p-Bis-(o-MethyIstyry()Benze ne, Useful Second a ry Fluor for Liquid Scintillation Counting
SIR: Our laboratory routinely runs several different radioassays for enzyme activity which require the use of a liquid scintillation counter. The solvent systems required for these assays are all different, but the primary and secondary solutes employed as fluors are common to all systems. Diamine oxidase activity (3) is assayed using toluene, monoamine oxidase activity (4) is assayed using anisole, and histidine decarboxylase activity (2) is assayed using a mixture of dioxane, anisole, and dimethoxyethane (DAD). The radioactive substrate for the various enzyme determinations is usually assayed in a toluene-ethanol mixture. The primary fluor used is 2,5-diphenyloxazole (PPO) and the secondary fluor is p-bis-[2(4 -methyl - 5 phenyloxazoly1)lbenzene (dimethyl POPOP). A new secondary fluor, p -bis - ( 0 -methylstyryl)benzene, (bis-MSB) has recently become available commercially. This secondary fluor was substituted directly in place of dimethyl POPOP in four of our liquid scintillation counting solvent systems and an increase in counting efficiency was observed in every case (Table I).
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EXPERIMENTAL
Materials. Tritiated water, 1.368 106 d.p.m./ml., tritiated toluene, 2.14 x 106 d.p.m./mi., and C14toluene, 3.83 x l o 5 d.p.m./ml. were obtained from New England Nuclear Corp. C14 - carboxyl - labeled DL - histidine (Merck, Sharpe, and Dohme, Canada) was made up to contain 1.91 X lo5 d.p.m./lO pg. m-histidine/O.l ml. aqueous solution. PPO and bis-MSB were obtained from Pilot Chemical, Inc.
x
Dimethyl POPOP was obtained from Arapahoe Chemicals, Inc. All solvents were reagent or analytical grade from Distillation Products or Fisher Scientific
co.
Apparatus. The liquid scintillation counters used were a Packard, Model 314 EX-2, and a Nuclear-Chicago, Model 725. For carbon-14 assay, one pair of discriminators (channel 1) was set so that approximately 50% of the carbon spectrum including the 156-k.e.v. beta emission was in the window. The second pair of discriminators (channel 2) was set so that approximately 75% of the carbon spectrum including the 156-k.e.v. beta emission was in the window. The ratio, channel l/channel 2, is an accurate monitor for absolute counting efficiencies between 15 to about 55% (:). For tritium assays, the discriminators were set for the best figure of merit (efficiency*/background) and the counting efficiency was determined by the addition of an internal standard. Solvents Systems. TOLUENE. Toluene contained 0.5% PPO and 0.01% of either dimethyl POPOP or bis-MSB. To 10 ml. of this solution was added either 38,300 d.p.m. of C14toluene (0.1 ml.) or 214,000 d.p.m. of tritiated toluene (0.1 ml.). ANISOLE. Anisole contained 0.6% PPO and O . O l ~ o of either dimethyl POPOP or bis-MSB. To 10 ml. of this solution was added either 38,300 d.p.m. of CI4toluene (0.1 ml.) or 214,000 d.p.m. of tritiated toluene (0.1 ml.). TOLUENE-ETHANOL. This mixture contained 7 parts toluene to 3 parts absolute ethanol (v./v.), 0.35% PPO, and O . O l ~ o of either dimethyl POPOP or bis-MSB. To 10 ml. of this mixture was added 0.2 ml. of water containing either 273,600 d.p.m. of tritium (as
water) or 191,000 d.p.m. of C14 as DL-histidine (10 pg.). DAD. This mixture contained 6 parts dioxane, 1 part anisole, 1 part DAD (v./v.), 0.7% PPO, and 0.005% of either dimethyl POPOP or bis-MSB. To 15 ml. of DAD was added 2 ml. of water containing either 191,000 d.p.m. of Cl4as Dbhistidine (10 pg.) or 273,600 d.p.m. of tritium (as water). DISCUSSION
Similar results were obtained with the Packard and the Nuclear-Chicago liquid scintillation counters. No attempt was made in this survey to determine optimum concentrations of the fluors used. In view of the fact that a modest improvement in counting efficiency was noted in every case by the direct substitution of bis-MSB for dimethyl POPOP in the radioassay solvent mixtures used in our laboratory, this observation is reported as a possible aid to others who may be seeking means of improving counting efficiency. It should be noted, however, that there was no apparent relationship between the improvement in counting efficiencies due to the bis-MSB and the absolute counting efficiencies. It is entirely possible that a systematic study of varying concentrations of primary and secondary fluors will lead to even greater gains than these reported here. ACKNOWLEDGMENT
The author gratefully acknowledges the technical assistance of Robert J. Seymour and Jacob Kupelian. LITERATURE CITED
(1) Baillie, L. A., Intern. J. Appl. Radiation Isotopes 8 , 1 (1960).
Table 1.
Effect of Substituting bis-MSB as a Secondary Fluor for Dimethyl-POPOP in Various Solvent Systems Used in Liquid Scintillation Counting
Carbon-14 Tritium Dimethyl Dimethyl Solvent bis-MSB, POPOP, Increase, bis-MSB, POPOP, Increase, system c.p.m. c.p.m. yo c.p.m. c.p.m. % 50,700 0.7 2.8 51,000 21,600 21,000 Toluene 38,600 12.7 13.4 43,500 13,400 Anisole 15,200 Toluene-EtOH 41,500 39,200 5.8 16,600 15,900 4.4 13,200 10.6 7.9 14,600 60,000" 55,600" DAD All counts are averages of duplicate samples counted a total of 30 minutes. a Counted at higher gain settings because of severe quenching.
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ANALYTICAL CHEMISTRY
(2) Kobayashi, Y., And. Biochem. 5 ,
284 (1963).
(3) Okuyama, T., Xobayashi, Y., Arch. Biochem. Biophys. 95, 242 (1961). (4) Otsuka, S., Kobayashi, Y., Biochem. Pharmucol. 13, 995 (1964).
YUTAKA KOBAYASHI Worcester Foundation for Experimental Biology 222 Maple Ave. Shrewsbury, Mass. 01545 Supported, in part, by Contract AT(30-1) 2085. with the U. S. Atomic Energy Commission.
