38, 1353. VAN DER LAARSE, J. D. (1967) Znt. J. Appl. R&at. Zsotop. 18, 485. DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404. VESTERBERG, 0. (1971) Biochim. Biophys. Acta 243, 345.

Biophys.