Rapid Laboratory Dialysis for Aminoacylation Assay of tRNA Hao-Chia Chen, Charles H. O'Neal,' and Lyman C. Craig The Rockefeller University, New York, N . Y . 10021 The use of a rapid dialyzer in assay procedures is described. As an example, it can substitute for trichloroacetic acid precipitation and washing in the aminoacylation assay of tRNA. Comparison with the method of precipitation on the filter paper disk indicates that the dialysis method gives more accurate results over a wide range of tRNA (0.5-200 gg) when it was used in the assay for the alanine accepting activity. The dialysis method offers the possibility of a continuous and automated assay procedure and the reusability of the tRNA. Bovine serum albumin is shown to be required as an entrapping agent at low concentration of tRNA in the precipitation method in order to achieve maximum sensitivity.
PRECIPITATION AND PRECIPITATION with precipitate adsorbed to an inert material are the commonly used methods for the separation of excess amino acids or mononucleotides and reagents from proteins or nucleic acids in assay procedures. Both methods employ denaturating agents such as TCA, perchloric acid and/or other concentrated acids to ensure precipitation. (Abbreviations used in this paper are: TCA, trichloroacetic acid; tRNA, transfer ribonucleic acid; BSA, bovine serum albumin; PEG, polyethylene glycol). These methods have at least two limitations: first, incomplete precipitation or adsorption at low concentrations of the polymerized product, a?d second, irreversible changes in conformation which in many cases result in the complete loss of biological activity. It is, therefore, desirable to investigate alternative methods for the efficient separation of small molecules from large molecules without changing the conformational state of the large molecule or at least to keep such change to its lowest level. The dialysis method, which separates molecules in a homogeneous phase by differential diffusion through an inert semipermeable membrane is ideal for this purpose. Although it has been used widely on the preparative scale, its application to microscale separations and analytical methods has been restricted because it is a slow process. Thin film dialysis, developed in recent years ( I ) , increases the speed and efficiency and thereby makes possible either a static dialyzer or a continuous dialyzer with countercurrent flow (2, 3). The usefulness of the dialyzer has been briefly discussed earlier (3). In this paper we describe its application as a substitute for TCA precipitation in the aminoacylation assay of tRNA. EXPERIMENTAL
Apparatus. The laboratory dialyzer (Figure 1) was essentially that described previously (3). Specific features of the apparatus pertinent to this report are mentioned here. The dimensions of the essential parts of the dialysis column were: length 90 cm, 0.d. of the inner tube 17 mm and i.d. of the outer tube 17.3 mm. These provided: the effective area of the membrane 480 cm2, volume inside the membrane Present address, Department of Biophysics, Medical College of Virginia, Richmond, Va. 23219. (1) L. C. Craig, Scieuce, 143, 687 (1964).
(2) L. C. Craig and K. Stewart, Biochemistry, 4, 2712 (1965). (3) L. C. Craig and H. C. Chen, ANAL.CHEW,41, 590 (1969).
3 ml, thickness of film solution inside the membrane 0.03 mm, and volume of diffusate outside the membrane 10 ml. Visking dialysis casting No. 20 was used. Chemicals. E. coli B unstripped tRNA was purchased from General Biochemicals. It was stripped from amino acids in 1.8M Tris-HC1, p H 8.0 a t 37 "C for 90 minutes and further fractionated with isopropanol t o yield 4s tRNA of more than 98% purity as judged by Sephadex G-100 gel chromatography in 1 M NaCl and 0.01M cacodylic acid-KOH p H 6.8. (14C)-~-alanine (uniformly labelled) and scintillator reagents were purchased from the New England Nuclear Corporation. The amino acids, labelled as chromatographically homogeneous, were purchased from Calbiochem. Polyethylene glycol 1540 (Carbowax 1540) was obtained from the Mann Research Laboratories. Reaction Mixture and Assay Methods. The reaction mixture for each assay in a volume of 0.3 ml contained : cacodylic acid-KOH buffer pH 6.8, 45 pmole, ATP (neutralized with K O H ) 1.5 pmole, MgClz 2 pmole, P-mercaptoethanol 1.2 pmole, NHICl 0.2 pmole, (1C)-alanine (10 pCi/pmole) 0.2 pCi and 0.02 ml of a solution of crude activating enzymes in 50% glycerol. The reaction was initiated by adding 0.3 ml of the reaction mixture to a small test tube which contained 0.2 ml of the t R N A solution in 0.05M cacodylic acid-KOH buffer, p H 6.8. After a 15-minute incubation at 37 "C, the reaction was stopped by immersing the test tube in dry ice-acetone. It was thawed in ice water just prior to use. The sample was divided into two 0.2-ml portions, one of which was pipetted onto a paper disk and washed by 5 % TCA as described in Method I. Another portion was applied t o the dialyzer in Method 11. METHODI. The method was essentially that described by Cherayil et al. ( 4 ) . Each paper disk (Whatman 3 M M 2.3 cm in diameter) was numbered and supported by a pin in a Styrofoam block. A series of these pins were linked by sticking them into a 45-pound soft nylon squidding line. T o each disk 0.2 ml of the assay sdmple and 0.05 ml of bovine serum albumin solution (5.0 mg/ml) were added. After drying under a ventilated hood, they were immersed gently into the following solutions in sequence at an interval of 10 minutes for the first wash of cold 5 TCA containing lO-4M nonradioactive alanine 5 minutes each for five times in cold 5 % TCA, twice in ethanol-diethylether mixture (3 :1 by volume) and once in anhydrous diethylether. After drying under a stream of warm dry air, each disk was transferred to a scintillation vial containing 10 ml of PPO and POPOP in toluene. METHOD11. A n assay sample of 0.2 ml with a washing of the same volume was pumped into the retentate stream at a rate of 0.4 ml/min. Between samples a 10-minute interval was spaced by pumping in a buffer solution (0.01M cacodylic acid-KOH p H 6.0). In the diffusate stream, the same buffer flowed downward at 3.0 ml/min. The retentate effluent was collected directly in a scintillation vial containing 10 ml of Bray's solution (5). After 2 minutes of collection, each vial contained 0.58 ml of the retentate in addition t o Bray's solution. All radioactivities were measured in a Tri Carb liquid scintillation spectrometer, Model 3320 or 3385 equipped with a n automatic external standardization. (4) J. D. Cherayil, A. Hampel, and R. M. Bock, Methods Enzymol., 12B, 166 (1968). ( 5 ) G. A. Bray, Anal. Biochem., 1, 279 (i960). ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
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Table I. Reproducibility of Precipitation and Dialysis Methods Radioactivity incorporated, cpm Std dev, ( n = 5 ) tRNA, p g Precipitation Dialysis Precipitation Dialysis 20 410 368 13.8 7.6 40 839 816 7.1 5.4 6.4 2.3 100 2490 2440 13.4 4.9 200 4310 4950
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Figure 2. Pattern of retentate effluent for different (14C> alanine assay solution with amounts of tRNA as shown RESULTS AND DISCUSSION
It has been shown from this laboratory (3) that NaCl and bacitracin (mol wt 1422) were virtually all removed from the retentate stream to leave a narrow and coherent band of the nondialyzable dextran blue when a pulse of solution containing these solutes was injected in the retentate stream of the dialyzer against a diffusate countercurrent stream. The essential parameters required to yield such a result are thought to be the thin film under the tension of the membrane and under the microagitation caused by the rotating outer tube. The profiles in Figure 2 demonstrate the result. When assay solutions containing tRNA after aminoacylation with [ "1alanine were successively passed through the dialyzer, peak heights of [14C]-alanine incorporation at the same concentration of tRNA are fairly even indicating a high degree of 1018
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Figure 3. Dose-response curves for tRNA assayed by the dialysis (e) and precipitation (A) methods. Vertical lines show ic Std div, n = 5
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ANALYTICAL CHEMISTRY, VOL. 43, NO. 8 , JULY 1971
Table 11. Clearance of Amino Acids by Rapid Dialysis Amount Amino Amount applied Method of remaining, acids in 0.5 ml analysis % Alanine 0 . 2 pCi Radioactivity
