A Fluorometric RICHARD
Department of Pathology, University of Tennessee Medical Units, and Department of Chemtitry, Southwestern at Memphis, Memphis, Tennessee 38103 Received February
Most of the methods currently available for the determination of RNase activity are dependent on the measurement of acid-soluble products released ‘by hydrolysis of RNA. The major advantages of these methods are that they are sensitive, reproducible, and convenient for large numbers of samples (l-3). The major disadvantage, however is that there is danger of nonenzymic degradation of the RNA during the incubation, requiring careful temperature control and rapid handling (1, 3). LePecq, Tot, and Paoletti (4) recently reported that the dye ethidium bromide forms a fluorescent complex with nonhydrolyzed RNA, but fails to fluoresce in the presence of hydrolyzed RNA, thereby forming the basis of a simple, rapid method for the determination of RNase activity by observation of the change in fluroescence as a standard amount of RNA is hydrolyzed by the RNase present in the sample. MATERIALS
Ethidium bromide (2,7-diamino-9-phenylphenanthridine-lo-ethyl bromide) was purchased from Calbiochem, Los Angeles, California, and Sephadex G-25 (100-270 mesh) was purchased from Pharmacia, Uppsala, Sweden. Yeast RNA purified by the method of Crestfield, Smith, and Allen (5) was purchased from Worthington Biochemical Corporation, Freehold, New Jersey. It was then extracted with 1.0 M NaCl and the salt-insoluble fraction resuspended in HzO, followed by reprecipitation with 2 vol of redistilled ethanol twice. The precipitate was then chromatographed on Sephadex G-25 equilibrated with 0.005 succinic buffer, pH 5.95. Crystalline bovine pancreatic RNase prepared by the method of Crestfield, Stein, and Moore (6) was purchased from Worthington and standardized by measuring the liberation of acid-soluble oligonucleotides using the method of Kalnitsky, Hummel, and Dierks (7) to 64 Kunitz units/lambda with one Kunitz unit being equal to 26.3 pg of RNase. 333
Working at room temperature (21” t 2°C)) we determined RNase activity by placing exactly 2.00 ml of ethidium bromide (15 r/ml) into a test tube with 1.00 ml of physiological saline (0.085% NaCl) and a known amount of RNase. At ‘time = 0 sec. exactly 1.00 ml of RNA solution (100 r/ml) was added and the contents of the test tube mixed by inversion. The test tube was then emptied into a 5 ml cuvet within 15 set and the fluorescence recorded using an Aminco-Bowman spectrophotofluorometer. The slits were set at 5 mm and the sensitivity at 1.0. The excitation wavelength was set to 546 -+ 1 nm and the emission wavelength was set to 590 & 1 nm and the spectrophotofluorometer connected to a Sargent SRLG strip recorder. The fluorescence was recorded over 240 set time period, The fluorescence at time = 0 see was then extrapolated and the change in fluorescence (AI) during the first 45 set of the incubation determined. Using this value, the RNase activity can be determined using Figure 2. RESULTS
Figure 1 shows the relationship of the fluorescent intensity (I) with time for various activity concentrations of RNase after the addition of the RNA to the ethidium bromide-RNase containing sample. As the RNA is hydrolyzed by the RNase present in the sample, there is at first a rapid drop in the fluorescence, which finally levels out as most of the the RNA is hydrolyzed. When the change in fluorescence is plotted against the RNase activity, as shown in Figure 2, it is found that there is a linear relationship between the two. As noted above, the relationship of the change in fluorescence to RNase at time = 45 set was arbitrarily taken for the standard calibration curve in subsequent data.
Fro. 1. Change in fluorescent intensity (I) with time centrations of ribonuclease : (A) 0 Kunitz units/ml; (B) 128 Kunitz units/ml; (D) 192 Kunitz units/ml;(E) 256 confidence limits with ten determinations being made at
for various activity con64 Kunitz units/ml; (C) Kunitz units/ml. I =95’% each point.
A CB D
“F g’ 5 3
FIO. 2. Variations of fluorescent intensity with enzyme concentration: (A) at time = 90 set; (B) at time = 75 set; (C) at time = 60 set; (D) at time = 45 sec. The fluorometric method was then compared to the method of Roth an ethanol, lanthanum, and HCl precipitation @), which utilized procedure. Table 1 shows that the values obtained with the fluorometric method are within the limits of experimental error of the values obtained by the method of Roth (8). However, the fluorometic method required only the mixing of the ethidium bromide, substrate, and sample compared to the lengthy hydrolysis, precipitation, centrifugation, and dilution employed by the other available methods. Comparison of Fluorometric
TABLE 1 Method with Method Ribonuclease
Sample No. 1 2 3 4 5 6 7 8 9 10
Fluorescence* 199 64 80 128 104 24 88 176 64 80
0 Expressed in Kunitz units/ml. b Represents results of five determinations.
f 8 + 2 f 3 f 5 f 3 + 1 z!z 3 f 6 zk 2 f 3
of Roth (8)
activity” Rothb 200 60 80 130 100 25 90 180 65 80
f ). f * f f f f rt *
6 2 3 5 4 1 2 7 2 3
The method described is based on the fluorescent complex formed between the dye ethidium bromide and nonhydrolyzed RNA. LePecq and Paoletti (9) have reported that complex biological fluids such as serum or plasma do not interfere with this reaction or with the linearity of the fluorescence as related to nonhydrolyzed RNA concentration. It appears, therefore, that the method described could be used for determining the RNase activity in biological fluids or tissue extracts. SUMMARY
A method is described for the rapid, simple determination of RNase activity by following the change in fluorescence of a solution containing a dye that forms a fluorescent complex with nonhydrolyzed RNA, but fails to fluoresce in the presence of hydrolyzed RNA. The values obtained with this method are comparable to those obtained by standard methods utilizing the measurement of acid-soluble products released during the action of RNase on RNA. ACKNOWLEDGMENT This study has been supported, Tl CA-5004 and 5TOl GMal296.
in part, by Public
REFERENCES C. B., AND WHITE, F. H., JR., in “The Enzymes” (P. D. Boyer, H. and K. Myrbbk, eds.), Vol. 5, p. 95. Academic Press New York, 1961. JOSEFSSON, L., AND LAGERSTEDT, S., in “Methods of Biochemical Analysis” (D. Click, ed.), Vol. 9, p. 39. Interscience, New York, 1962. ROTH, J. S., in “Methods in Cancer Research” (H. Brusch, ed.), Vol. 3, p. 153. Academic Press, New York, 1967. LEPECQ, J. B., YOT, P., AND PAOLETTI, C., Compt. Rend. Acad. Sci. Paris 259, 1786 (1964). CRESTFIELD, A. M., SMITH, K. C., AND ALLEN, F. W., J. Biol. Chem. 216, 185 (1955). CRESTFIELD, A. M., STEIN, W. H., AND MOORE, S., J. Biol. Chem. 238, 618 (1963). KALNITSEY, G., HARMEL, J. P., AND DIERKS, C., J. Bid. Chem. 234, 1512 (1959). ROTH, J. S., Biochim. Biophys. Acta 61, 903 (1962). LEPECQ, J. B., AND PAOLETTI, C., Anal. Biochem. 17, 100 (1966).
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